THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE PROTEINS, DIVERSE FUNCTIONS

A DISSERTATION

SUBMITTED TO THE DEPARTMENT OF CHEMICAL AND SYSTEMS BIOLOGY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Natalia Gomez-Ospina

May 2010

http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/np206cw1776

© 2010 by Natalia Gomez-Ospina. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.

ii

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Richard Dolmetsch, Primary Adviser

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Thomas Clandinin

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Gerald Crabtree

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Tobias Meyer

Approved for the Stanford University Committee on Graduate Studies.

Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.

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iv

ABSTRACT

Voltage-gated calcium channels are an important route of calcium entry into cells and

are essential for converting electrical activity into biochemical events. In neurons

these channels are vital for synaptic vesicle release and have been implicated in almost

every activity-dependent process including survival, dendritic arborization, synaptic

plasticity, and gene expression. One of the ways in which these channels regulate

cellular behavior is by regulating gene expression but the mechanisms that link

calcium channels to the transcription machinery are not well understood. In this thesis

I show that a C-terminal fragment of CaV1.2, an L-type voltage-gated calcium

channel, translocates to the nucleus and regulates transcription. I show that this

calcium channel associated transcription regulator (CCAT), binds to a nuclear protein,

associates with an endogenous promoter, and regulates the expression of a variety of

endogenous genes that are important for the function of neurons and muscle cells. The

nuclear localization of CCAT is regulated by changes in intracellular calcium on a

time scale of minutes, suggesting that CCAT integrates information about the

electrical activity of the cell. Together these findings reveal an entirely unexpected

function for a well-characterized calcium channel.

This works also addresses the question of how CCAT is generated. I show that CCAT

is not released from proteolysis of full-length Cav1.2 channel but is generated from an

mRNA that is transcribed from the 3’ end of the Cav1.2 gene (CACNA1C). Consistent

with this, I find that CCAT expression is independent of full-length channel protein.

v

Furthermore, Exon 46 of the CACNA1C gene contains a promoter whose

transcriptional activity is required for the expression of CCAT. Activity at this

promoter, and consequently CCAT expression, is regulated spatially and temporally in

the brain having highest expression during embryonic stages and in regions of the

brain rich in inhibitory neurons. Analysis of 5’ transcriptional starts from CACNA1C

and Cap Analysis of Gene Expression (CAGE) tags from genome-wide studies show

at least two mRNAs one of which encodes CCAT in vivo and a second transcript that

is predicted to encode a membrane bound CCAT containing a voltage sensor. These

findings reveal an unexpected mechanism by which CCAT is generated in neurons

and provide a unique example by which two proteins with distinct biologic functions

can be derived from a single gene. Such transcriptional phenomena may be at play in

many other genes throughout the genome and has far reaching implications for

prediction of gene products and interpretation of phenotypes in gene mutations and

knockout studies.

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ACKNOWLEDGEMENTS

Over the past (not so few but enjoyable) years several people helped me reach

the completion of this work. First and foremost, I must thank my advisor Ricardo. He

has consistently supported this project and my learning through intellectual advice,

financial support, and fostering a nourishing work environment that developed

independence, creativity, and camaraderie. Through a series of improbable

coincidences I became his first graduate student and never looked back. In every

situation, Ricardo is always personable, always available, and always has the utmost

confidence in his students.

My foundation as a scientist also rests on the tutelage of my previous mentors--

Dr Andrew Staehelin and Dr Tomas Giddings who first gave me the opportunity to

work in a lab. Dr Staehelin allowed me to join his when I spoke broken English, when

I was new to biology, and when I had never used a word processor. Later he would tell

me how much he had watched my “growth” and thereby let me know that he truly

understood the distance I had traveled. Tom, who was one of the kindest persons I

have ever met and who opened opportunities for me by entrusting me with difficult

projects for several accomplished scientists.

I also would like to acknowledge my colleagues in the Dolmetsch lab with

whom I am proud to have spent these formative years working side-by-side—Jocelyn,

Eric, Jake, Matthieu, Fuminori, Chan, Agatha and Georgia will be lifelong friends.

I owe a warm thank you to my family, in particular my mother Cielo. She followed

me to California and supported me in all those small ways that make the world go

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around at a smooth pace without creaking or faltering. Only with her help did I have

the luxury of dedicating so much time to my research. I have yet to explain the

importance of calcium channels or CCAT to her but it does not matter.

Thanks to Anil, who can make anything fun and believes in me more that I do. In a

world of unexpected things, difficult choices and constant compromise he gives me

the certainty that at least one thing is right and always better than I could predict or

imagine.

Lastly, none of this would have been possible without the support of

Stanford’s Medical Scientist Training Program.

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TABLE OF CONTENTS

List of Tables……………....…………………………………………………………..x List of Figures…………………………………………………………………………xi Chapter 1: Activity-dependent L-type channel regulation of gene expression Abstract….……………………………………………………………………..2 L-type calcium channels.………………………………………………………2 L-type channels and c-fos: The beginning.………….…………………………4 L-type channels and CREB.……………………………………………………5 What mechanisms link LTCs to CREB? .……………………...………………8

A. Biophysical Properties………………………….…………….……9 B. Localization…………………………………….…………………11 C. L-type calcium channels and nuclear calcium.…………….…..…12 D. Local calcium signaling………………………..…………………14

Isoform Specific Considerations……………...………………………………18 Chapter 2: The C-terminus of the L-type voltage-gated calcium channel Cav1.2 encodes a novel transcription factor

Summary……………………………………………………………………...20 Introduction……………………………………………………………...……20 Results

CCAT is found in the nucleus of neurons in the brain………………..22 The concentration of CCAT is regulated by intracellular calcium.…..26 Nuclear CCAT is regulated developmentally………………………...28 CCAT binds to a nuclear protein……………………………………..29 CCAT activates transcription………………………………..………..29 CCAT regulates transcription of endogenous genes…….……………32 CCAT bind and regulates the promoter of Cx31.1………….………..33 Endogenous Cav1.2 and CCAT regulate transcription of Cx31.1……36 CCAT expression promoted neurite growth………………….………38 Discussion…………………………………………………………..………...39 Experimental Procedures……………………………………………………..45 Future Experiments………………………………………………….………..56 Figures………………………………………………………………………...62 Supplementary Figures……………………………………………………….69 Tables…………………………………………………………………………73 Figure legends………………………………………………………………...78

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Chapter 3: An independent promoter in the CACNA1C channel gene generates the transcription factor CCAT

Summary……………………………………………………………………...88 Introduction……………………………………………………………...……89 Results

CCAT is not generated by Proteolytic Cleavage of Exogenously Expressed Channels…………………………………………………..92 Cav1.2 Channel Protein is not necessary for CCAT Expression In Vivo……………………………………………………………………………95 CCAT is translated from a Separate Transcript from the cDNA………………………………………………………………....97 An Exonic Promoter Drives CCAT Expression…………………………………………………………….98 CCAT is Translated from a Separate Transcript In Vivo whose Expression is Spatiotemporally Regulated in the Brain……………..101 CACNA1C has Multiple TSS Predicting Multiple Proteins…………104

Discussion…………………………………………………………..…….....108 Experimental Procedures…………………………………………………....117 Future Experiments………………………………………………….………130 Figures……………………………………………………………………….133

Supplementary Figures……………………………………………………...137 Tables………………………………………………………………………..141 Figure legends……………………………………………………………….142 List of References………………………………………………………..………….150

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List of Tables Chapter 2 Table 1 Genes Significantly Up-regulated by CCAT versus CCAT∆TA…………..………...73 Table 2 CCAT versus GFP Up-regulated genes…………………..…………………………..74 Table 3 CCAT versus GFP Down-regulated genes…………………………...…………...75-76 Table 4 Genes Regulated by CCAT in All Experiments………………...……………………77 Chapter 3 Table 1 Summary of transcription start sites and nearby CAGE tags………………………..141

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List of Figures Chapter 1 Figure 1 Schematic Representation of VGCC Showing the Topology of the Pore-forming α1C Subunit, and β, α2δ Accessory Subunits……………………………..………………..3 Figure 2 Schematic Representation of Signaling pathways from LTCs to CREB……………17 Chapter 2 Figure 1 The C Terminus of Cav1.2 Is Found in the Nucleus of Neurons…………...…….…..62 Figure 2 Ectopically Expressed CCAT Localizes to the Nucleus of Neurons via a Nuclear Retention Domain…………………………………………………………………….63 Figure 3 The Nuclear Localization of CCAT Is Regulated by Intracellular Calcium and by Developmental Processes in the Brain………………………………………………..64 Figure 4 The C Terminus of Cav1.2 Binds to Nuclear Proteins and Activates Transcription…65 Figure 5 CCAT Regulates Endogenous Genes………………………………………………...66 Figure 6 Endogenous CCAT Regulates Transcription Driven by the Cx31.1 Promoter………67 Figure 7 CCAT Regulates Neurite Growth in Primary Neurons……………...………….……68 Figure S1 CCAT’s Nuclear Localization and TA Domain are Conserved Among Cav1.2 Channels in Vertebrates……………………………………………...……………….69 Figure S2 CCAT Derived from Cav1.2-YFP channels is Regulated by Depolarization…….…..70 Figure S3 CCAT Regulates Expression of Endogenous Genes: Summary of Microarray Data...71

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Figure S4 sh-RNA Knockdown of Endogenous Cav1.2 in Neurons Decreases CREB Activation Induced by K+ ………………………………………………………...………………72 Chapter 3 Figure 1 CCAT is Not Generated by Proteolytic Cleavage of Exogenously Expressed or Endogenous Cav1.2 Channels…………………………………………………….…133 Figure 2 CCAT is Translated from a Separate Transcript Driven by an Exonic Promoter.…134 Figure 3 CCAT is Translated from a Separate Transcript whose Expression is Cell-type and Developmentally Regulated In Vivo……………………………………………...…135 Figure 4 CACNA1C has Multiple Transcriptional Start Sites Predicting Multiple Proteins Including CCAT……………………………………………………………………..136 Figure S1 CCAT staining in Cav1.2 knockout embryos……………………………………….137 Figure S2 Multiple Sequence alignment of Cav1.2 C-termini from multiple species………….138 Figure S3 Developmental CCAT staining……………………………………………………...139 Figure S4 Leuzine Zipper mutations and Multiple Sequence alignment from Cav1 channels...140

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Chapter 1:

Activity-dependent L-type channel regulation of gene expression

2

Abstract

Calcium-regulated transcription plays a key role in converting electrical

activity at the membrane into long-lasting structural and biochemical changes in

excitable cells. Although several calcium influx pathways contribute to the

intracellular calcium rise that follows membrane depolarization in neurons, a

preponderance of data suggest that calcium entry through voltage-gated L-type

calcium channels and NMDA receptors is particularly important in activating gene

expression. In this chapter, we review seminal work implicating L-type channels in

the induction of gene expression in response to neuronal activity and discuss some of

the mechanisms that explain the dependence of activity-induced transcription on

LTCs. We will focus our discussion on studies that explore the biophysical, structural,

and cell biological features of LTCs that allow them to activate CREB-dependent

transcription.

L-type Calcium Channels

Voltage-gated calcium channels (VGCC) are an important route of calcium

entry into neurons and are essential for converting electrical activity into biochemical

events in excitable cells (Catterall et al., 2005). All VGCCs have a common ability to

carry calcium in response to depolarization of the membrane but they differ in their

subcellular localization, biophysical properties and in their ability to regulate specific

3

biochemical processes. VGCCs are classified into L, N, P/Q, R and T types based on

their pharmacological and biophysical properties and are composed of four protein

subunits: a pore forming α1 subunit, and β, α2δ and γ subunits that modulate gating

and trafficking (Tsien and Tsien, 1990) (Figure 1). Neuronal L-type channels contain

one of three α1 subunits: Cav1.2, Cav1.3 or Cav1.4. Cav1.2 and Cav1.3 form the

predominant LTCs in the brain and have been implicated in a wide variety of neuronal

functions including promoting survival, increasing dendritic arborization and

regulating synaptic plasticity (Galli et al., 1995; Moosmang et al., 2005; Redmond et

al., 2002).

Figure 1: Schematic representation of VGCC showing the topology of the pore forming α1C subunit,

and β, α2δ accessory subunits

LTCs have a number of features that set them apart from other types of

VGCCs. They exhibit high sensitivity to dihydropyridines (DHP), are activated by

strong depolarization and have slow activation and inactivation kinetics1 (Tsien and

1 Some LTCs including Cav1.3 can be low voltage-activated and have fast kinetics of activation Lipscombe, D., Helton, T.D., and Xu, W. (2004). L-type calcium channels: the low down. J Neurophysiol 92, 2633-2641, Xu, W., and Lipscombe, D. (2001).

4

Tsien, 1990). LTCs are localized in the cell body, dendrites and postsynaptic

membranes of adult neurons, making them ideally poised to control the signal

transduction pathways that are activated post-synaptically (Hell et al., 1993;

Westenbroek et al., 1990). Finally, LTCs have been shown to be particularly effective

at activating gene expression in response to electrical activity. A key question,

however, is what features of LTCs allow them to activate the signaling pathways that

lead to the nucleus.

L-type calcium channels and c-fos: The beginning

Morgan and Curran first reported this peculiar link between LTCs and the

nucleus more than two decades ago. They discovered that depolarizing concentrations

of potassium provoked an influx of calcium ions via VGCCs that led to the

transcription of the immediate-early gene c-fos (Morgan and Curran, 1986). DHPs

and calmodulin (CaM) inhibition were found to block this effect suggesting a role for

LTC activity and the ubiquitous calcium sensor, CaM, in the expression of c-fos in

response to neuronal activity. Contemporaneous studies implicated LTCs downstream

of nicotinic receptors in the ensuing induction of c-fos and actin expression in the

same cells (Greenberg et al., 1986). In another influential study, Murphy and

colleagues showed that blocking and activating LTCs respectively eliminated and

increased basal c-fos expression in spontaneously active neuronal cultures (Murphy et

Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 21, 5944-5951.

5

al., 1991). This implied that LTCs play a role in the induction of c-fos expression in

response to endogenous electrical activity. To examine the possibility that this

observed LTC specificity was related to a greater ability of LTCs to elevate

intracellular calcium during neuronal activity, the authors measured the relative

contributions of LTCs and other ligand-gated glutamate receptors to the synaptically

evoked calcium rise. They found that LTCs contributed less than 20% of the

synaptically-induced calcium elevation, significantly less than NMDA or kainate

receptors, suggesting that the route of calcium entry rather than the absolute amplitude

of the calcium rise was important for the activation of c-fos. This implied that specific

mechanisms other than bulk calcium elevations must exist that link these channels to

the nucleus. Thenceforth, much effort has been underway to uncover such

mechanisms.

L-type channels and CREB

Dissection of the c-fos promoter by a number of groups identified two main

calcium-regulated response elements, the calcium response element (CRE) and the

serum response element (SRE) (Miranti et al., 1995; Sheng et al., 1988). The CRE

binds to the transcription factor CREB and the SRE binds to serum response factor

(SRF) both of which are activated by calcium influx in neurons. CREB has emerged as

a major regulator of calcium signaling in the brain and has been implicated in neuronal

development, survival and plasticity (Lonze and Ginty, 2002). Early studies by Sheng

and Greenberg first demonstrated that calcium influx through LTCs is particularly

6

effective at activating CREB-dependent transcription (Sheng et al., 1990). Blockers of

LTCs potently block the activation of CREB reporter genes, and calcium influx

through LTCs in developing cortical neurons is substantially more effective at

activating CREB than equivalent calcium elevations through NMDA receptors,

suggesting that LTCs are specifically linked to CREB activity (Bading et al., 1993).

The most compelling illustration of the central role of LTCs in activating CREB is a

study of LTC knockout mice. Eliminating Cav1.2 specifically in the hippocampus and

cortex of mice using CRE recombinase-mediated recombination resulted in a loss of

CREB phosphorylation in response to electrical activity, a reduction in an LTC-

dependent form of long term potentiation, and in learning deficits (Moosmang et al.,

2005). This result demonstrates the importance of LTCs in activating CREB and in

regulating neuronal plasticity of neurons in vivo.

While the mechanisms that link calcium influx through LTCs to the activation

of CREB are not completely understood, a great deal is known about how intracellular

calcium elevations can activate CREB-dependent transcription. Activation of CREB-

dependent transcription is a multi-step process that involves both the recruitment of

CREB to CRE elements, the phosphorylation of CREB and the recruitment of other

co-activators. Recent studies suggest that CREB does not constitutively occupy CRE

sites and that activation of CREB involves its recruitment to CREs via a nitric oxide-

dependent cascade (Riccio et al., 2006). At the same time as CREB is recruited to

CRE elements, it is also phosphorylated at Ser133 which enhances its transactivation

potential. This phosphorylation event is strongly calcium-dependent and is absolutely

required for the activation of CREB-dependent transcription (Gonzalez and

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Montminy, 1989). Phosphorylation of Ser133 allows CREB to recruit the CREB

binding protein (CBP), which acts as a transcriptional co-activator by means of its

intrinsic histone acetyl transferase activity and by promoting binding to basal

transcriptional machinery (Chrivia et al., 1993; Kwok et al., 1994). CBP is also

subject to regulation by calcium via two calcium-inducible transactivation domains

(Hu et al., 1999) and via calcium induced phosphorylation by CamKIV (Impey et al.,

2002). LTCs promote CamKIV mediated phosphorylation of CBP suggesting that

LTC-specific recruitment of co-activators can help explain the need for LTC activity

in the transcriptional activation of CREB (Hardingham and Bading, 1999). Other

CREB co-activators such as the Transducers of Regulated CREB activity (TORCs)

translocate to the nucleus is response to intracellular calcium elevations (Conkright et

al., 2003; Impey et al., 2002). In addition to phosphorylation at Ser133, CREB is also

phosphorylated at several other serines including Ser142 and Ser143 although how

these phosphorylation events regulate transcription has not been elucidated yet

(Kornhauser et al., 2002). Thus CREB is subject to calcium-dependent regulation at

many different points during its activation.

Calcium influx through LTCs simultaneously activates several signaling

pathways culminating in Ser133 phosphorylation. Two of these signaling systems, the

calcium Calmodulin (CaM) activated kinases CaMKIV and CamKI and the mitogen

activated kinases (MAPK), seem to be particularly important for linking CREB to

calcium influx through LTCs. CamKIV, downstream of the calcium-calmodulin

dependent kinase pathway, and RSK2, downstream of the canonical Ras/MAPK

pathway, are thought to be the major players in phosphorylating CREB in response to

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depolarization in neurons (Lonze and Ginty, 2002). CaMKIV is activated by the

concerted action of Calcium-bound calmodulin and CaMKK phosphorylation

(Tokumitsu et al., 1994). The canonical MAP kinase cascade includes Ras, Raf, MEK,

ERK, and the nuclear kinases RSK1, RSK2 and MSK1, which phosphorylate CREB

on Ser133. Phosphorylation of CREB and CREB-dependent transcription are defective

in mice lacking CaMKIV (Ho et al., 2000), or MAP kinase MSK1 (Arthur et al., 2004;

Wiggin et al., 2002) and in cells whose CaMKI levels have been reduced using

siRNAs (Wayman et al., 2006), showing that activation of these signaling molecules is

important for CREB-mediated transcription. Furthermore, the importance of the LTC

induced activation of the Ras/MAPK pathway is highlighted by the markedly reduced

MAPK activation observed in the hippocampus and cortex specific L-type channel

knockouts (Moosmang et al., 2005). Activation of LTCs therefore leads to the

activation of several signaling cascades that result in phosphorylation of CREB.

Precisely what role each of these kinases plays in regulating the activation of

CREB is still a subject of controversy. It has been proposed that the kinetics of

activation of each of these kinases results in specific CREB phosphorylation profiles.

The CaM kinases, for instance, are activated rapidly and transiently in response to

calcium influx, whereas the MAP kinase cascade is activated more slowly and is more

sustained. CaMK activation therefore leads to rapid, transient CREB phosphorylation,

whereas activation of MAP kinase allows CREB to remain phosphorylated for a

prolonged period of time (Wu et al., 2001).

What mechanisms link LTCs to CREB?

9

Despite a wealth of information on the biochemical signaling pathways that

regulate CREB phosphorylation in response to depolarization, the mechanisms that

specifically link calcium influx through LTCs to the activation of CREB are not

completely understood. It is likely that multiple features of LTCs contribute to their

ability to activate CREB. At least three features of LTCs seem to be important for

their ability to activate transcription: their biophysical properties, their localization in

the dendrites and cell bodies of neurons, and their association with signaling proteins

that activate nuclear signaling cascades.

A. Biophysical Properties

Two distinct biophysical properties make LTCs particularly well suited to

activate CREB: their high voltage of activation and their slow activation/inactivation

kinetics (Tsien and Tsien, 1990; Xu and Lipscombe, 2001). In other words, LTCs

open relatively slowly and thus require sustained bursts of action potentials or

continuous depolarization for maximal activity (Deisseroth et al., 1996; Nakazawa and

Murphy, 1999)2. Consistent with this, depolarization using concentrated potassium or

strong electrical stimulation3 has been observed to trigger a far more sustained CREB

phosphorylation response than bath stimulation of NMDA receptors4 (Bito et al.,

2 Interestingly LTCs and the pathways leading to CREB activation can be recruited by somatic action potentials if these are delivered as tetha burts, suggesting you may not need signaling evoked from synaptic NMDA receptors. 3 Prolongued vs Transient: 180s vs. 18s 5Hz electrical stimulation or 90mM K+ 3min vs. 1min bath depolarization 4 Synaptic stimulation of NMDA receptors can also trigger sustained phosphorylation (Hardingham, 2002).

10

1996; Sala et al., 2000). Conceivable, their selective recruitment during enhanced

activity can explain their privileged pathway to CREB. In order to investigate whether

selective activation of LTCs takes place during more complex or physiologically

relevant patterns of neuronal activity, Liu and colleagues looked at how different

VGCCs respond to waveforms that mimic synaptic stimuli in the form of gamma and

theta frequency stimulation (Liu et al., 2003). They found that these type of stimuli

lead to the inactivation of non-L-type VGCCs leading to a calcium current mostly of

L-type suggesting that these channels by virtue of their activation/inactivation kinetics

are selectively recruited and conduct most of the calcium during strong neuronal

activity. Hence the observed importance of these channels in transcriptional induction

during electrical activity.

Though many stimuli can cause CREB phosphorylation, not all can lead to

transcriptional activation. This is in part because the stimulus must cause sustained

phosphorylation that persists for at least 30 minutes. To maintain this sustained

phosphorylation, calcium levels must be elevated for prolonged periods of time. In

contrast to other types of VGCCs, LTCs inactivate slowly and incompletely and so

they contribute a disproportionate amount of the calcium current under conditions of

tonic electrical stimulation (Liu et al., 2003). Consistent with this mutations that slow

voltage dependent inactivation of channels, such as in timothy syndrome, lead a faster

more sustained phosphorylation of CREB ((Splawski et al., 2004) and unpublished

data). A sustained calcium rise would best engage signaling molecules in the locality

of the channel that would normally deactivate quickly after a drop in calcium

concentration. In principle, a prolonged calcium rise could also lead to the selective

11

inactivation of a negative pathway that prevents long-lasting CREB phosphorylation.

A reported example of a molecule involved in such negative feedback mechanism is

the calcium regulated phosphatase, calcineurin. Inhibition of calcineurin with FK506

leads to prolonged CREB phosphorylation and transcription even under conditions

where phosphorylation would be transient and insufficient for transcriptional

activation (Bito et al., 1996; Liu and Graybiel, 1996). Whether LTC activation

promotes calcineurin inactivation and calcineurin ultimately leads to the

dephosphorylation of CREB has not been elucidated.

B. Localization

The biophysical properties of LTCs, however, do not account entirely for the

ability of these channels to activate CREB-dependent transcription. In developing

cortical and hippocampal neurons, sustained calcium elevations mediated by NMDA

receptors or generated by the addition of calcium ionophores are significantly less

effective at activating CREB-dependent transcription than calcium influx through

LTCs (Bading et al., 1993). This suggests that there are additional features of LTCs

that link them to the signaling pathways that activate transcription. Another feature of

LTCs that may be involved in their ability to activate transcription is their subcellular

localization.

Early immunohistochemical studies described LTCs as concentrated at the

soma and basal dendrites of neurons (Ahlijanian et al., 1990; Hell et al., 1996; Hell et

al., 1993; Westenbroek et al., 1990). Other VGCCs such as Cav2.1 (P/Q) and Cav2.2

12

(N) are thought to be presynaptic and primarily involved in synaptic vesicle release.

Intuitively, one could posit that their proximity to the nucleus and their strategic

position to summate and respond to depolarizing activity that reaches the soma

explains at least in part why LTCs can more effectively convey signals to the nucleus.

Somatic localization of the channels could imply that LTCs could be more effective at

elevating somatic calcium following depolarization or alternatively that they activate

relevant signaling molecules which are closer to the nucleus and poised for activation

by calcium influx through these channels. However, in several systems it has been

clearly demonstrated that P/Q and N-type channels are also abundant in the soma

where they contribute significantly and in fact more to the somatic calcium rise than

the L-types (Deisseroth et al., 1998; Dolmetsch et al., 2001; West et al., 2001). In

addition, subsequent immunolabeling studies revealed that LTCs also localize to the

synapse and co-localize with synaptic markers and their synaptic localization may

enhance their ability to signal to CREB (Zhang et al., 2006). Consequently, somatic

localization of these channels does not entirely explain the observed L-type channel

specificity.

C. L-type calcium channels and nuclear calcium

Cytoplasmic calcium transients are normally accompanied by large nuclear

calcium rises. Under some conditions, elevations in nuclear calcium have been shown

to be required for CREB activation. Nuclear microinjection of the non-diffusible

calcium chelator, BAPTA-dextran, blocks expression mediated by the CRE element in

13

response to depolarization suggesting that nuclear calcium is necessary for CREB-

dependent transcription (Chawla et al., 1998; Hardingham and Bading, 1998). Both

CaMKK and CaMKIV are localized in the nucleus, and therefore a nuclear calcium

elevation would lead to activation of these signaling proteins and phosphorylation of

CREB. However, it is unclear how BAPTA-dextran works in these experiments given

that BAPTA’s chelating activity is exhausted within seconds (given the large amounts

of calcium that enters the nucleus) and the fact the stimulation is in the order of

several minutes. Furthermore, loading cells with the calcium buffer EGTA, which

prevents nuclear calcium elevation, has no effect on activity–induced CREB

phosphorylation (Deisseroth et al., 1996), suggesting that if nuclear calcium plays a

role, it does so in other stages of CREB activation beyond Ser133 phosphorylation.

Another line of evidence for the role of nuclear calcium is the observation that isolated

nuclei can support CREB phosphorylation (). However, it is unknown whether this

phosphorylation would be sustained and whether isolated nuclei would support

transcriptional activation of CREB. In general, the activation of CREB is response to

LTC activation cannot be solely explained on the basis of nuclear calcium. First, other

calcium channels elevate nuclear calcium as or even more effectively than LTCs

(Deisseroth et al., 1998; Dolmetsch et al., 2001). Secondly, activation of the serum

response factor SRF, which also happens downstream of LTC activity is entirely

independent of nuclear calcium (Deisseroth et al., 1996; Hardingham et al., 1997).

Together, the data suggest the nuclear calcium is not sufficient to lead to CREB-

dependent transcription but suggest a role for other nuclear, calcium regulated players.

14

D. Local calcium signaling

Studies by Deisseroth and colleagues showed that loading neurons with EGTA,

a slow calcium buffer that prevents calcium elevation in the cell body and nucleus but

allows calcium elevations close to the membrane, does not inhibit CREB

phosphorylation in response to depolarization (Deisseroth et al., 1996). On the other

hand loading neurons with BAPTA, a fast calcium buffer that chelates calcium close

to the mouth of the channels, blocks CREB phosphorylation. This suggested a role for

calcium and calcium sensor molecules near the mouth of the channels in triggering the

signaling pathways that lead to the nucleus. Furthermore, mutations of LTCs that do

not alter their ability to carry calcium or to elevate nuclear calcium prevent LTCs from

inducing CREB phosphorylation and CREB-dependent transcription (Dolmetsch et al.,

2001). To investigate the features of LTCs that specifically couple them to the

activation of CREB, a functional knock-in technique was developed where DHP

resistant recombinant channels could be introduced into neurons and thus their

behavior could be distinguished from their endogenous/DHP-sensitive counterpart.

Using this functional knock-in approach it was found that point mutations that disrupt

Calmodulin binding to the LTC prevent LTC activation of CREB. These mutations

did not affect the ability of the LTC to activate the CaMK signaling pathway but

prevented activation of MAP kinase, suggesting that local calcium elevations around

LTCs activate MAP kinase signaling that is necessary for CREB-dependent

transcription. Together, these findings clearly demonstrated that the coupling of LTCs

to signaling pathways that activate gene expression goes beyond the calcium conduit

15

properties of the channel. They have led to the idea that global calcium elevations

(including elevations of nuclear calcium) are required for activation of the CaMK

signaling cascade, but that full activation of CREB-dependent transcription requires

the activation of MAP kinase by signaling molecules close to the mouth of LTCs.

CaM binding to the channels cannot constitute the molecular basis of

specificity since other VGCCs contain IQ motifs and are regulated by calmodulin5.

By means of the functional knock-in approach, other structural domains in the channel

have been found to impinge on the channels ability to signal to CREB. PDZ motifs in

the structures of Cav1.2 and Cav1.3 proteins have also been shown to be necessary for

signaling CREB. Inhibition of the interaction of Cav1.2’s PDZ motif with its

endogenous binding proteins attenuated CREB phosphorylation and CRE-dependent

transcription following depolarization (Weick et al., 2003). In the case of Cav1.3 an

association with post-synaptic density protein shank is necessary for CREB

phosphorylation in response to Cav1.3 activation (Zhang et al., 2006; Zhang et al.,

2005). Taken together these data further provides support for the idea that calcium

responses at the mouth of calcium channels are centrally important for transcriptional

regulation by LTCs.

The identity of the molecule that transmits the signal from the nucleus is not

known. Two candidates have been proposed, CaM itself and elements of the

Ras/MAPK signaling cascade. Because CREB activation depends on CaM-binding

proteins such as CaMKK and CaMKIV, and CaM is found enriched in the vicinity of

5 In addition, calmodulin regulates the channels activation and inactivation kinetics, and thus perturbation of calmodulin binding may have effects on the dynamics of the local calcium concentration.

16

the channels (Mori et al., 2004), calmodulin has been proposed as the herald of LTC

activation to the nucleus. Consistently with this hypothesis CaM translocates to the

nucleus of neurons in response to increases in intracellular calcium and this

translocation seems to be, under conditions of synaptic stimulation, L-type dependent

and NMDA receptor dependent (Deisseroth et al., 1998). However, CaM is also found

in high levels in the nucleus of resting neurons so CaM is unlikely to be the only

signal that conveys information from LTCs to the nucleus.

As discusses earlier, LTCs also activate the MAPK pathway and this signaling

cascade seems to be important for the prolonged phosphorylation of CREB that is

required for CREB-dependent transcription. The importance of the LTC induced

activation of the Ras/MAPK pathway is highlighted by markedly reduced MAPK

activation in response to strong LTC stimuli in the hippocampus and cortex of specific

L-type channel knockouts (Moosmang et al., 2005). Surprisingly, nothing is known

about how calcium influx through LTCs lead to sustained ERK activation. In

mammalian cells calcium can trigger Ras activity via Pyk2 a calcium regulated

tyrosine kinase/scaffolding protein (Lev et al., 1995), via calcium-sensitive K-Ras

(Villalonga et al., 2002) or calcium-regulated Ras-guanine nucleotide exchange factors

(GEFs) including RAS-GRF (Farnsworth et al., 1995), RAS-GRP (Ebinu et al., 1998),

CAPRI (Lockyer et al., 2001), and RASL (Liu et al., 2005). However, which and how

any of these proteins are preferentially activated by LTCs remains to be discovered.

Despite more than 20 years of study, the signaling molecules that connect

LTCs to the activation of CREB-dependent transcription have not been defined. It is

likely that there is a complex of proteins around LTCs that senses calcium and

17

converts calcium elevations into activation of the MAP kinase signaling cascade (Fig.

2). This complex includes calmodulin, which binds directly to the LTC. Calmodulin

has multiple effects on LTCs, and mediates both calcium-dependent inactivation and

calcium-dependent potentiation of the channel in addition to connecting the channels

to activation of CREB. Calmodulin therefore alters the conformation of LTCs in

response to local calcium elevations, and this conformational change might activate

signaling proteins bound to the channel leading to CREB-dependent transcription.

Understanding the molecular mechanisms that connect calmodulin to the activation of

CREB-dependent transcription is a critical question in channel signaling.

Figure 2: Schematic representation of signaling pathways from LTCs to CREB

18

ISOFORM SPECIFIC CONSIDERATIONS

In neurons, Cav1.3 is co-expressed with Cav1.2 where they share

somatodendritic and postsynaptic localization (Hell et al., 1993). Among VGCCs

Cav1.2 and Cav1.3 channels share the highest sequence similarity and have both been

implicated in mediating signaling from the membrane to the nucleus (Zhang et al.,

2006). Some important differences do exist which impact how we view the

contributions of these channels to transcriptional regulation.

Although the traditional view of L-type channels is that they are high-voltage

activated, have slow activation kinetics and are highly sensitive to DHP inhibition,

there is increasing evidence that channels composed by the Cav1.3 subunit have in fact

fast activation kinetics and low activation thresholds (Xu and Lipscombe, 2001).

Consistent with the biophysical properties of clones Cav1.3 channels it has been

reported that Cav1.3 preferentially mediates CREB phosphorylation at low (20mM

KCL 30sec and 5Hz 30sec) but not high levels of stimulation (Zhang et al., 2006).

Therefore it is possible that under low levels of stimulation, perhaps spontaneous

activity Cav1.3 may carry out most of the signaling to the nucleus.

19

Chapter 2:

The c-terminus of the l-type voltage-gated calcium channel Cav1.2 encodes a

transcription factor

20

SUMMARY

Voltage-gated calcium channels play a central role in regulating the electrical and

biochemical properties of neurons and muscle cells. One of the ways in which

calcium channels regulate long-lasting neuronal properties is by activating signaling

pathways that control gene expression, but the mechanisms that link calcium channels

to the nucleus are not well understood. We report that a C-terminal fragment of

CaV1.2, an L-type voltage-gated calcium channel (LTC), translocates to the nucleus

and regulates transcription. We show that this calcium channel associated

transcription regulator (CCAT), binds to a nuclear protein, associates with an

endogenous promoter, and regulates the expression of a wide variety of endogenous

genes important for neuronal signaling and excitability. The nuclear localization of

CCAT is regulated both developmentally and by changes in intracellular calcium,

suggesting that CCAT integrates information about the developmental history and

electrical activity of the cell. These findings provide the first evidence that voltage-

gated calcium channels can directly activate transcription, and suggest a novel

mechanism linking voltage-gated channels to the function and differentiation of

excitable cells.

INTRODUCTION

Changes in intracellular calcium regulate many cellular events including

synaptic transmission, cell division, survival, and differentiation. Voltage-gated

21

calcium channels are an important route of calcium entry and are essential for

converting electrical activity into biochemical events in excitable cells (Catterall et al.,

2005). Among the ten different types of neuronal voltage gated calcium channels, L-

type channels (LTC), encoded by the Cav1.2 and Cav1.3 pore forming subunits are

particularly effective at inducing changes in gene expression that underlie plasticity

and adaptive neuronal responses (Bading et al., 1993). Calcium influx through LTCs

activates transcription factors such as CREB, MEF, and NFAT (Graef et al., 1999;

Mao et al., 1999; Sheng et al., 1990) that lead to the expression of genes such as c-fos

and BDNF (Morgan and Curran, 1986; Murphy et al., 1991; Zafra et al., 1990). Two

mechanisms link LTCs, particularly CaV1.2, to the activation of transcription factors

such as CREB. Calcium entering through the channels can diffuse to the nucleus and

activate nuclear calcium-dependent enzymes, such as CaMKIV, that regulate the

activity of transcription factors and co-regulators (Hardingham et al., 2001). In

addition, calcium entering cells through LTCs can activate calcium-dependent

signaling proteins around the mouth of the channel which propagate the signal to the

nucleus (Deisseroth et al., 1998; Dolmetsch et al., 2001).

In this study we have identified a new mechanism by which calcium channels

control gene expression. We report that neurons produce a C-terminal fragment of

CaV1.2 that can regulate transcription and which we call the calcium channel

associated transcriptional regulator or CCAT. CCAT is located in the nucleus of

many inhibitory neurons in the developing and adult brain, and its production and

nuclear localization are regulated developmentally. In addition, calcium influx

through LTCs and NMDA receptors causes CCAT export from the nucleus. In the

22

nucleus, CCAT interacts with the transcriptional regulator p54(nrb)/NonO and can

activate transcription of both reporter and endogenous genes. Using microarrays and

real-time PCR, we show that CCAT affects the transcription of a many neuronal genes

including a gap junction, an NMDA receptor subunit, and the sodium calcium

exchanger. CCAT binds to the enhancer of the Connexin 31.1 gene (Cx31.1) and

directly regulates both the expression of a Cx 31.1 reporter gene and the expression of

the endogenous gene. Finally, we show that CCAT expression can cause an increase

in neurite extension in primary neurons. This is the first example of a calcium channel

having a dual function as an ion pore and a transcription factor.

RESULTS

CCAT Is Found in the Nucleus of Neurons in the Brain

Experiments in neurons and cardiac myocytes have suggested that the C-

terminus of CaV1.2 is proteolytically cleaved, yielding a truncated channel and a

cytoplasmic C-terminal fragment (De Jongh et al., 1994; Gerhardstein et al., 2000).

To investigate the function of the C-terminal fragment we developed an antibody to a

fourteen-amino acid peptide in the C-terminus of CaV1.2 (a.a. 2106-2120) and used it

to probe HEK 293T cells expressing CaV1.2. The C-terminal antibody (anti-CCAT)

recognizes both the intact channel and a short cleavage product that corresponds to the

C-terminal fragment. In contrast, an antibody recognizing an epitope in the II-III

23

cytoplasmic loop of CaV1.2 (anti-II-III loop) detects full-length and C-terminally

truncated channels only (Figure 1A).

To determine where CCAT is localized in cells in the brain, we purified

nuclear, cytoplasmic and membrane fractions of postnatal day 7 (P7) rat brain cortex

and used western blotting to probe them with the anti-CCAT antibody (Figure 1B).

Surprisingly, we found that the nuclear extracts contained high levels of CCAT

suggesting that the C-terminus of CaV1.2 is localized in the nucleus of cells in the

brain. In contrast the N–terminal portion of the channel was localized in the membrane

and cytoplasmic fractions as expected for an ion channel. To provide further

evidence that CCAT is indeed nuclear in neurons or glial cells, we examined its

localization by immunostaining primary cortical cultures. The anti-CCAT antibody

stained the cell body and dendrites of neurons weakly (Figure 1D), suggesting that the

anti-CCAT antibody recognizes some intact CaV1.2 channels. Importantly, however,

a significant number of neurons (10 ± 5%) exhibited very strong nuclear CCAT

staining (Figure 1C). In contrast, the II-III loop antibody stained the cell bodies and

dendrites of neurons but was excluded from the nucleus, suggesting that the full-length

channel is not nuclear (Figure 1E).

To investigate which types of neurons have nuclear CCAT, we co-stained

neurons with anti-CCAT and with antibodies that stain precursor cells (nestin), glial

cells (GFAP), excitatory neurons (NR2A) or inhibitory neurons (GAD65) in the

cortex. We found that cells that have strong nuclear CCAT also expressed glutamic

acid decarboxylase (GAD65), suggesting that CCAT is strongly nuclear in inhibitory

neurons that produce GABA (Figure 1F). To determine if CCAT is also in the nucleus

24

of neurons in vivo, we used the anti-CCAT antibody to stain P30 rat brain sections. A

subset of cells in the thalamus (data not shown), inferior colliculus (Figure 1G),

inferior olivary nucleus (Figure 1H), and in the olfactory bulb (Figure 1I) displayed

prominent nuclear CCAT staining. In the cortex and the hippocampus, CCAT was

nuclear in a small number of neurons, consistent with its localization in a subset of

GAD65 positive neurons in cortical cultures (data not shown). Taken together, these

experiments indicate that CCAT is localized in the nucleus of inhibitory neurons, in

culture and in restricted regions of the brain in vivo.

To provide further evidence that CCAT can translocate to the nucleus, we

fused yellow fluorescent protein (YFP) to the C-terminus of full-length CaV1.2

(CaV1.2-YFP). We observed cytoplasmic and nuclear fluorescence when CaV1.2-YFP

was expressed in neurons (Figure 2A), cardiac myocytes (data not shown), or

Neuro2A glioblastoma cells (Figure S2). In contrast, in neurons expressing CaV1.2

tagged at its N-terminus with YFP, the channel was localized in the membrane and in

the endoplasmic reticulum (Figure 2B). We did not observe nuclear fluorescence in

HEK 293T cells expressing CaV1.2-YFP, consistent with previous reports that in HEK

293T cells the C-terminus of CaV1.2 remains associated with the plasma membrane

following cleavage (Gao et al., 2001; Gerhardstein et al., 2000; Hulme et al., 2005).

However, a fusion of YFP and the last 503 amino acids of CaV1.2 was nuclear and

formed distinct nuclear punctae in neurons, myocytes and HEK 293T cells (c503

Figure 2C, E). Interestingly, this punctate pattern did not seem to be the result of

overexpression, as it was also observed in some neurons by confocal imaging of

endogenous CCAT staining (Figure 2D) and it was enhanced by incubation in low

25

calcium media (Figure 3B). These experiments provide further evidence that CCAT is

indeed nuclear, and suggest that formation of punctae by endogenous CCAT is

modulated by signaling events in the cell.

Nuclear CCAT does not contain a canonical nuclear localization sequence

suggesting that it enters the nucleus via an alternative pathway, perhaps as has been

described for Stat1 protein where nuclear import is mediated by direct interaction with

nucleoporins (Marg et al., 2004). To identify the regions of CCAT that are necessary

for its nuclear localization, we made truncations of the 503-YFP protein and

introduced them in HEK 293T cells. Deletion of the carboxyl end of CCAT and of

amino acids 1642-1814 of CaV1.2 (c330) had little effect on the protein’s localization.

In contrast, deletion of amino acids 1814-1864 (c280) decreased nuclear retention and

abolished punctae formation (Figure 2E and F). Comparison of the CaV1.2 sequence

from other vertebrates indicates that this nuclear retention domain is conserved

evolutionarily (Figure S1A) suggesting that it plays an important role in the function

of CaV1.2 and CCAT proteins.

Endogenous CCAT is predicted to be a 75 kD protein; therefore, nuclear

translocation of CCAT is likely to involve an active process rather than passive

diffusion across nuclear pores. To estimate the rate of CCAT import into the nucleus,

we used fluorescence recovery after photobleaching (FRAP) and time-lapse

microscopy of Neuro2A cells expressing CaV1.2-YFP. After photobleaching of

nuclear CCAT, nuclear fluorescence recovered over the course of 300 seconds with a

single exponential time course (t=48 +/-16 sec; n=11), while cytoplasmic fluorescence

declined over the same time period (Figure 2G-H). In control cells expressing YFP

26

alone, we observed an almost instantaneous recovery of nuclear fluorescence after

photobleaching concomitant with a decrease in cytoplasmic fluorescence, consistent

with the observation that YFP diffuses rapidly through nuclear pores. The slow rate of

recovery of CCAT-YFP nuclear fluorescence suggests that this protein is actively

imported into the nucleus at a rate similar to that of NFAT, another transcription factor

that translocates to the nucleus (Shibasaki et al., 1996). Measurements of CCAT

export by bleaching cytoplasmic fluorescence indicate that CCAT returns to the

cytoplasm with a time course of approximately 400 seconds (t=62 +/-21 sec; n=5)

(data not shown). These results are consistent with the idea that CCAT is

constitutively transported into the nucleus, and that CCAT shuttles between the

cytoplasm and the nucleus of unstimulated cells.

The Concentration of Nuclear CCAT Is Regulated by Intracellular Calcium

To determine whether the nuclear localization of CCAT is regulated by

changes in intracellular calcium, we assessed the distribution of CCAT by

immunocytochemistry in cortical neurons following treatment with agents that affect

intracellular calcium levels. Decreasing free extracellular calcium using 2.5mM

EGTA caused a robust increase in nuclear CCAT fluorescence (Figure 3A, C), and

caused CCAT to aggregate into punctae in the nucleus of many neurons (Figure 3B).

Conversely, treatment with 65mM KCl, which mimics tonic electrical activity by

increasing the activity of VGCCs, and treatment with 100µM glutamate caused a

significant decrease in the nuclear fluorescence (Figure 3A and 3C). The decrease in

27

nuclear CCAT could be reliably detected after five minutes and reached a maximum

after 30 minutes of stimulation with either depolarization or glutamate, although

depolarization had a more pronounced effect at earlier time points (Figure 3D). The

nuclear fluorescence of Neuro2A cells expressing the CaV1.2-YFP also declined with

tonic depolarization, providing further evidence that electrical activity leads to a net

decrease of CCAT from the nucleus (Figure S2A-B). The decrease in nuclear CCAT

triggered by depolarization was blocked by removing extracellular calcium or by

treating cells with the CaV1.2 blocker nimodipine. Application of NMDA receptor

blocker MK-801 partially blocked the activity-induced decrease in nuclear CCAT but

treatment with the AMPA receptor inhibitor NBQX had no effect (Figure 3E),

suggesting that NMDA but not AMPA receptors can also influence the export of

CCAT from the nucleus of cortical neurons.

The decrease in nuclear CCAT observed in response to high intracellular

calcium could be due to a net export from the nucleus or to selective degradation of

CCAT in the nucleus. To determine if CCAT is degraded following a rise in

intracellular calcium, we measured total CCAT immunoreactivity before and after

depolarization. We found that depolarization had no effect on the total CCAT staining

in neurons, or on the levels of CCAT-YFP expressed in Neuro2A cells (Figure 3F).

Furthermore, addition of the proteosome inhibitor lactacystin failed to block the

depolarization-induced decrease in nuclear CCAT (Figure 3G). The lack of a decrease

in total CCAT levels in depolarized neurons and Neuro2A cells and the lack of effect

of lactacystin on CCAT nuclear localization argue that the decrease in CCAT

following depolarization is not due to protein degradation.

28

Nuclear CCAT Is Regulated Developmentally

The levels of nuclear CCAT vary considerably among neurons in the

developing brain (Figure 1G-I). Since neurons in the central nervous system

differentiate at different rates, we considered whether the levels of CCAT in the

nucleus could be regulated developmentally. To investigate this possibility, we

assessed the levels of nuclear and total CCAT found in brains taken from embryonic

day eighteen (E18), postnatal day one (P1), three-week old (P21), and adult rats. The

levels of CCAT immunoreactivity in the nuclear fractions increased substantially with

age (Figure 3H; middle panel), whereas the amount of CCAT-containing channel at

the membrane appeared to decrease (Figure 3H; upper panel). This is consistent with

increasing cleavage of CaV1.2 during development. The total levels of CaV1.2, as

determined by immunoreactivity of the CaV1.2 internal loop antibody, were also

regulated developmentally. CaV1.2 levels were low at E18 and increased through P8

before declining in P21 and adult brains (Figure 3I; upper panel). Interestingly, early

in development a long and a short form of CaV1.2 could be detected whereas only the

short form of the channel and a new, 150 kD band were observed in both p21 and

adult brains, suggesting that there is increasing cleavage and possibly different

cleavage events in older brains. Together, these results indicate that the levels of

CCAT in the nucleus, the cleavage of CaV1.2, and the levels of CaV1.2 are regulated

independently to yield a complex pattern of channel and transcription factor

expression.

29

CCAT Binds to a Nuclear Protein

To get an indication of CCAT’s function, we looked for proteins that interact

with CCAT in the nucleus. We expressed CCAT or a mutant form lacking the nuclear

localization domain in Neuro2A cells, immunoprecipitated them via epitope tags, and

identified interacting proteins by mass spectrometry. One of the proteins that co-

immunoprecipitated with full length CCAT was p54(nrb)/NonO, a nuclear protein that

plays a role in regulating transcription downstream of the neuronal Wiscott Aldrich

Protein (Wu et al., 2006), the retinoic acid receptor, and the thyroid hormone receptor

(Mathur et al., 2001). We verified the interaction of p54 (nrb)/NonO with CCAT by

co-immunoprecipitation followed by Western blotting against endogenous p54

(nrb)/NonO (Figure 4A). These results indicate that CCAT is associated with a

nuclear protein that participates in transcriptional regulation and regulates mRNA

splicing, and suggest a role for the C-terminus of CaV1.2 in the nucleus.

CCAT Activates Transcription

Based on its nuclear localization and its binding to p54 (nrb)/NonO, we

hypothesized that CCAT might regulate transcription. To investigate whether CCAT

can activate transcription when recruited to a promoter by a heterologous DNA

binding domain, we made a C-terminal fusion of the intact channel and the Gal4 DNA

30

binding domain from yeast (CaV1.2-Gal4, Figure 4B). The Gal4 DNA binding

domain recognizes the UAS DNA sequence but requires a transcriptional activation

domain to activate transcription. We introduced CaV1.2-Gal4 into neuro2A cells

along with a UAS luciferase reporter gene and measured luciferase expression. We

found that CaV1.2-Gal4 activated transcription approximately 80 times better than

Gal4 alone or than the channel lacking the Gal4 DNA binding domain (Figure 4C).

These results suggest that the C-terminus of CaV1.2 is produced as a soluble protein in

cells, that it translocates to the nucleus, and that it activates transcription when

recruited to a heterologous gene.

To identify the domains of CaV1.2 that are required for transcriptional

activation, we made a family of proteins containing fragments of the C-terminus of

CaV1.2 fused to Gal4 and tested them in primary neurons for their ability to activate

the expression of a UAS luciferase reporter gene. A fragment containing 503 amino

acids of the CaV1.2 C-terminus fused to Gal4 activated transcription almost as well as

a CREB-Gal4 fusion protein, and about 130 times better than the Gal4-DNA binding

domain alone (Figure 4D). Deleting 170 amino acids from the N-terminus of this C-

terminal CaV1.2 fragment (c330-Gal4) reduced but did not completely abolish the

ability of the C-terminus to activate transcription. In contrast deletion of a second

domain consisting of the most C-terminal 133 amino acids (c503∆133-Gal4)

completely eliminated the ability of CCAT to activate transcription (Figure 4D).

Deletion of these final 133 amino acids in the full length CaV1.2-Gal4 also produced a

channel unable to activate transcription (Figure 4C; bar 4) suggesting that this domain

is required for transcriptional regulation by the intact channel. These experiments

31

suggest that CCAT has two domains that are necessary to activate transcription: an N-

terminal domain that modulates transcriptional activation, and a C-terminal domain

that is essential for transcription (red and blue boxes in Figure 4B). Significantly, both

transactivation domains are highly conserved in vertebrates (Figure S1B and D), and

the N-terminal transactivation domain has 42% similarity and 27% identity to a

conserved transactivation domain of the transcription factor GATA4, suggesting that it

has a bona fide role in transcriptional regulation (Figure S1C).

Because recruiting proteins to DNA via Gal-4 DNA binding domains can

produce ectopic transcriptional regulators, we also fused various other calcium

channel C-terminal domains to Gal4 and expressed these with the UAS reporter gene.

We found that the C-termini of CaV1.3 and CaV2.1 when fused to Gal4 had no effect

on transcription, suggesting that CaV1.2’s C-terminal domain is specific in its ability

to activate transcription in neurons (Figure 4E).

In earlier experiments we observed that the amount of CCAT in the nucleus

decreased in response to tonic electrical activity. To determine whether this activity-

induced decrease in nuclear CCAT has functional relevance, we depolarized cells

expressing CaV1.2-Gal4 and measured activation of the UAS luciferase reporter

(Figure 4F). Prolonged depolarization led to a 30% decline in transcription from the

reporter gene, and removing extracellular calcium blocked this effect. These results

provide evidence that the nuclear localization of CCAT is important for its activation

of transcription and are consistent with the observation that nuclear CCAT

concentration is regulated by electrical activity.

32

CCAT Regulates Transcription of Endogenous Genes

To determine whether CCAT regulates transcription of endogenous genes, we

used oligonucleotide microarrays to identify mRNAs that are transcriptionally

regulated by CCAT over-expression. We built two plasmids that encode either full

length CCAT or a CCAT∆TA that lacks the N-terminal transcriptional activation

domain. Both plasmids also contain a GFP gene driven by a separate promoter that

was used to identify transfected cells. We introduced these plasmids into Neuro2A

cells and used fluorescence activated cell sorting (FACS) to select transfected cells.

We then compared the mRNA expression profile of cells expressing full-length CCAT

to cells expressing either CCAT∆TA or GFP alone, using Agilent mouse whole

genome arrays. In three independent experiments, we found 23 mRNAs that were up-

regulated more than two fold (p<0.005) in cells expressing CCAT relative to cells

expressing CCAT∆TA, and 22 genes that were down-regulated more than two fold by

CCAT relative to CCAT∆TA (Table S1). Because we subsequently discovered the

CCAT∆TA still activates transcription albeit at a much lower level than full length

CCAT (see Figure 4D), we also compared mRNA expression profiles of cells

expressing CCAT and GFP to cells expressing GFP alone. In three additional

experiments, we found 66 mRNAs up-regulated more than 1.8 fold (p<0.005) in cells

expressing CCAT relative to those expressing GFP (Figure S1 and Table S2). The

genes that were up-regulated by CCAT include the genes for the gap junction protein

Connexin 31.1 (Cx31.1), the axon guidance factor Netrin4, the regulator of G protein

signaling RGS5, the tight junction protein claudin19 and a broad array of other genes

33

(Figure 5A). Approximately 206 genes were repressed more than 0.55 fold (p<0.005)

by CCAT, including the sodium calcium exchanger, the cation channel TRPV4, the

potassium channel Kcnn3, and the transcription factor GATA6 (Figure 5A, Figure S3

and Table S3; Raw data available at http://ncbi.nlm.nih.gov/geo; account:

Dolmetsch_rev; password: reviewer; series #: GSE4180). Combining the results from

all six of our micro-array experiments (CCAT vs. CCAT∆TA and CCAT vs. GFP)

revealed that 16 mRNAs were significantly up-regulated (Table S4) and 31 genes

were significantly down-regulated by CCAT. These results suggest that CCAT can

both increase and decrease the expression of a wide set of genes that regulate neuronal

differentiation, connectivity, and function.

To verify the results of the microarray experiments, we measured changes in

mRNA expression due to CCAT expression using RT-PCR (Figure 5B). CCAT

changed the expression of all seven mRNAs tested, in accordance with the results

from the array experiments. As normalizing controls we used β-actin and GAPDH,

which showed no detectable change in response to overexpression of CCAT. These

data provide independent evidence that CCAT regulates expression of endogenous

genes, some of which are important for the function of excitable cells.

CCAT Binds and Regulates the Promoter of Cx31.1

The microarray and RT-PCR experiments suggested that connexin 31.1

(Cx31.1) was strongly regulated by CCAT in cells. To study the regulation of Cx31.1

by CCAT in more detail, we constructed a reporter gene consisting of the 2 Kb

34

promoter/enhancer region of Cx31.1 in front of the firefly luciferase coding sequence.

We introduced this Cx31.1 luciferase reporter gene into neurons along with either the

full length CCAT or CCAT∆TA, a version of CCAT lacking the C-terminal

transcriptional activation domain. Full length CCAT increased the expression of the

Cx31.1 reporter by 3.4 ± 0.4 fold (n=12) relative to a control vector or to CCAT∆TA

(Figure 5C) providing additional evidence that CCAT regulates the expression of

Cx31.1.

CCAT could affect the transcription of Cx31.1 either by regulating the

transcriptional machinery in the nucleus directly or by modifying signaling proteins in

the cytoplasm of cells that lead to changes in transcription. To determine if CCAT

acts in the nucleus, we fused CCAT to the ligand binding domain of a modified

estrogen receptor (ER) that binds 4-hydroxytamoxifen (4OHT) but not endogenous

estrogen (Littlewood et al., 1995). When expressed in Neuro2A cells, ER-CCAT is

largely excluded from the nucleus but brief treatment with 4OHT causes ER-CCAT to

move into the nucleus (Figure 5D). Treatment of cells expressing ER-CCAT with

4OHT caused a fifty-fold increase in the transcription of Cx31.1 relative to untreated

cells (Figure 5E). 4OHT had no effect on cells expressing ER alone, and caused a

much smaller effect in cells expressing ER-CCAT∆TA. These results provide

compelling evidence that CCAT regulates the transcription of Cx31.1 when it is in the

nucleus of cells.

To identify regions of the Cx31.1 promoter that are important for its regulation

by CCAT, we made a series of deletions of the Cx31.1 promoter and placed them

upstream of the firefly luciferase gene (Figure 5F). We introduced this library of

35

deletion mutants of the Cx31.1 promoter into Neuro2A cells along with full length

CCAT and measured luciferase activity in these cells. CCAT regulation of the Cx31.1

promoter was critically dependent on 148 base pairs at the 3’ end of the Cx31.1

promoter. Deletion of this domain eliminated the ability of CCAT to activate

transcription of the Cx31.1 reporter gene, and this domain alone was sufficient to

confer CCAT regulation on to a reporter gene (Figure 5F). Together, this data

suggests that CCAT regulates the expression of Cx31.1 in a sequence-specific manner,

and that the CCAT recognition element lies in the final 148 base pairs of the Cx31.1

promoter sequence.

In the nucleus, CCAT could affect transcription directly by binding to a

complex of proteins on the promoter of genes, or indirectly by binding to other

proteins in the transcriptional activation pathway. We used chromatin

immunoprecipitation (ChIP) to determine whether CCAT binds to the promoter of Cx

31.1 directly. We introduced an epitope-tagged CCAT into cells, crosslinked the

protein to the DNA and immunoprecipitated CCAT from these cells, and used PCR to

determine if any region in the promoter of the Cx 31.1 gene was co-

immunoprecipitated by CCAT. We found that CCAT could reproducibly

immunoprecipitate a fragment of the endogenous Cx31.1 promoter approximately 1

Kb upstream of the transcriptional start site but not other regions, suggesting that the

CCAT is bound close to this region of the Cx 31.1 promoter (Figure 5G). These

results suggest that CCAT regulates transcription by binding, either directly or through

protein-protein interactions, to the promoter of Cx31.1, providing further evidence that

CCAT is a transcriptional regulator.

36

Endogenous CaV1.2 and CCAT Regulate Transcription of Cx31.1

We have provided evidence that exogenous expression of CaV1.2 leads to the

production of CCAT, which in turn affects transcription. To determine whether

endogenous CaV1.2 regulates transcription by generating CCAT, we asked whether

reducing the levels of endogenous CCAT in the nucleus by depolarization had an

effect on expression of the Cx31.1 reporter gene or of the endogenous Cx 31.1 gene.

Depolarization of cortical neurons reduced activation of the Cx31.1 reporter gene by

2.12 ± 0.12 fold (Figure 6A) and caused a 2.4 fold decrease in the expression of the

Cx31.1 mRNA levels as measured by RT-PCR (Figure 6B). The effects of

depolarization on Cx31.1 mRNA levels were also apparent in Neuro2As and in

cultured thalamic neurons suggesting that CCAT regulates the expression of Cx 31.1

in multiple cell types (Figure 6B). These results support the conclusion that CCAT-

dependent transcription of the Cx31.1 gene requires nuclear localization of CCAT.

Because CCAT is derived from CaV1.2, we also asked whether Cx31.1

expression depends on the expression of endogenous CaV1.2. We designed several

short hairpin RNAs (shRNAs) and asked whether introducing these shRNAs into

neurons reduced the expression of Cx31.1. Two shRNAs targeting the rat CaV1.2

(RCav1.2 sh6410 and RCaV1.2 sh6500) reduced the expression of rat CaV1.2

expressed in Neuro2A cells, whereas an shRNA targeting the mouse CaV1.2 sequence

had no effect on the expression of the rat channel (Figure 6C; lanes 1-3). The shRNAs

37

targeting the rat CaV1.2 also reduced CaV1.2-dependent signaling to CREB in rat

cortical neurons, suggesting that these shRNAs reduce the expression of endogenous

CaV1.2 and prevent activation of CREB-dependent transcription (Bading et al., 1993;

Dolmetsch et al., 2001; Murphy et al., 1991) (Figure S4A). We next introduced the

shRNAs targeting the rat CaV1.2 into cortical neurons and measured the activation of

the Cx31.1 reporter. Both rat shRNAs decreased the expression of Cx31.1 by

approximately six-fold, indicating that CaV1.2 regulates the expression of Cx31.1 in

neurons. (Figure 6D). CaV1.2 knockdown had no effect on Renilla luciferase

expression from the control vector, suggesting that the decrease in Cx31.1 reporter

activity was not due to decreased viability. To assess whether the effect of the

shRNAs targeting CaV1.2 on the transcription of Cx31.1 was the result of the loss of

calcium influx through the channel, we tested whether L-type calcium channel

blockers affected Cx31.1 transcription. Twenty-four hour (24h) treatment of neurons

with 10µM nimodipine had no effect on the expression of Cx31.1 in the presence or

absence of CCAT, suggesting that Cx31.1 is not regulated by calcium influx through

CaV1.2 in unstimulated cells (Figure S4B). To determine if the inhibitory effects of

CaV1.2 shRNAs on the Cx31.1 promoter are due to reduction of CCAT, we

constructed a version of CCAT that is insensitive to the rat CaV1.2 shRNA (CCAT*;

Figure 6E) and expressed it in cells along with the shRNA targeting rat CaV1.2.

Expression of CCAT* rescued the effect of knocking down the endogenous CaV1.2 on

the expression of the Cx 31.1 gene (Figure 6F, n=6). In contrast, CCATDTA* that

lacked the transcriptional activation domain did not rescue the effects of the CaV1.2

shRNA on Cx 31.1 expression. This suggests that CCAT alone can restore expression

38

of Cx 31.1 in cells in which CaV1.2 has been reduced by an shRNA, and that this

effect depends on the transcriptional activation domain of CCAT. We also made a

version of CaV1.2 that is insensitive to the rat CaV1.2 shRNA (CaV1.2*) (Figure 6G)

and asked whether this channel can rescue Cx31.1 expression in cells lacking

endogenous CaV1.2 (Figure 6H). Expression of CaV1.2* in neurons partially rescued

the effect of the CaV1.2 shRNA on Cx31.1 expression while a form of CaV1.2* that

lacks the C-terminal transcriptional activation domain did not restore the effects of

CaV1.2 knockdown on Cx31.1 expression. Together these results support the

conclusion that endogenous CaV1.2 modulates transcription of the Cx31.1 gene, and

that this transcriptional regulation depends on the production of CCAT from the C-

terminus of CaV1.2.

CCAT Expression Promotes Neurite Growth

Our microarray and RT PCR experiments suggested that CCAT regulates the

transcription of a number of genes important in neuronal function and excitability. To

explore the cell biological functions of CCAT, we measured the effect of expressing

CCAT on the morphology and survival of cerebellar granule neurons. We selected

these cells because they are a largely homogenous population of neurons that have low

basal levels of CCAT and that have well-characterized survival and dendritic

arborization patterns. Expression of CCAT or CCAT∆TA did not significantly affect

granule cell survival, but it did cause a dramatic change in the length of neurites

(Figure 7A and B). Full-length CCAT doubled the average length of neurites to 10

39

µm (Figure 7C; bottom panel and 7D) whereas the CCAT∆TA decreased the average

length of neurites to approximately 2.7 um (Figure 7C; top panel and 7D). There was

also a small but statistically significant effect of CCAT on the number of neurites,

suggesting that under some circumstances CCAT could affect the growth and

formation of new dendrites (Figure 7E). Interestingly, expressing CCAT in other cell

types such as Neuro2As also caused a change in the morphology of the cells, causing

an increase in the production of filopodial extensions (data not shown). This data

suggests that CCAT-dependent transcription can lead to rearrangement of the

cytoskeleton and may contribute to changes in the connectivity of neurons during

development.

DISCUSSION

Neurons and myocytes generate characteristic patterns of electrical activity and

intracellular calcium that are essential for cell function. The reliability of the calcium

signal requires a delicate balance of proteins that import and export calcium from the

cytoplasm – proteins whose individual expression is regulated independently in

response to cellular function. The expression of voltage gated calcium channels is

closely coordinated with the expression of other ion channels, pumps and signaling

proteins that regulate membrane excitability and calcium homeostasis. In this paper

we describe a novel mechanism by which cells coordinate the expression of voltage

gated calcium channels with the expression of other molecules. LTCs generate a

transcription factor that integrates information both about the number of calcium

40

channels and the electrical activity of a cell. CCAT is generated from the L-type

channel, and its nuclear localization is negatively regulated by the electrical activity of

the cell, it is therefore in a privileged position to integrate information about the

number of channels with information about the calcium history of a cell.

Several laboratories have reported that LTCs are cleaved at their C-terminus,

and the site of cleavage of Cav1.1, the homologous LTC in skeletal muscle, was

recently identified (Hulme et al., 2005). The cleaved channel carries more calcium,

so channel cleavage could have profound effects on the electrical properties of a

neuron by changing the properties of the LTC. The proteolytically processed C-

terminal domain is also thought to bind to truncated channels, where it exerts an

inhibitory effect on channel function (Hulme et al., 2006b). This hypothesis does not

preclude the idea that the C-terminus of CaV1.2 also acts as a transcription factor. By

analogy with the potassium channel-binding protein KChip/DREAM, which is also a

calcium-sensitive transcriptional repressor, we propose that CCAT both regulates

transcription and reduces calcium influx through CaV1.2 (An et al., 2000; Carrion et

al., 1999). This hypothesis is appealing in light of the observation that CCAT is

exported from the nucleus by elevations in intracellular calcium, suggesting that under

conditions of tonically elevated calcium, CCAT would both alter the transcription of

specific genes and inhibit the activity of CaV1.2. Thus CCAT may be an important

part of a negative feedback pathway regulating both gene expression and calcium

influx in the neurons.

In addition to CaV1.2, it has also been reported that CaV1.3 (Hell et al., 1993),

CaV2.1 (Kubodera et al., 2003), and CaV2.2 (Westenbroek et al., 1992) are cleaved in

41

neurons. In the case of CaV2.1, the cleavage product is also approximately 75 kD and

has been localized to the nucleus of Purkinje neurons in the cerebellum (Kordasiewicz

et al., 2006). This suggests that C-terminal cleavage is a general feature of CaV

channels and that other members of this family may also be transcriptional regulators.

In our studies we did not find that the C-terminal domains of CaV1.3 or CaV2.1

activated transcription in cortical neurons, but it is possible that the C-terminal

domains of other channels may act in other types of neurons or may be transcriptional

repressors or regulators of chromatin structure. This would be consistent with our

finding that in addition to activating transcription CCAT also represses the

transcription of many genes.

Despite more than a decade of experiments, the stimuli and mechanisms that

lead to cleavage of CaV1.2 remain enigmatic. It has been reported that cleavage of

CaV1.2 is triggered by NMDA stimulation in hippocampal slices (Hell et al., 1996),

and CaV1.2 cleavage has also been reported to occur in response to sex hormone

stimulation of uterine muscle (Helguera et al., 2002). We did not observe any obvious

increase in CCAT following stimulation of neurons in culture with NMDA or

potassium chloride, however it is possible that CaV1.2 cleavage only occurs in the

context of hippocampal slices. In cortical neurons, cerebellar granule cells, cardiac

myocytes, Neuro2A cells and PC12 cells exogenous CaV1.2 appears to be cleaved

constitutively to yield nuclear and cytoplasmic CCAT. While the production of

CCAT did not appear to be regulated, its nuclear localization and its transcriptional

effects on the Cx31.1 gene were strongly regulated by changes in cytoplasmic

calcium. Therefore, we favor the idea that CCAT is produced in proportion to the

42

number of CaV1.2 channels in cells and that cytoplasmic calcium levels regulate its

nuclear localization and transcriptional activity. In addition to being regulated by

calcium, nuclear CCAT levels were also regulated in a cell-specific manner and its

appearance in brain nuclear fractions increased substantially over the course of

postnatal development. In cultured neurons, CCAT levels were highest in GABAergic

inhibitory neurons, while in brain slices CCAT staining was particularly strong in the

inferior colliculus, inferior olive and thalamus. These data suggest that CCAT may

play an important role in the development of neurons and in regulation of neuronal

properties in specific cell types.

Our studies have identified many interesting genes regulated by CCAT, and

these genes offer clues to understanding CCAT’s physiologic function. CCAT

regulates the expression of several gap junction proteins, a glutamate receptor, several

potassium channels, a sodium-calcium exchanger and of signaling proteins such as

RGS5, Formin and Nitric Oxide Synthase. One of the main targets of CCAT in the

nucleus is the gap junction protein Cx31.1. Cx31.1 is expressed in the retina

(Guldenagel et al., 2000), in developing embryos (Davies et al., 1996), and in

GABAergic striatal output neurons of the thalamus (Venance et al., 2004). Our array

and RT PCR studies suggest that Cx31.1 is also well expressed in neuroblastoma cells

and in thalamic neurons. Transcription of the Cx31.1 gene correlates well with the

amount of endogenous CCAT in the nucleus and depolarization, which reduces the

amount of nuclear CCAT, also decreases the amount of Cx 31.1 transcript suggesting

that these two are correlated. Finally CCAT binds to the promoter of Cx 31.1

providing compelling evidence that CCAT is a regulator of Cx 31.1 expression in

43

neurons. Connexins play a key role in forming electrical connections between

developing neurons and form conduits for signaling molecules that can regulate a

developing tissue. The expression of Cx 31.1 during development in response to

changes in CCAT could thus play an important role in regulating the electrical

coupling of neurons and the overall excitability of the brain.

We have found that CCAT expression in neurons increases dendritic length.

This effect is blocked by CCAT lacking a transcriptional activation domain. There are

many possible mechanisms for this effect of CCAT on neuronal morphology. The

observation that CCAT up-regulates Cx31.1, formin, claudin 19, procolagen type XI

and an α-catenin-like protein suggests that it might promote the formation of adhesion

complexes or junctional contacts between neurons and the extracellular matrix.

Alternatively, since CCAT increases the production of Netrin4 and of two chemokines

that regulate axonal and dendritic growth, it could lead to increases in neurite length

via these mechanisms (Adler and Rogers, 2005; Barallobre et al., 2005). Finally, by

down-regulating a potassium channel and a sodium calcium exchanger, CCAT could

increase the excitability of neurons and thus regulate their morphology indirectly.

Understanding how CCAT modulates dendritic length might help uncover the

mechanisms by which L-type calcium channels regulate neuronal morphology.

We provide strong evidence that CaV1.2 encodes a transcription factor that can

regulate expression of a variety of genes that are important for the function of neurons

and muscle cells. This finding reveals an entirely unsuspected function for a well-

characterized calcium channel that plays an essential role in electrical tissues. This

44

new function of CaV1.2 will be a rich area for future study in ion channel physiology

and neurobiology.

45

EXPERIMENTAL PROCEDURES

Materials

Nimodipine, MK-801, NBQX and 4OHT were purchased from Sigma. Lactacystin

was from Calbiochem and L-glutamate was from Tocris Bioscience.

Anti-CCAT was used 1:1000 for western blots. Anti-CaV1.2 II-III loop (1:1000) was

purchased from Chemicon or BD Biosciences, anti-CREB (1:1000) was from Upstate

Biotechnologies, anti-p54nrb/NonO (1:1000) and anti-DsRed (1:400) from BD

Biosciences, anti-b-actin (1:2000) and anti-GAPDH (1:2000) were from Ambion, anti-

gal4 (1:500) and anti-GST (1:500) were from Santa Cruz Biotechnology. Anti-flag M2

(1:1000) was purchased from Sigma.

Cell culture and transfection

HEK 293T cells, Neuro2A and PC12 cells were cultured in Dulbecco’s Minimal

Essential Media (DMEM) containing 10% fetal bovine serum (FBS; 15% for PC12s),

penicillin, streptomycin (P/S) and L-glutamine (LQ). Cortical neurons were

dissociated from E17-19 Sprague Dawley rats as described (Xia et al., 1996) and

maintained for 6 to14 days in culture in Basal Medium Eagle with 5% FBS, P/S, LQ

and 1% glucose or in Neurobasal medium containing B27 supplement (Invitrogen).

Cardiac myocytes were cultured from P0-P1 rats using a neonatal myocyte isolation

kit (Cellutron Life Technology) and maintained in DMEM with 10% FBS, P/S, LQ

and 0.1mM BRDU for 3 to 4 days. Cerebellar granule cells were cultured from P5

46

Sprague Dawley rats and grown as described elsewhere (Dudek et al., 1997). For

description of thalamic neuron cultures see Supplemental experimental procedures.

HEK 293T (24h) cells, cortical and granule neurons (72h) were transfected using a

standard calcium phosphate method at a concentration of 2 µg of DNA/106 cells.

Neuro2As (24h), cortical neurons (96h), cardiac myocytes (24h) and PC12s (24h)

were transfected using lipofectamine 2000 according to manufacturer’s instructions.

For luciferase reporter gene experiments and see Supplemental experimental

procedures.

Thalamic Neuron cultures

Thalamic neurons were dissected from E17-19 Sprague Dawley rats in ice-cold Hank's

Balanced Salt Solution without Ca++ and Mg++ (HBSS, Gibco). Thalami were

enzymatically digested using trypsin (Worthington, 10mg/ml), DNase (Sigma, 200

U/ml) in HBSS at room temperature for 5 min. Thalami were washed 3x in Basal

Medium Eagle with 5% FBS, P/S, LQ and 1% glucose and gently triturated in the

same media. Neurons were plated at 25,000/cm2.

Plasmid construction

Construction of the dihydropyridine resistant (DHP- CaV1.2) in the pcDNA4/HisMax

vector has been previously described (Dolmetsch et al., 2001). CaV1.2-YFP and

CaV1.2-Gal4 fusion proteins were constructed by the insertion of the YFP and Gal4

DBD coding sequences into the AfeI/ NotI sites in DHP- CaV1.2. The plasmid

encoding the N-terminal tagged YFP-CaV1.2 was generated using Gateway

47

technology (Invitrogen) by first cloning the CaV1.2 coding sequence from the DHP-

CaV1.2 plasmid into the TOPO sites of the pCR8 entry vector and subsequently

transferring the CaV1.2 coding sequence into a destination vector called pDEST-

pGWYFP that contains a CMV promoter and an N-terminal YFP in frame with the

ATTR acceptor sequences. CaV1.2-flag was made by PCR amplification of CaV1.2

coding sequence with the tag in the 3’ primer and topo cloning into pcDNA4/HisMax

vector (Invitrogen). The CCAT YFP fusion proteins were constructed by inserting

PCR amplified portions of the CaV1.2 C-terminal tail into the HindIII and Kpn1 sites

of pcDNA3.0-YFP, a C-terminal YFP fusion vector. Plasmids encoding the following

amino acids of the CaV1.2 (accession # AAA18905) fused to YFP were generated:

1642-2143, 1642-2011, 1814-2143, 1864-2143, 1841-2101, 1814-2051, 1814-2000,

accession number AAA18605. N-terminal Gal4 DBD fusion proteins encompassing

the following amino acids of CaV1.2 1642-2143, 1642-2011, 1814-2143, 1864-2143

were generated by PCR cloning into BamHI/HindIII sites of PFA-CMV vector

(Stratagene). For microarray experiments and granule cell morphology assays the

sequence encoding amino acids 1642-2143 and 1642-2011 was cloned into the

HindIII/Kpn1 site of the PA1 expression vector, which was a kind gift from Dr.

Michael Lin. CCAT-ER fusions were made by inserting PCR amplified sequences

1642-2143 and 1642-2011 into BglII site of pCS4-myc. The ER sequence was

amplified from pBlu-ER-KS vector (kind gift from Dr. Ann Brunet) and inserted as a

C-terminal fusion into BamHI and EcorI sites.

Cx31.1-luciferase reporter was constructed by PCR amplification of a 2Kb promoter

segment (chr4 (-): 126860469-126862515) from mouse tail DNA using the primers:

48

Cx31.1 2Kb fwd: 5’-AGAGGAGCCCCAGGTAACACAG-3’ and Cx31.1-2kb rev:

5’-AGCCCAGGCGTGTCCTGTTGG-3’. The promoter was first cloned into

pCR8/GW/TOPO and later subcloned into XhoI, HindIII sites of the pGL3-Basic

vector (Promega).

Rat CaV1.3 and Human CaV2.1 were kind gifts from Dr. Diane Lipscombe and Dr.

Richard Tsien respectively. Gal4 fusions were made by PCR cloning into PFA-CMV

of C-terminal domains including amino acids 1669-2203 for CaV1.3 (NP_058994)

and 1975-2505 for CaV2.1 (AAB64179).

For shRNA knockdown experiments, the following 21mer oligonucleotides sequences

were selected using Dharmacon and Invitrogen’s design tools at

www.dharmacon.com/sidesign and rnaidesigner.invitrogen.com/rnaiexpress:

RCav1.2 6410 GGGACAGTTTGCTCAAGATCC, RCav1.2 6500

CGCCGCAGACAACATCCTC and MCav1.2 6203

GCTCAAGATCCCAAGTTTATC; Accession numbers, RATRBCII and AY728090,

for rat and mouse respectively.

Short hairpin Oligonucleotides were designed and inserted into RNAi-Ready pSIREN-

DNR-DsRed-Express vector (Clontech) by ligation into the BamHI and EcoRI sites.

RNAi resistant CCAT and CaV1.2 were made by introducing four silent mutations in

CaV1.2-flag and CCAT-pcDNA3.0 vectors using site-directed mutagenesis

(Stratagene). The resulting sequence was CGCAGCCGATAATATCCTC.

The CRE-luciferase reporter has been previously described (Dolmetsch et al., 2001).

Antibody generation

49

Peptides of the following sequence DPGQDRAVVPEDES were synthesized

(Covance) coupled to KLH (Pierce), injected into rabbits and affinity purified as

previously described (Datta et al., 1997).

Subcellular fractionation and Western blotting

The brain was rapidly removed and homogenized in 320 mM sucrose and 20 mM

HEPES homogenization buffer, pH 7.2, containing 1 mM EDTA, 1 mM dithiothreitol,

Complete protease inhibitors (Roche Applied Science), and calpain inhibitors (A.G.

Scientific). The homogenate was centrifuged for 10 min at 1000g to obtain the nuclear

fraction. The supernatant was then centrifuged for 30 min at 100,000g at 4 °C to

obtain the cytoplasmic and membrane fractions. The nuclear pellet was extracted

using the Dignam method (Dignam et al., 1983). For membrane-bound channel

visualization, proteins were extracted as described previously (Haase et al., 2000).

Western blotting was conducted using standard protocols. Antibodies and dilutions are

included in Supplemental experimental procedures. Protein concentration was

measured by the BCA method (Pierce).

Immunofluorescence

6-day old cortical cultures cells were fixed in 4% paraformaldehyde/2% sucrose,

permeabilized, and blocked with 3% BSA in PBS. Neurons were stained with either

rabbit anti-CCAT or rabbit anti-Cav1.2 II-III loop (each diluted 1:100) and anti-

GAD65 followed by 1:500 dilutions of Alexa 594-conjugated anti-mouse and Alexa

488-conjugated anti-rabbit antibodies (Molecular Probes). Nuclei were stained using

50

Hoechst 33258 (Molecular Probes). Neuro2A cells expressing CCAT-ER fusions were

stained with mouse anti-myc tag (Upstate). P30 rat brain sections were a gift from Dr.

Ben Barres. Sections were blocked and permeabilized 30 min using 10% goat serum,

0.25% triton X-100 in PBS. Primary and secondary antibody incubations were done as

described above. Slides were visualized by conventional epifluorescence microcopy

using a cooled CCD camera (Hamamatsu) coupled to an inverted Nikon Eclipse

E2000-U microscope. Confocal images were obtained using the Volocity grid

confocal microscope (Improvision Inc).

Quantitative Image analysis

Images were analyzed using OpenLab 4.0.4 software (Improvision, Inc). For

measurements of nuclear and cytoplasmic fluorescence, nuclear and whole cell regions

of interest (ROI) were generated by density slicing the Hoechst and anti-CCAT

images respectively and cytoplasmic ROIs were obtained by subtraction. Fluorescence

measurements were analyzed using Igor Pro software (Wavemetrics).

Fluorescence Recovery after Photobleaching (FRAP)

FRAP experiments were conducted at 37° C using a Zeiss Axiovert 200M inverted

microscope coupled to a Coolsnap cooled CCD camera controlled by Slidebook

software. Bleaching was achieved with a 100 ms long 488 nm laser pulse. Images

were captured every 400 ms.

51

Luciferase assays

Channels were transfected in a ratio of 1:1:1 CaV1.2, b1b and a2d subunits. For

luciferase assays, channels were transfected in a ratio of 2:1:1:0.5 CaV1.2, b1b, Firefly

luciferase reporter, and Renilla luciferase reporter and C-term constructs were

transfected at a ratio of 2:1:0.5, C-term, Firefly luciferase reporter and Renilla

luciferase reporter.

For shRNA experiments neurons and PC12s were transfected at a ratio of 1:1:0.5

shRNA vector, channel or c-term construct, Firefly luciferase reporter for 2.5-4 days.

PC12s were arrested and differentiated by switching them to 0.5% media with

50ng/ml NGF.

Most luciferase assays were performed 24 hours after transfection using the Dual-Glo

luciferase assay kit from Promega. For shRNA experiments, assays were performed 72

hours post-transfection. A Veritas 96 well luminometer (Turner biosystems) was used

to measure light emission. CREB-Gal4, constitutively active PKA, and PFA-CMV

constructs were obtained as part of the Path-Detect Trans-Reporting system from

Strategene. Data sets were analyzed using Igor Pro and Prism4 software. Two-paired

t-tests were performed between relevant conditions.

Immunoprecipitation and mass spectrometry

HEK 293T cells were transfected with c503-Gal4 or c280-Gal4. 24 hours after

transfection, immunoprecipitations were carried out using the ProFound Co-

Immunoprecipitation Kit (Pierce) and mouse or rabbit anti-Gal4 antibodies (Santa

Cruz Biotechnology). SDS-PAGE gels were silver stained using the SilverQuest

52

system from Invitrogen. Individual bands were analyzed by Stanford Mass

Spectrometry facility using LC-MS/MS as previously described (Shevchenko et al.,

1996).

RNA isolation and oligonucleotide microarrays

Neuro2A cells were transfected using the CCAT-PA1, CCAT∆TA-PA1 or PA1

control vectors. Twenty-four (24) hours after transfection, cells were trypsinized and

resuspended in fresh media without phenol red and GFP positive cells were sorted

using FACS. RNA was isolated from 2 x 106 cells using an RNAeasy kit from Qiagen.

RNA was hybridized to Agilent whole mouse oligo microarrrays by Mogene, Inc

(Saint Louis). Expression data was analyzed using GeneSpring GX 7.3 software

(Agilent).

Real-Time PCR

First strand synthesis was conducted using the first-strand cDNA synthesis kit from

Invitrogen. 500 ng of cDNA was used as a template for RT PCRs performed using an

Mx3000P Real-Time System (Stratagene), and the reactions were carried out using

Quantitec SYBR green PCR master mix (Qiagen). For a list of primers see

Supplemental experimental procedures.

Cycling parameters were 95°C for 10 min, followed by 45 cycles of 95°C for 30 s,

55°C for 1 min, 72°C for 30 s. Fluorescence intensities were analyzed using the

53

manufacturer’s software, and relative amounts were obtained using the 2–∆∆Ct method

(Livak and Schmittgen, 2001).

The following primers were used:

Mouse Cx31.1 (NM_010291) (F)5’- TTCTGATGCTTGCTGAACCCC -3’

(R)5’- GGAGTCCCTCAAAAACACTCC -3’;

Rat Cx31.1 A: (F) 5’- TTTTGATGCTTGCTGAACCCC-3’

(R)5’- GGAGCCCCTCAAAGACGCTCC-3’

Rat Cx3.1 B: (F)5’- CTGAGTGTGCACCAGCGAAGAGACC-3’

(R)5’-CGAGGGCGATCAGGTAACAGAGGTG-3’

Fmn (NM_010230) (F)5’-GGTCACCCCCAGCTATGTGTTT-3’

(R)5’-GCGGTGGGATTGATTTTCTTTG-3’;

Ppef2 (NM_011148) (F)5’-ATGCATTGCCAGGGTAGTCGG-3’

(R)5’-CAGGCACCTAACCCATGTCTG-3’;

Rgs5 (BC005656) (F)5’-TCACTTGCTCCCCCTTCCTC-3’

(R)5’-TCCTGGAATGTCTGGCAAGC-3’;

SCL8A1 (AF115505) (F)5’-CCTCGGTGCCAGACACATTTG-3’

(R)5’-GCCGGGGGACACTTTGAACT-3’;

Artn (NM_009711) (F)5’-TAGGTGGCAGTCAGCCTGGT-3’

(R)5’-GGGGTCGCAGGGTTCTTTC-3’;

NOS1 (NM_008712) (F)5’-CTCCCACCCTGCACCATCTT-3’

(R)5’-ATTCCTGAAGCCCCTTGCTG-3’;

b-actin (F)5’-CTTTGCAGCTCCTTCGTTGCC-3’

(R)5’-CGATGGAGGGGAATACAGCC-3’.

54

ChIP

ChIP was carried out as described in the Upstate Biotechnology protocol. Briefly 107

cells were transfected with CCAT-GST or CCAT-Gal4, or GST or Gal4 alone as a

controls. Proteins were crosslinked to DNA with 1% paraformaldehyde for 10

minutes and the nuclei were isolated by centrifugation. The DNA was sheared by

sonication to generate DNA fragments between 200 and 1000 bp. The C-terminal

fusion proteins were immunoprecipitated, washed and uncrosslinked by adding high

salt and incubating at 65° overnight. DNA was recovered by phenol/chloroform

extraction and ethanol precipitation and used as a template for PCR. Reaction

products were visualized by agarose gel electrophoresis.

Primers used analyze the immunoprecipitated DNA were to CX31.1 promoter

(Genbank accession number NM_010291):

(-30 to -201) 5’TGGGGGTGAAAGGTCAAAGTGT3’;

5’GTGTGTGTGTGGGAGGAGCTGT3’

(-201 to -351) 5’GCAATGGAGGAGGAGGGAAGAG3’;

5’AACCTTGTGTGGGGATGAAACG3’

(-455 to -610) 5’GGTGTGTGGCAGATAGGCTTCA3’;

5’CTCTCCAGTCCCTGCATTTGCT3’

(-589 to -761) 5’CCCATCCTTCATTTCCCTGGTT3’;

5’TGAAGCCTATCTGCCACACACC3’

(-859 to -1081)

5’ATGGTGGCCGTTCATACAGAGC3’;5’GCTTGGAGTTGGGAGACAGGAG3’

55

For the 3’UTR, (-1060 to -1217) 5’ CCAACCAGCCTTTCCTCTCCAT3’;

5’RGCTCTGTATGAACGGCCACCAT3’

Dendritic arborization assays

Cerebellar granules cells were imaged 24-48h post-transfection using the ImageXpress

500A system (Molecular devices). Dendrites were analyzed employing ImageJ and

NeuroJ programs.

56

FUTURE EXPERIMENTS

The findings described in this chapter beg further investigation on the molecular

mechanisms by which CCAT influences transcription. Several models can explain

how CCAT can influence gene expression. On one hand, CCAT can bind DNA

directly by binding to specific cis-regulatory DNA sequences such as the transcription

factors CREB and NFAT. The identification of this binding motif could point to

endogenous target genes and would help us to better understand CCAT’s function. If

CCAT cannot bind DNA on its own, an alternative model must be considered where

the CCAT-DNA interaction is mediated by DNA binding partners. These binding

partners could be themselves sequence-specific transcription factors or components of

the basal transcriptional machinery. Here I describe a series of experiments that would

help shed some light on these questions

Does CCAT bind DNA and what is its target sequence?

To begin to understand how CCAT regulates transcription I searched for

sequence homology with previously known DNA-binding proteins. Of the 53 DNA-

binding domains for which Pfam has Hidden Markov models none can be predicted

from CCAT’s sequence. While this suggests that CCAT does not bind DNA it is

possible that it employs a yet uncharacterized domain for interacting with DNA.

Interestingly, the C-terminus of Cav2.1, a P/Q type channel, contains several NLSs and

a predictable AT-hook motif commonly found in many nuclear proteins (Aravind and

57

Landsman, 1998). In the absence of sequence homology with previously known DNA

binders, I turned to prediction methods based on amino acid composition and

secondary structure (Ahmad et al., 2004). Such methods predict DNA-binding

properties for the c-termini of Cav1.2, 1.3 and 2.1, which is likely due to the high

concentration of basic residues within these sequences. These predictions, in the

absence of structural or sequence conservation information, need to be taken

cautiously. Together, sequence analysis although suggestive provides little definite

information on CCAT’s DNA-binding properties.

We have already identified a 148 bp region in the CX31.1 promoter to be

necessary and sufficient for CCAT-dependent transcriptional activation. In chapter 2,

we showed that CCAT regulates transcription of Cx31.1 by acting directly at the

promoter. Deletion of this sequence eliminated the ability of CCAT to activate

transcription and insertion of this domain alone was sufficient to confer CCAT

regulation on to a reporter gene (Figure 5). This sequence can be used as starting point

to investigate CCAT’s ability to bind DNA.

In future experiments the 148 bp sequence can be further narrowed down by

deletion analysis. Once the target DNA sequence has been sufficiently reduced point

mutations can be made to determine which bases are important for CCAT

transcriptional activity. Subsequently, binding of CCAT to isolated sequences can be

evaluated using mobility shift assays and labeled probes from the target sequence

identified above. In these experiments, the migration of labeled oligos is compared

between reactions containing CCAT or control proteins. Because CCAT may bind to

58

DNA non-specifically, it is important to test control oligos for which binding is not

expected and additionally, perform a competitive assay using unlabeled probe.

To provide independent evidence of binding and to demonstrate that this

binding is sufficient to confer CCAT-dependent transcription reporter genes can be

constructed of several repeats of the candidate motif by inserting them in front of the

luciferase coding sequence and a basal promoter. These vectors can be used for

transcriptional assays where luciferase expression can be compared in the presence

and absence of CCAT.

Alternatively a non-biased approach can be used. PCR-assisted binding site

selection can be used to determine the optimal DNA binding site for CCAT. In this

approach, a random library of oligonucleotides (20nt) is incubated with purified

CCAT-GST fusion proteins, the CCAT-DNA complexes are then isolated via their

GST tags and bound oligos are recovered. Our library of oligos has been constructed

so that T7 forward and T3 reverse primers flank the 5’ and 3’ ends of each oligo

respectively, and can thus be randomly amplified using PCR. After several rounds of

binding, recovery and amplification, the oligomers can be cloned and examined by

sequencing. The sequences recovered will hopefully provide the consensus DNA

binding domain for CCAT.

Assuming that the above experiments will yield a sequence or family of related

sequences for CCAT, a subsequent experiment will be to use a bioinformatic approach

to determine endogenous target genes by searching putative regulatory regions in the

genome for the presence of such motifs. Because such an approach would likely yield

many false positives (given the short size of the DNA motifs), it is prudent to restrict

59

the initial search to genes previously identified in our microarray experiments

(Gomez-Ospina et al., 2006). Confirmatory experiments involve building reporters

using upstream regulatory regions from likely candidates. Deletion studies can be

used to prove necessity.

Nuclear binding partners

The absence of predictable DNA-binding domains in CCAT’s sequence has

led us to consider that CCAT may bind DNA via a specialized DNA-binding protein

or scaffolding protein. One possible mechanism involves a motif-specific transcription

factor. Such mechanism would be reminiscent to the one employed by Notch, another

membrane protein that is cleaved and encodes a transcriptional regulator within its

intracellular domain (ICD). ICD lacks a DNA-binding ability and so uses a DNA

bound transcription factor called CSL to induce transcription of target genes

(Schroeter et al., 1998). However, these DNA-binding partners need not be sequence-

specific. It is possible that CCAT influences transcription by binding to chromatin or

basal transcription factors. CCAT could promote either their recruitment or activation

on target promoters such is the case for the RNA poly II carboxyl-terminal domain

phosphatases (Meinhart et al., 2005).

Our lab has begun to develop and utilize several proteomic approaches for the

identification of nuclear partners for CCAT. In pilot experiments, we expressed

CCAT or a mutant form lacking the nuclear localization domain in Neuro2A cells,

immunoprecipitated them via epitope tags, and identified interacting proteins by mass

60

spectrometry. This approach has identified several binding proteins. As described in

chapter 2, one protein immunoprecipitated by full length CCAT is P54/nrb/NonO

(NonO), a DNA and RNA-binding protein that appears to play roles in transcriptional

regulation as Ill as RNA processing (Shav-Tal and Zipori, 2002). We verified the

interaction by co-immunoprecipitation followed by western blotting against

endogenous NonO (Figure 4). This confirmation suggests that such biochemical

screen could be a productive approach toward identifying nuclear partners for CCAT.

The next step is to determine how these proteins relate to CCAT’s mechanism of

action.

In the case of NonO, the literature gives us some interesting clues regarding

possible mechanisms. NonO has been demonstrated to associate with the Carboxyl-

terminal domain of RNA pol II (Emili et al., 2002) and has been recently shown to

couple Neuronal Wiskott-Aldrich syndrome protein (N-WASP) with RNA polymerase

II to regulate transcription (Wu et al., 2006). Thus, one could hypothesize that NonO

could acts as an adaptor protein between CCAT and the transcriptional apparatus.

Future experiments can be designed to test this hypothesis. To begin, one can search

for the presence of RNA pol II in immunoprecipitated complexes of epitope tagged

CCAT along with NonO. Subsequent experiments can be aimed at substantiating the

need for NonO in CCAT-dependent transcription.

The question whether NonO is necessary for CCAT’s observed transcriptional

activation can be tested by knocking-down endogenous NonO and replacing it with a

NonO incompetent to bind CCAT. Towards this aim, a NonO protein unable to bind

CCAT and resistant to RNAi needs to be constructed. This modified NonO can be

61

included in transcriptional assays using CCAT-Gal4 to determine if it can block or

reduce transcription. According to my hypothesis, luciferase expression should be

decreased and this effect should be rescuable by full-length NonO insensitive to

RNAi. Considering NonO’s pleotropic role in the nucleus, it is possible that

knockdown and rescue with modified NonO will have a global negative impact on

transcription and it would not be specific for CCAT. Consequently, it is critical to

control for this possibility by co-expressing a constitutively expressed reporter, which

should report for overall decreases in transcription in the same cells.

62

Unt Unt HEK293T Cortex

240

75

Unt CytNuc

75

43

A B

C

240 anti-CCAT

anti-CREB

anti-CCATanti-II-III loop

EM ergedH oechstC C AT

II-III loop H oechst

H oechstC C AT

M erged

M ergedG AD 45

F

Mem

240 anti-II-III loop

anti-GAPDH38

C C AT Hoechst Merged

400X 600X

G H

D

M ergedH oechstC C AT

CB

IC IO

BSM ergedH oechstC C AT

I

H oechstC C AT M ergedG AD 45

400X

100X

Cav1.2 Cav1.2 Cav1.2

A

YFP-Cav1.2 Hoechst MergedHoechst Merged

B

Myocyte HEK 293TNeuron

Hoechst Mergedc280

Hoechstc330 Merged

D

F

1.0

2.0

3.0

c503 c330 c280 c330-50

c330-100

Nucle

ar/c

ytop

lasm

ic ra

tioE

C

**

Hoechst Mergedc503-133

HoechstCCAT-ConfocalCCAT-Epi

G

Nucleus

L

TC-Y

FP

Fluo

resc

ence

(A.U

.)

Time (s)

Cytoplasm

190

180

170160

50 100 150 200 250 300

t=0 s t=250 st=75 st=-50 s

H

Cav1.2-YFP

c503 YFP1642-2143 aac503-133 1642-2011 aac330 1814-2143 aac280 1864-2143 aac330-50 1841-2101 aac330-100 1814-2051 aa

YFP

YFP

YFP

YFP

YFP

63

A

Hoechstanti-CCAT Merged

5mM K+

60mM K+

C

F

E18 P1 P21 Adult

anti-CREB

75

50

E18 P1 P21 AdultP8

anti-CCAT

anti-II-III loop

anti- ß-actin

210

150

240

42

H

I

anti-CCATMembrane

Nuclear

240

EGTA

60

50

40

30

20

10

02.52.01.51.00.5

Num

ber o

f cel

ls

Nuclear/cytoplasmic ratio

Hoechstanti-CCAT MergedB

0

0.5

1.0

1.5

5 K+ EGTA

60 K+

Nuc

lear

/cyt

opla

smic

ratio

***

EGTA Nimo MK-801 NBQX

D

E

-40

-20

0

% d

ecre

ase

5 10 15 20 25 30time (min)

5 K+

60 K+

EGTA

60 K+

Glutamate

EGTA

Glutamate

Glutamate

500

1000

1500

M

ean

cell

body

fluor

esce

nce

(A.U

.)

60 K+5 K+

Nucle

ar/cy

topla

smic

ratio

0

0.4

1.2

0

0.8

60 K+5 K+

Lactacystin

60mM K+

*

G

64

a

IQ Gal4

c503

c330

c280IQ Gal4

c503

c330

c280

Gal4

Cav1.2

Cav1.2

-Gal4

0.5

1.0

3.5

3.0

2.5

2.0

1.5UA

S T

rans

crip

tion

(Fi

refly

/Ren

illa)

0

C

F

Gal4

CREB-Gal4

(+P

KA)c5

03-G

al4

c330

-Gal4

c280

-Gal4

A

Blot: α-p54/nrb

Blot: CCAT

Blot: α-p54/nrbIP CCAT

90

20

60

60

B

Cav1.2∆

133-G

al4

c503-CCAT

c280-CCAT

1

2

3

4

5

6

7

0

0

1

2

3

4

60mM K+

+++

---2.5mM EGTA

**

D

∆133

UA

S T

rans

crip

tion

(Fi

refly

/Ren

illa)

UA

S T

rans

crip

tion

(Fi

refly

/Ren

illa)

5

****

**

0.5

1.0

3.53.02.52.0

1.5UA

S T

rans

crip

tion

(Fi

refly

/Ren

illa)

0

E

Gal4

Cav1.2

c503

Gal4

Cav1.3

c537

Gal4Cav

2.1

c530

Gal4

3.5

c503∆133

-G

al4

****

**

65

Connexin 31.1 (Gjb5)

Formin (Fmn)

Peroxisomal membrane protein (Mpv17)Regulator of G-protein signaling (Rgs5)

Diflavin reductase (Ndor1)

Vomeronasal 1 receptor H13 (V1rh13)

Catenin alpha like 1 (Catna1)SET-binding protein (Sbf1)

Schlafen (Slfn)

FBJ osteosarcoma oncogene (Fos)

LIM and SH3 protein 1 (Lasp1)

GATA binding factor 6 (Gata6)SRY-box containing gene 2 (Sox3)

Phosphatase EF hand containing (Ppef2)

Paralemmin 2 (Palm2)

TRP channel (TrpV4)

Potassium channel (Kcnn3)

Forkhead box E3 (Foxe3)

Protease 26S subunit ATPase (Psmc5)

Activating transcription factor (Atf7)

Serine/threonine/tyrosine kinase (Styk1)

Myosin IG (Myo1G)

Nitric oxide synthase 1, neuronal (Nos1)

Sodium/Calcium exchanger 1 (Scl8A1)

Glutamate receptor NMDA2D (Grin2d)Artemin (Artn)

Glucokinase (Gck)

Chemokine (C-X3-C motif) ligand 1(Cx3cl1)

Leucine-rich pentatricopeptide repeat (Lrpprc)

Claudin 19 (Cldn19)

MYC-CCAT-ER MergedHoechst

Hoechst MergedMYC-CCAT-ER

IP

IP

IP

IP

C C

C C

I I

I I

350

350

250

250

150

150

50

3' UTR 3' UTR

450

0

86420

12108642

10 12-6 -4 -2Fold Change

Rel

ativ

e m

RN

A ex

pres

sion

1

0

345

-1-2-3

-4

FmnRgs

5Gjb5

nNOS1

Scl8A1

Artn Ppef2

+ 4O

HT

1h

- 4O

HT

Cx3

1.1

trans

crip

tion

Fire

fly (A

.U.)

10

3

4

5

6

2

7

2

pcDNA3 pcDNA3 CCAT

pcDNA3CCAT∆TA

**

Cx3

1.1

trans

crip

tion

(Fol

d In

duct

ion

+4O

HT/

-4O

HT)

10

0

30

40

50

60

20

ER CCAT-ER CCAT∆TA-ER

5’ promoter 3’ promoter

Cx3

1.1

trans

crip

tion

Fire

fly (A

.U.)

5

0

15

20

25

10

A B C D

E F G H

A B C D E F G H2K

A

B D F

C E G

66

A

B

C

D

E

F

G

H

0

1

2

3

4

5mM K+ 65mM K+

****

Cx3

1.1

trans

crip

tion

F

irefly

(A.U

.)

CCAT CCAT*

RCav1.2

sh65

00 MCav1.2

sh62

03 RCav1.2

sh65

00

anti-flag M2

anti-DsRed

240

30

Rel

ativ

e C

x31.

1 m

RN

A ex

pres

sion

65mM K+ 0 Ca+2

Neuro2A

Cortical

Thalamic

0

1

2

-1

-4

-3

-2

-5

Cx3

1.1

trans

crip

tion

(F

irefly

/Ren

illa)

0

****1

2

3

4

5

6

sh-scr MCav1.2 sh6203

RCav1.2 sh6410

RCav1.2 sh6500

MCav1.2

sh62

03

65

30

Cx3

1.1

trans

crip

tion

F

irefly

(A.U

.)

anti-CCAT

anti-DsRed

0

1

2

3

4

5

6

****

sh-scrVector CCAT* CCAT∆TA

RCav1.2 sh6500

Cav1.2-flag

RCav1.2

sh64

10 RCav1.2

sh65

00MCav1.2

sh62

03

Cav1.2∆TA-flag

RCav1.2

sh65

00

Cav1.2-flag Cav1.2-flag*

RCav1.2

sh65

00MCav1.2

sh62

03 RCav1.2

sh65

00 MCav1.2

sh62

03

240

30

anti-flag M2

anti-DsRed

Cx3

1.1

trans

crip

tion

F

irefly

(A.U

.)

0

1

2

3

4

5

6

7 ** **

Vector Cav1.2* Cav1.2∆TA

Vector

sh-scr RCav1.2 sh6500Vector

67

60

40

20

0

% of cells

50403020100

60

40

20

0

% of cells

50403020100Neurite length (µm)

50403020100

Number of Primary Neurites

CCA∆TA Vector CCAT0.0

0.5

1.0

1.5

2.0

2.5

CCAT∆TA Vector CCAT0.0

2.5

5.0

7.5

10.0

12.5Av

g.N

eurit

e Le

ngth

(µm

)Av

g. #

of P

rimar

y N

eurit

es

% o

f Cel

ls%

of C

ells

CCAT∆TA

Vector

CCAT

**

A B

CD

E

CCAT∆TA CCAT CCAT

**

Neurite length (µm)

Neurite length (µm)

60

40

20

0

% of cells

% o

f Cel

ls

Length of Primary Neurites

**

**

68

69

A CCAT’s nuclear localization domain is conserved among vertebrates channels CAC1C_RAT 1794 VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVSQDMFPDETRSSVRLSEEVEYCSE CAC1C_MOUSE 1791 VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVNQEIFPDETR-SVRMSEEAEYCSE CAC1C_HUMAN 1790 VEGHGPPLSPAIRVQEVAWKLSSNRCHSRESQAAMAGQEETSQDETYEVKMNHDTEACSE CAC1C_RABBIT 1821 VEGHGSPLSPAVRAQEAAWKLSSKRCHSQESQIAMACQEGASQDDNYDVRIGEDAECCSE CAC1C_ZEBRAFISH 1808 ----GPPLT-TIPLPRPTWCFPNKSSDSSDSRLPIIRREEASTDETYDETFLDE----RD CAC1C_RAT 1854 PSLLSTDILSYQDDENRQLTCLEEDKREIQ CAC1C_MOUSE 1851 PSLLSTDMFSYQEDEHRQLTCPEEDKREIQ CAC1C_HUMAN 1850 PSLLSTEMLSYQDDENRQLTLPEEDKRDIR CAC1C_RABBIT 1881 PSLLSTEMLSYQDDENRQLAPPEEEKRDIR CAC1C_ZEBRAFISH 1858 QAMLSMDMLEFQDEESKQLAPMVE------

B CCAT’s N-terminal transcription activation domain is conserved among vertebrates channels

CAC1C_RAT 1642 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_MOUSE 1639 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_RABBIT 1669 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_HUMAN 1687 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_ZEBRAFISH 1684 LVAKIPPKTALSLQAGLRTLHDMGPEIRRAISGDLTVEEELERAMKETVCAASEDDIFRR CAC1C_RAT 1702 AGGLFGNHVSYYQ-SDSRSNFPQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSS CAC1C_MOUSE 1699 AGGLFGNHVTYYQ-SDSRGNFPQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSS CAC1C_RABBIT 1729 AGGLFGNHVSYYQ-SDSRSAFPQTFTTQRPLHISKAGNNQGDTESPSHEKLVDSTFTPSS CAC1C_HUMAN 1747 AGGLFGNHVSYYQ-SDGRSAFPQTFTTQRPLHINKAGSSQGDTESPSHEKLVDSTFTPSS CAC1C_ZEBRAFISH 1744 SGGLFGNHVNYYHQSDGHVSFPQSFTTQRPLHISKSGS-PGEAESPSHQKLVDSTFTPSS CAC1C_RAT 1762 YSSTGSNANINNANNTALG-RFPHPAGYSSTVSTVEGH-GPPLSPAVRVQEAAWKL CAC1C_MOUSE 1759 YSSTGSNANINNANNTALG-RFPHPAGYSSTVSTVEGH-GPPLSPAVRVQEAAWKL CAC1C_RABBIT 1789 YSSTGSNANINNANNTALG-RLPRPAGYPSTVSTVEGH-GSPLSPAVRAQEAAWKL CAC1C_HUMAN 1807 YSSTGSNANINNANNTALG-RLPRPAGYPSTVSTVEGH-GPPLSPAIRVQEVAWKL CAC1C_ZEBRAFISH 1803 YSSSGSNANINNANNTAIGHRYPKP-----TVSTVDGQTGPPLT------------

C CCAT’s N-terminal transcription activation domain is homologous to GATA4’s C-terminal domain GATA4_HUMAN 313 IQTRK-RKPKNLNKSKTPAAPSGSESLPPASGASSNSSNATTSSS--EEMRPIKTEPGLS GATA4_RAT 311 IQTRK-RKPKNLNKSKTPAGPPG-ESLPPSSGASSNSSNATSSSSSSEEMRPIKTEPGLS GATA4_CHICK 264 IQTRK-RKPKNLNKTKTPAGPSSSESLTPTTSSTSSSSSATTT----EEMRPIKTEPGLS CAC1C-RAT 1722 PQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSSYSSTG---SNANINNANNTAL consensus ** ::* :**: : : * * :: * ::: : ::: GATA4_HUMAN 370 SHYGHSSSVSQTFSVSAMSGHGPSIHPVL----SALKLSPQGYASPVSQSPQTS GATA4_RAT 372 SHYGHSSSMSQTFST--VSGHGSSIHPVL----SALKLSPQGYPSPVSQTSQAS GATA4_CHICK 320 SHYGHPSPISQAFSVSAMSGHGSSIHPAI----SALKLSPQAYQSAISQSPQAS CAC1C-RAT 1779 GRFPHPAGYSSTVST--VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVS consensus :: * : * ::* : ***: : *:: :* *** * ** :*

D CCAT’s C-terminal transcription activation domain is conserved among vertebrate channels CAC1C_RAT 2009 SSMARRARPVSLTVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_MOUSE 2005 SSMARRARPVSLTVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_RABBIT 2035 SSAARRARPVSLTVPSQAGAQGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_HUMAN 2087 SSAARRVRPVSLMVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_ZEBRAFISH 2017 NHSGRAQRPVSLTVPPVTRRDSISLAHGSAGSLVEAVLISEGLGRYAHDPSFIQVAKQEI CAC1C_RAT 2059 ADACDMTIEEMENAADNILSGGAQQSPNGTLLPFVNCRDPGQDRAVVPE-DESCVYALGR CAC1C_MOUSE 2064 ADACDMTIEEMENAADNILSGGAQQSPNGTLLPFVNCRDPGQDRAVAPE-DESCAYALGR CAC1C_RABBIT 2094 ADACDLTIEEMENAADDILSGGARQSPNGTLLPFVNRRDPGRDRAGQNEQDASGACAPGC CAC1C_HUMAN 2146 ADACDMTIEEMESAADNILSGGAPQSPNGALLPFVNCRDAGQDRAGGEE-DAGCVRARG- CAC1C_ZEBRAFISH 2077 AEACDMTMEEMENAADNILNANAPPNANGNLLPFIQCRDTGSQ-------ESRCSLSLGL CAC1C_RAT 2127 GRSEEALPDSRSYVSNL CAC1C_MOUSE 2123 GRSEEALADSRSYVSNL CAC1C_RABBIT 2153 GQSEEALADRRAGVSSL CAC1C_HUMAN 2204 APSEEELQDSRVYVSS- CAC1C_ZEBRAFISH 2130 SPATGSDGALEAELEESEGAGQRNSPLMEDEDMECVTSL

Figure S1. CCAT’s nuclear localization and transcription activation domains are conserved among CaV1.2 channels in vertebrates

70

Figure S2. CCAT derived from CaV1.2-YFP channel is regulated by depolarization

71

Supplemental Figure 3. CCAT regulates expression of endogenous genes

72

Figure S4.

7.32

18.

968

(5.6

87 to

13.

15)

1.22

5 (1

.205

to 1

.267

)G

jb5

NM

_010

291

Mus

mus

culu

s ga

p ju

nctio

n m

embr

ane

chan

nel p

rote

in b

eta

5 (G

jb5)

, mR

NA

[NM

_010

291]

3.56

93.

812

(2.9

5 to

5.2

09)

1.06

8 (1

.055

to 1

.08)

Mpv

17N

M_0

0862

2M

us m

uscu

lus

Mpv

17 tr

ansg

ene,

kid

ney

dise

ase

mut

ant (

Mpv

17),

mR

NA

[NM

_008

622]

3.55

13.

487

(2.8

67 to

3.9

91)

0.98

2 (0

.961

to 0

.998

)C

ol11

a2N

M_0

0992

6M

us m

uscu

lus

proc

olla

gen,

type

XI,

alph

a 2

(Col

11a2

), m

RN

A [N

M_0

0992

6]3.

107

3.57

6 (2

.796

to 4

.337

)1.

151

(1.1

39 to

1.1

72)

2310

021P

13R

ikBC

0265

04;

Mus

mus

culu

s R

IKEN

cD

NA

2310

021P

13 g

ene,

mR

NA

(cD

NA

clon

e M

GC

:570

92 IM

AGE:

6489

956)

, com

plet

e cd

s. [B

C04

9362

]2.

924

2.46

8 (1

.758

to 3

.041

)0.

844

(0.8

04 to

0.8

76)

Cat

nal1

NM

_018

761

Mus

mus

culu

s ca

teni

n al

pha-

like

1 (C

atna

l1),

mR

NA

[NM

_018

761]

2.73

52.

606

(2.4

15 to

2.7

12)

0.95

3 (0

.884

to 1

.03)

Slco

5a1

AK04

1736

1M

us m

uscu

lus

solu

te c

arrie

r org

anic

ani

on tr

ansp

orte

r fam

ily, m

embe

r 5A1

(Slc

o5a1

), m

RN

A [N

M_1

7284

1]2.

709

2.14

3 (1

.848

to 2

.63)

0.79

1 (0

.591

to 1

.044

)Pp

ef2

NM

_011

148

Mus

mus

culu

s pr

otei

n ph

osph

atas

e, E

F ha

nd c

alci

um-b

indi

ng d

omai

n 2

(Ppe

f2),

mR

NA

[NM

_011

148]

2.47

21.

955

(1.3

76 to

3.5

44)

0.79

1 (0

.712

to 0

.936

)Bt

g4AB

0509

83M

us m

uscu

lus

B-ce

ll tra

nslo

catio

n ge

ne 4

(Btg

4), m

RN

A [N

M_0

1949

3]2.

420

1.99

4 (1

.392

to 3

.077

)0.

824

(0.7

66 to

0.8

62)

His

t1h1

tN

M_0

1037

7M

us m

uscu

lus

hist

one

1, H

1t (H

ist1

h1t),

mR

NA

[NM

_010

377]

2.35

11.

855

(1.3

16 to

2.6

14)

0.78

9 (0

.708

to 0

.974

)Ta

f3AK

0155

34M

us m

uscu

lus

TAF3

RN

A po

lym

eras

e II,

TAT

A bo

x bi

ndin

g pr

otei

n (T

BP)-a

ssoc

iate

d fa

ctor

, mR

NA

(cD

NA

clon

e IM

AGE:

3066

0628

), pa

rtial

cds

[BC

0890

30]

2.29

313

.65

(9.9

36 to

16.

02)

5.95

4 (5

.237

to 6

.534

)Ifi

t1N

M_0

0833

1M

us m

uscu

lus

inte

rfero

n-in

duce

d pr

otei

n w

ith te

tratri

cope

ptid

e re

peat

s 1

(Ifit1

), m

RN

A [N

M_0

0833

1]2.

237

1.87

9 (1

.412

to 2

.502

)0.

84 (0

.712

to 0

.891

)BI

8554

34;

BI85

5434

603

3825

13F1

NC

I_C

GAP

_Mam

6 M

us m

uscu

lus

cDN

A cl

one

IMAG

E:54

1488

3 5'

, mR

NA

sequ

ence

[BI8

5543

4]2.

205

5.29

5 (3

.177

to 1

0)2.

401

(2.0

16 to

3.0

73)

Usp

18N

M_0

1190

9M

us m

uscu

lus

ubiq

uitin

spe

cific

pro

teas

e 18

(Usp

18),

mR

NA

[NM

_011

909]

2.17

71.

885

(1.4

16 to

3.0

9)0.

866

(0.8

6 to

0.8

71)

Cld

n19

AK03

2743

Mus

mus

culu

s cl

audi

n 19

(Cld

n19)

, mR

NA

[NM

_153

105]

2.17

61.

756

(1.5

46 to

2.1

89)

0.80

7 (0

.721

to 0

.992

)R

assf

6AK

0054

72M

us m

uscu

lus

Ras

ass

ocia

tion

(Ral

GD

S/AF

-6) d

omai

n fa

mily

6 (R

assf

6), m

RN

A [N

M_0

2847

8]2.

157

2.03

(1.6

35 to

2.4

93)

0.94

1 (0

.916

to 0

.959

)M

cf2

NM

_133

197

Mus

mus

culu

s m

cf.2

tran

sfor

min

g se

quen

ce (M

cf2)

, mR

NA

[NM

_133

197]

2.14

96.

673

(6.1

28 to

7.0

73)

3.10

5 (2

.969

to 3

.234

)Bn

ip3

NM

_009

760

Mus

mus

culu

s BC

L2/a

deno

viru

s E1

B 19

kDa-

inte

ract

ing

prot

ein

1, N

IP3

(Bni

p3),

mR

NA

[NM

_009

760]

2.10

12.

067

(1.7

34 to

2.2

96)

0.98

4 (0

.742

to 1

.566

)N

tn4

NM

_021

320

Mus

mus

culu

s ne

trin

4 (N

tn4)

, mR

NA

[NM

_021

320]

2.08

05.

608

(3.9

57 to

7.7

53)

2.69

6 (2

.529

to 2

.933

)C

cl5

NM

_013

653

Mus

mus

culu

s ch

emok

ine

(C-C

mot

if) li

gand

5 (C

cl5)

, mR

NA

[NM

_013

653]

2.07

51.

89 (1

.44

to 2

.727

)0.

911

(0.9

02 to

0.9

29)

Rgs

5BQ

9488

84;

Mus

mus

culu

s cD

NA

clon

e IM

AGE:

3710

250,

com

plet

e cd

s. [B

C00

5656

]2.

059

1.97

9 (1

.156

to 2

.622

)0.

961

(0.8

77 to

1.0

53)

Stk3

AK08

7803

;M

us m

uscu

lus

2 da

ys p

regn

ant a

dult

fem

ale

ovar

y cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:E

3300

23A1

5 pr

oduc

t:unk

now

n ES

T, fu

ll in

sert

sequ

ence

[AK0

8780

1]1.

998

1.90

6 (1

.704

to 2

.132

)0.

954

(0.8

57 to

1.0

61)

Cyl

n2N

M_0

0999

0M

us m

uscu

lus

cyto

plas

mic

link

er 2

(Cyl

n2),

mR

NA

[NM

_009

990]

0.15

62.

053

(0.8

03 to

3.3

6)13

.15

(10.

87 to

16.

45)

V1rh

13N

M_1

3423

8.1

Mus

mus

culu

s vo

mer

onas

al 1

rece

ptor

, H13

(V1r

h13)

, mR

NA

[NM

_134

238]

0.25

72.

286

(1.7

35 to

2.6

75)

8.89

3 (4

.627

to 1

2.7)

Egln

3N

M_0

2813

3M

us m

uscu

lus

EGL

nine

hom

olog

3 (C

. ele

gans

) (Eg

ln3)

, mR

NA

[NM

_028

133]

0.35

12.

74 (2

.015

to 3

.857

)7.

813

(5.9

65 to

9.3

03)

Cam

pN

M_0

0992

1M

us m

uscu

lus

cath

elic

idin

ant

imic

robi

al p

eptid

e (C

amp)

, mR

NA

[NM

_009

921]

0.36

10.

285

(0.1

95 to

0.4

42)

0.78

9 (0

.775

to 0

.813

)AU

0187

78BC

0134

79M

us m

uscu

lus

expr

esse

d se

quen

ce A

U01

8778

(AU

0187

78),

mR

NA

[NM

_144

930]

0.37

50.

331

(0.1

4 to

0.7

84)

0.88

3 (0

.846

to 0

.921

)BQ

1751

90U

nkno

wn

0.40

32.

698

(2.5

68 to

2.8

87)

6.69

4 (6

.483

to 6

.805

)Lm

lnAK

0304

63M

us m

uscu

lus

leis

hman

olys

in-li

ke (m

etal

lope

ptid

ase

M8

fam

ily) (

Lmln

), m

RN

A [N

M_1

7282

3]0.

411

5.24

3 (4

.109

to 8

.328

)12

.75

(11.

47 to

14.

12)

2310

001H

13R

ikAK

0307

66M

us m

uscu

lus

mR

NA

for m

KIAA

1042

pro

tein

[AK1

2242

6]0.

418

3.15

5 (2

.222

to 4

.735

)7.

553

(5.0

87 to

9.3

97)

Swap

70N

M_0

0930

2M

us m

uscu

lus

SWA-

70 p

rote

in (S

wap

70),

mR

NA

[NM

_009

302]

0.42

51.

909

(1.7

32 to

2.0

71)

4.49

(4.2

74 to

4.9

19)

LOC

4325

85BB

6675

59PR

EDIC

TED

: Mus

mus

culu

s th

yroi

d ho

rmon

e re

cept

or a

ssoc

iate

d pr

otei

n 1

(Thr

ap1)

, mR

NA

[XM

_109

726]

0.44

71.

92 (1

.55

to 2

.384

)4.

296

(3.3

93 to

6.1

76)

Pip5

k2a

NM

_008

845

Mus

mus

culu

s ph

osph

atid

ylin

osito

l-4-p

hosp

hate

5-k

inas

e, ty

pe II

, alp

ha (P

ip5k

2a),

mR

NA

[NM

_008

845]

0.46

40.

456

(0.3

65 to

0.5

77)

0.98

3 (0

.919

to 1

.052

)49

2150

9F24

Rik

AK01

4852

Mus

mus

culu

s ad

ult m

ale

test

is c

DN

A, R

IKEN

full-

leng

th e

nric

hed

libra

ry,c

lone

:492

1509

F24

prod

uct:h

ypot

hetic

al G

lyco

side

hyd

rola

se fa

mily

35 c

onta

inin

g pr

otei

n,[A

K014

852]

0.46

92.

637

(2.3

67 to

2.7

97)

5.62

2 (5

.46

to 5

.81)

BC01

0711

Mus

mus

culu

s cD

NA

clon

e IM

AGE:

5010

343,

par

tial c

ds. [

BC07

1254

]0.

479

7.83

7 (5

.104

to 9

.72)

16.3

7 (1

4.58

to 1

7.78

)Fm

n1N

M_0

1023

0M

us m

uscu

lus

form

in 1

(Fm

n1),

mR

NA

[NM

_010

230]

0.47

92.

046

(2.0

07 to

2.0

7)4.

273

(4.1

86 to

4.3

41)

Pa2g

4N

M_0

1111

9M

us m

uscu

lus

prol

ifera

tion-

asso

ciat

ed 2

G4

(Pa2

g4),

mR

NA

[NM

_011

119]

0.48

00.

426

(0.3

31 to

0.5

72)

0.88

7 (0

.801

to 1

.08)

Prss

32BC

0249

03M

us m

uscu

lus

prot

ease

, ser

ine,

32

(Prs

s32)

, mR

NA

[NM

_027

220]

0.48

41.

889

(1.6

74 to

2.0

85)

3.89

9 (3

.38

to 4

.395

)C

lasp

1AJ

2769

62M

us m

uscu

lus

13 d

ays

embr

yo fo

relim

b cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:5

9304

24F1

3 pr

oduc

t:CLI

P-as

soci

atin

g pr

otei

n C

LASP

1, fu

ll in

sert

seq

[AK0

3118

1]0.

493

0.42

4 (0

.345

to 0

.496

)0.

86 (0

.789

to 0

.997

)AK

0320

63M

us m

uscu

lus

adul

t mal

e m

edul

la o

blon

gata

cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:6

3305

68D

16 p

rodu

ct:u

ncla

ssifi

able

, ful

l ins

ert s

eque

nce.

[AK0

3206

3]0.

512

0.36

7 (0

.347

to 0

.394

)0.

717

(0.6

93 to

0.7

35)

Grin

2dN

M_0

0817

2M

us m

uscu

lus

glut

amat

e re

cept

or, i

onot

ropi

c, N

MD

A2D

(eps

ilon

4) (G

rin2d

), m

RN

A [N

M_0

0817

2]0.

519

0.47

5 (0

.255

to 0

.885

)0.

916

(0.7

96 to

1.0

57)

Muc

10N

M_0

0864

4M

us m

uscu

lus

muc

in 1

0, s

ubm

andi

bula

r gla

nd s

aliv

ary

muc

in (M

uc10

), m

RN

A [N

M_0

0864

4]0.

572

0.46

6 (0

.399

to 0

.521

)0.

815

(0.6

2 to

1.0

23)

Ccl

12N

M_0

1133

1M

us m

uscu

lus

chem

okin

e (C

-C m

otif)

liga

nd 1

2 (C

cl12

), m

RN

A [N

M_0

1133

1]0.

575

0.48

5 (0

.333

to 0

.84)

0.84

3 (0

.508

to 1

.495

)Fi

gla

NM

_012

013

Mus

mus

culu

s fa

ctor

in th

e ge

rmlin

e al

pha

(Fig

la),

mR

NA

[NM

_012

013]

0.58

60.

468

(0.2

68 to

0.6

2)0.

798

(0.7

7 to

0.8

3)Pc

dha5

NM

_009

959

Mus

mus

culu

s pr

otoc

adhe

rin a

lpha

5 (P

cdha

5), m

RN

A [N

M_0

0995

9]

Gom

ez-O

spin

a, T

suru

ta, B

arre

to-C

hang

, Hu

and

Dol

met

sch

Supp

lem

enta

ry T

able

1

CC

AT/C

CAT

TASy

nony

ms

Gen

bank

Des

crip

tion

Gen

es s

igni

fican

tly re

gula

ted

by C

CAT

CC

AT/U

ntra

nsfe

cted

CC

AT <

CC

ATTA

CC

ATTA

/Unt

rans

fect

ed

CC

AT/C

CAT

TA

CC

AT >

CC

ATTA

CC

AT/U

ntra

nsfe

cted

CC

ATTA

/Unt

rans

fect

edSy

nony

ms

Gen

bank

Des

crip

tion

Tab

le 1

. Gen

es S

igni

fican

tly u

preg

ulat

ed b

y C

CA

T o

vere

xpre

ssio

n

73

Gene name Description Fold Changenovel proteins-32 genesNAP050965-1 Unknown 12.6AK046243 adult male corpora quadrigemina cDNA, RIKEN full-length enriched library, clone:B230359I22 product:hypothetical LIM domain, Villin headpiece domain containing protein, full insert sequence. [AK046243] 2.416AK036079 16 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:9630032O13 product:unknown EST, full insert sequence [AK036079] 2.219530076H17 unknown EST [9530076H17] 2.187BC026942 RIKEN cDNA 2610036N15 gene, (cDNA clone MGC:30353 IMAGE:5011571), complete cds. [BC026942] 2.116AK032743 12 days embryo male wolffian duct includes surrounding region cDNA, RIKEN full-length enriched library, clone:6720426C15 product:CLAUDIN-19 (FRAGMENT), full insert sequence. [AK032743] 2.011D130050C24 unclassifiable [D130050C24] 2.004NM_145463 RIKEN cDNA 9430059P22 gene (9430059P22Rik), [NM_145463] 1.976NAP071275-1 Unknown 1.903NAP104086-1 for mKIAA1398 protein. [AK129349] 1.896B930011K02 unclassifiable [B930011K02] 1.892E030017C01 hypothetical protein [E030017C01] 1.872A130026M04 unclassifiable [A130026M04] 1.821NAP051804-1 Unknown 1.819B930091H02 unknown EST [B930091H02] 1.8C430019P20 unknown EST [C430019P20] 1.788A930002G02 hypothetical SAM domain (Sterile alpha motif) containing protein [A930002G02] 1.787NAP034432-1 Unknown 1.776E230011A12 unknown EST [E230011A12] 1.77BC006743 cDNA clone MGC:12079 IMAGE:3708702, complete cds. [BC006743] 1.724AK019190 10 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2610510H03 product:hypothetical protein, full insert sequence. [AK019190] 1.619NM_025832 RIKEN cDNA 1300019C06 gene (1300019C06Rik), [NM_025832] 1.605AK085030 13 days embryo lung cDNA, RIKEN full-length enriched library, clone:D430026K21 product:hypothetical ARM repeat structure containing protein, full insert sequence. [AK085030] 1.594AK003565 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110008E08 product:hypothetical protein, full insert sequence. [AK003565] 1.556BC023450 cDNA sequence BC035291, (cDNA clone IMAGE:5054193), with apparent retained intron. [BC023450] 1.535NAP029182-1 Unknown 1.511NM_028170 RIKEN cDNA 1700030K09 gene (1700030K09Rik), [NM_028170] 1.491AK005714 adult male testis cDNA, RIKEN full-length enriched library, clone:1700007H20 product:hypothetical Heat shock hsp20 (alpha crystallin) proteins family containing protein, full insert sequence. [AK005714] 1.478C330007P06 hypothetical protein [C330007P06] 1.476NM_029532 RIKEN cDNA 6330548G22 gene (6330548G22Rik), [NM_029532] 1.457AK030240 adult male testis cDNA, RIKEN full-length enriched library, clone:4933429B21 product:zinc finger protein 13, full insert sequence. [AK030240] 1.453NAP030565-1 Unknown 1.452Transcription -8 genesNM_013926 chromobox homolog 8 (Drosophila Pc class) (Cbx8), [NM_013926] 1.499NM_010377 histone 1, H1t (Hist1h1t), [NM_010377] 1.509NM_010234 FBJ osteosarcoma oncogene (Fos), [NM_010234] 1.681NAP056339-1 S79410 nuclear localization signal binding protein {}, partial (12%) [TC1084061] 2.378E430001J03 MYC PROTO-ONCOGENE PROTEIN (C-MYC) [E430001J03] 1.51E030018P21 general transcription factor II I [E030018P21] 2.077D130023D18 TAFII140 PROTEIN (FRAGMENT) homolog [] [D130023D18] 1.532ENSMUST00000036004 HNRNP A1 (FRAGMENT). [Source:SPTREMBL;Acc:P70370] [ENSMUST00000036004] 1.499Signaling molecules- 6 genesNM_031256 pleckstrin homology domain-containing, family A (phosphoinositide binding specific) member 3 (Plekha3), [NM_031256] 1.727NM_008813 ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1), [NM_008813] 1.477NM_007913 early growth response 1 (Egr1), [NM_007913] 1.587C530047D22 transducer of ERBB2, 2 [C530047D22] 1.5BC005656 regulator of G-protein signaling 5, (cDNA clone IMAGE:3710250), complete cds. [BC005656] 3.089AB015614 for SET-binding protein (SEB), partial cds. [AB015614] 2.686Receptor and adhesion molecules - 6 genesX12905 for properdin (AA 5 - 441). [X12905] 1.471NM_010688 LIM and SH3 protein 1 (Lasp1), [NM_010688] 2.308NM_009142 chemokine (C-X3-C motif) ligand 1 (Cx3cl1), [NM_009142] 1.827NAP040979-1 CKR2_C-C chemokine receptor type 2 (C-C CKR-2) (CC-CKR-2) (CCR-2) (CCR2) (JE/FIC receptor) (MCP-1 receptor). [Mouse] {}, complete [TC968512] 1.874BC059909 gene model 793, (NCBI), (cDNA clone IMAGE:6825008), partial cds. [BC059909] 3.971AF068258 EY-cadherin precursor, , partial cds. [AF068258] 1.9229330162O16 inferred: sprouty homolog 4 (Drosophila) [9330162O16] 1.607cytoskeleton - 5 genes NM_010230 formin (Fmn), [NM_010230] 1.591NAP026121-1 MYOSIN HEAVY CHAIN, FAST SKELETAL MUSCLE, EMBRYONIC (FRAGMENT). [Source:SWISSPROT;Acc:P13541] [ENSMUST00000007302] 1.461M74753 myosin heavy chain , 3' flank. [M74753] 1.481ENSMUST00000068404 ACTIN RELATED PROTEIN 2/3 COMPLEX, SUBUNIT 5; ACTIN RELATED PROTEIN 2/3 COMPLEX, SUBUNIT 5 (165 KDA). [Source:RefSeq;Acc:NM_026369] [ENSMUST00000068404] 1.455BC014809 tropomyosin 2, beta, (cDNA clone MGC:18587 IMAGE:3497670), complete cds. [BC014809] 1.501Metabolic enzymes -3 genesNM_018788 exostoses (multiple)-like 3 (Extl3), [NM_018788] 1.556NAP045478-1 AF199509 NADPH-dependent FMN and FAD containing oxidoreductase {Homo sapiens}, partial (17%) [TC1078483] 6.414BC028276 demethyl-Q 7, (cDNA clone IMAGE:5376178), partial cds. [BC028276] 1.557Translation - 2 genesNAP108110-1 AF337055 lysyl tRNA synthetase {Methanosarcina barkeri}, partial (3%) [TC960574] 1.457NM_007906 eukaryotic translation elongation factor 1 alpha 2 (Eef1a2), [NM_007906] 1.456Proteases- 2 genesNAP031150-1 AF302077 neprilysin-like peptidase gamma {}, partial (5%) [TC1010010] 1.5182700060P05 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 [2700060P05] 1.483Ion channel and Transporters- 2 genesNM_010291 gap junction membrane channel protein beta 5 (Gjb5), [NM_010291] 14.36NM_009579 solute carrier family 30 (zinc transporter), member 1 (Slc30a1), [NM_009579] 1.496Others- 2 genesNM_008622 Mpv17 transgene, kidney disease mutant (Mpv17), [NM_008622] 8.499NAP037317-1 T2_Octapeptide-repeat protein T2. [Mouse] {}, partial (25%) [TC951701] 1.782

CCAT vs GFP upregulated genes

Table 2. CCAT versus GFP upregulated genes

74

Gene name Description Fold Changenovel proteins-175 genesC230075P17 unclassifiable [C230075P17] -7.6921700034G24 unknown EST [1700034G24] -5.348BC024416 mRNA similar to RIKEN cDNA 9030624G23 gene (cDNA clone MGC:36379 IMAGE:4988668), complete cds [BC024416] -4.2196530405F15 unknown EST [6530405F15] -3.268NM_177210 RIKEN cDNA D830044D21 gene (D830044D21Rik), mRNA [NM_177210] -3.0584631411J20 unknown EST [4631411J20] -2.9241110005G03 unclassifiable [1110005G03] -2.849NM_026690 RIKEN cDNA 0610012D14 gene (0610012D14Rik), mRNA [NM_026690] -2.762A130066H07 unknown EST [A130066H07] -2.6184732470K04 weakly similar to CDNA FLJ25135 FIS, CLONE CBR06974 [Homo sapiens] [4732470K04] -2.4884933412F11 inferred: RIKEN cDNA 4933412F11 gene [4933412F11] -2.481AK016814 adult male testis cDNA, RIKEN full-length enriched library, clone:4933415A04 product:hypothetical protein, full insert sequence. [AK016814] -2.463NAP027343-1 Unknown -2.451B930066C19 unclassifiable [B930066C19] -2.415NP580029 BAC29978.1 unnamed protein product [] [NP580029] -2.315NAP065110-1 Unknown -2.2529630032J19 unknown EST [9630032J19] -2.2171700091C04 unknown EST [1700091C04] -2.198D830035G22 hypothetical Hypothetical protein HI1434 (YbaK homologue) structure containing protein [D830035G22] -2.1653110001P10 unclassifiable [3110001P10] -2.151ENSMUST00000049565 RIKEN cDNA 9530080O11 gene (9530080O11Rik), mRNA [NM_175680] -2.151ENSMUST00000052692 RIKEN cDNA 1110006O24 gene (1110006O24Rik), mRNA [NM_021417] -2.137AK033606 adult male cecum cDNA, RIKEN full-length enriched library, clone:9130025L13 product:similar to FGF RECEPTOR 4B [Homo sapiens], full insert sequence. [AK033606] -2.137NAP037294-1 Unknown -2.1324833401H05 unclassifiable [4833401H05] -2.1327030417J21 hypothetical protein [7030417J21] -2.110NAP012952-001 Unknown -2.083AK014285 17 days embryo head cDNA, RIKEN full-length enriched library, clone:3200001I04 product:hypothetical Cysteine-rich flanking region, C-terminal/Leucine-rich repeat/Leucine-rich repeat, typical subtype containing protein, full insert sequence -2.079NM_177850 RIKEN cDNA 9230105K17 gene (9230105K17Rik), mRNA [NM_177850] -2.058ENSMUST00000056761 adult male testis cDNA, RIKEN full-length enriched library, clone:4930505N22 product:hypothetical protein, full insert sequence. [AK015708] -2.058NAP051032-1 UI-M-HB0-clk-h-02-0-UI.r1 NIH_BMAP_HB0 cDNA clone IMAGE:30619849 5', mRNA sequence [CF742218] -2.033B830034B11 unknown EST [B830034B11] -2.012BC020151 cDNA sequence BC020151, mRNA (cDNA clone MGC:28382 IMAGE:4021767), complete cds. [BC020151] -2.008D230050L11 unclassifiable [D230050L11] -2.000AK015919 adult male testis cDNA, RIKEN full-length enriched library, clone:4930527J03 product:hypothetical Alanine-rich region containing protein, full insert sequence. [AK015919] -1.9928430403D15 unclassifiable [8430403D15] -1.988BC025887 open reading frame 34, mRNA (cDNA clone IMAGE:5252967), partial cds. [BC025887] -1.976BI151098 BI151098 602917037F1 NCI_CGAP_Lu29 cDNA clone IMAGE:5067368 5', mRNA sequence [BI151098] -1.953B230379C01 unknown EST [B230379C01] -1.946C530030P08 hypothetical protein [C530030P08] -1.946ENSMUST00000051080 adult male testis cDNA, RIKEN full-length enriched library, clone:4930516E05 product:hypothetical Gram-positive cocci surface protein 'anchoring' hexapeptide containing protein, full insert sequence. [AK015803] -1.942NAP014274-001 Unknown -1.938D030041L05 unclassifiable [D030041L05] -1.934E130309O17 hypothetical protein [E130309O17] -1.927AK005010 adult male liver cDNA, RIKEN full-length enriched library, clone:1300015B04 product:similar to CDNA FLJ32009 FIS, CLONE NT2RP7009498, WEAKLY SIMILAR TO FIBULIN-1, ISOFORM A PRECURSOR [Homo sapiens], full insert sequence [AK005010]-1.923AK049933 adult male hippocampus cDNA, RIKEN full-length enriched library, clone:C630012M08 product:SNRNA ACTIVATING PROTEIN COMPLEX 50 KDA SUBUNIT(PROXIMAL SEQUENCE ELEMENT-BINDING TRANSCRIPTION FACTOR BETA -1.923C030002C18 unknown EST [C030002C18] -1.919AK077155 adult male testis cDNA, RIKEN full-length enriched library, clone:4933414G08 product:weakly similar to HYPOTHETICAL 68.7 KDA PROTEIN [Macaca fascicularis], full insert sequence. [AK077155] -1.919ENSMUST00000060815 Unknown -1.916NAP062636-1 UI-M-CG0p-bdb-d-11-0-UI.s1 NIH_BMAP_Ret4_S2 cDNA clone UI-M-CG0p-bdb-d-11-0-UI 3'. [BE981373] -1.912A930019J01 unknown EST [A930019J01] -1.905E330009A12 unknown EST [E330009A12] -1.901NAP068274-1 RIKEN cDNA 6030419C18 gene (6030419C18Rik), mRNA [NM_176921] -1.901AK029554 adult male testis cDNA, RIKEN full-length enriched library, clone:4921526F01 product:weakly similar to PROTEIN C21ORF13 [Homo sapiens], full insert sequence. [AK029554] -1.8989430062F24 unclassifiable [9430062F24] -1.8946720477H23 unknown EST [6720477H23] -1.890AK003303 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110002J03 product:unknown EST, full insert sequence [AK003303] -1.890AK037260 16 days neonate thymus cDNA, RIKEN full-length enriched library, clone:A130001I01 product:inferred: RIKEN cDNA 2810488G03 gene, full insert sequence. [AK037260] -1.890NP573446 BAC36495.1 unnamed protein product [] [NP573446] -1.883NM_177051 RIKEN cDNA C730014E05 gene (C730014E05Rik), mRNA [NM_177051] -1.883ENSMUST00000055270 2 days pregnant adult female ovary cDNA, RIKEN full-length enriched library, clone:E330039O16 product:unknown EST, full insert sequence. [AK087910] -1.883NAP103733-1 Unknown -1.880A430101J24 unclassifiable [A430101J24] -1.880NAP071390-1 RIKEN cDNA B830017H08 gene (B830017H08Rik), mRNA [NM_001002790] -1.876AK018840 adult male testis cDNA, RIKEN full-length enriched library, clone:1700034E13 product:hypothetical Zinc finger, C2H2 type containing protein, full insert sequence. [AK018840] -1.876NAP035983-1 Unknown -1.873NAP097891-001 Unknown -1.862AK015956 adult male testis cDNA, RIKEN full-length enriched library, clone:4930533K18 product:hypothetical protein, full insert sequence. [AK015956] -1.859ENSMUST00000059184 adult male testis cDNA, RIKEN full-length enriched library, clone:4930423F13 product:hypothetical protein, full insert sequence. [AK019581] -1.859NAP050809-1 Unknown -1.855A130057H05 unknown EST [A130057H05] -1.842C630018D16 unknown EST [C630018D16] -1.835E030043F12 RIKEN cDNA 2210414H16 gene [E030043F12] -1.828AK016492 adult male testis cDNA, RIKEN full-length enriched library, clone:4931430N09 product:unclassifiable, full insert sequence [AK016492] -1.828Gene name Description Fold Change5033423K11 hypothetical protein [5033423K11] -1.821AK003577 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110008I14 product:hypothetical SEA domain containing protein, full insert sequence. [AK003577] -1.818NM_026808 RIKEN cDNA 1110028A07 gene (1110028A07Rik), mRNA [NM_026808] -1.805AI467211 vd74h04.x1 Beddington embryonic region cDNA clone IMAGE:806359 3'. [AI467211] -1.802AK015063 adult male testis cDNA, RIKEN full-length enriched library, clone:4930403J07 product:hypothetical Double-stranded RNA binding (DsRBD) domain/Adenosine-deaminase (editase) domain containing protein, full insert sequence. [AK015063] -1.7921500031H18 unknown EST [1500031H18] -1.786E130307J04 hypothetical protein [E130307J04] -1.786E130318F11 unknown EST [E130318F11] -1.779AA738637 vv59h10.r1 Soares_thymus_2NbMT cDNA clone IMAGE:1226755 5'. [AA738637] -1.7731110038B08 inferred: homologue to bA12M19.1.3 (novel protein) {Homo sapiens} [1110038B08] -1.773NAP063026-1 similar to hypothetical protein FLJ38281 (LOC382019), mRNA [XM_356088] -1.773AK041206 adult male aorta and vein cDNA, RIKEN full-length enriched library, clone:A530090G11 product:R KAPPA B homolog [Homo sapiens], full insert sequence. [AK041206] -1.7732900090F08 unknown EST [2900090F08] -1.770BC021614 cDNA sequence BC021614, mRNA (cDNA clone MGC:37914 IMAGE:5102505), complete cds. [BC021614] -1.764AK011391 10 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2610014F08 product:hypothetical SAM domain (Sterile alpha motif)/Modified RING finger domain/G-protein beta WD-40 repeats containing protein, full insert sequence. [A-1.764C230075A15 unclassifiable [C230075A15] -1.761AK006745 adult male testis cDNA, RIKEN full-length enriched library, clone:1700049J03 product:hypothetical protein, full insert sequence. [AK006745] -1.757E130120I19 unclassifiable [E130120I19] -1.754AK034394 adult male diencephalon cDNA, RIKEN full-length enriched library, clone:9330185P08 product:CDNA FLJ10704 FIS, CLONE NT2RP3000841 homolog [Homo sapiens], full insert sequence. [AK034394] -1.7424932416K20 hypothetical protein [4932416K20] -1.736AK035222 adult male urinary bladder cDNA, RIKEN full-length enriched library, clone:9530003A11 product:hypothetical Copper amine oxidase containing protein, full insert sequence. [AK035222] -1.736AK006033 adult male testis cDNA, RIKEN full-length enriched library, clone:1700016H13 product:hypothetical protein, full insert sequence. [AK006033] -1.736AK081177 10 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:B930096O19 product:CGI-40 PROTEIN homolog [Homo sapiens], full insert sequence. [AK081177] -1.730NM_026208 RIKEN cDNA 1700019N19 gene (1700019N19Rik), mRNA [NM_026208] -1.724AK016497 adult male testis cDNA, RIKEN full-length enriched library, clone:4931431F19 product:hypothetical Ubiquitin domain containing protein, full insert sequence. [AK016497] -1.724A730045E23 unknown EST [A730045E23] -1.721NM_021447 ring finger protein 30 (Rnf30), mRNA [NM_021447] -1.718NAP106603-1 Unknown -1.712NM_030890 open reading frame 31 (ORF31), mRNA [NM_030890] -1.712NAP029297-1 Unknown -1.709NM_206973 RIKEN cDNA A930009H15 gene (A930009H15Rik), mRNA [NM_206973] -1.704D830005K03 unknown EST [D830005K03] -1.698NM_025584 RIKEN cDNA 2410026K10 gene (2410026K10Rik), mRNA [NM_025584] -1.695NM_023258 PYD and CARD domain containing (Pycard), mRNA [NM_023258] -1.695AK006273 adult male testis cDNA, RIKEN full-length enriched library, clone:1700023F06 product:hypothetical protein, full insert sequence. [AK006273] -1.695NAP044523-1 Unknown -1.6862210403N08 hypothetical protein [2210403N08] -1.686NAP033236-1 Unknown -1.681Transcription -34 genesENSMUST00000053071 TRANSCRIPTION FACTOR GATA-6 (GATA BINDING FACTOR-6). [Source:SWISSPROT;Acc:Q61169] [ENSMUST00000053071] -2.494AK043538 10 days neonate cortex cDNA, RIKEN full-length enriched library, clone:A830006N08 product:hypothetical Ankyrin repeat region circular profile/Yeast DNA-binding domain containing protein, full insert sequence. [AK043538] -2.475NM_008814 insulin promoter factor 1, homeodomain transcription factor (Ipf1), mRNA [NM_008814] -2.353NM_183298 forkhead box E1 (thyroid transcription factor 2) (Foxe1), mRNA [NM_183298] -2.247NM_146065 activating transcription factor 7 (Atf7), mRNA [NM_146065] -2.174AY364010 NALP12 mRNA, partial cds. [AY364010] -2.165AB010307 mRNA for mszf2, partial cds. [AB010307] -2.041X51959 Brn-3 gene POU-box region. [X51959] -2.016NM_009237 SRY-box containing gene 3 (Sox3), mRNA [NM_009237] -1.992NM_015758 forkhead box E3 (Foxe3), mRNA [NM_015758] -1.972AK077696 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730530B02 product:weakly similar to HYPOTHETICAL 30.1 KDA PROTEIN (TGF BETA INDUCIBLE NUCLEAR PROTEIN TINP1) (HAIRY CELL LEUKEMIA -1.908NM_009576 zinc finger protein of the cerebellum 4 (Zic4), mRNA [NM_009576] -1.869AK040404 0 day neonate thymus cDNA, RIKEN full-length enriched library, clone:A430091O22 product:hypothetical RNA-binding region RNP-1 (RNA recognition motif) containing protein, full insert sequence. [AK040404] -1.845NM_152947 zinc finger protein 339 (Zfp339), transcript variant B, mRNA [NM_152947] -1.825NM_178192 histone 1, H4a (Hist1h4a), mRNA [NM_178192] -1.799AK017633 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730441M18 product:hypothetical Zinc finger, C2H2 type containing protein, full insert sequence. [AK017633] -1.779

CCAT vs GFP downregulated genes

Table 3. CCAT versus GFP downregulated genes

75

ENSMUST00000023456 YIPPEE-LIKE PROTEIN 1 (DGL-1) (MDGL-1). [Source:SWISSPROT;Acc:Q9ESC7] [ENSMUST00000023456] -1.748NAP048862-1 ets homologous factor (Ehf), mRNA [NM_007914] -1.742AY080897 onecut 3 mRNA, complete cds. [AY080897] -1.695E130320J01 runt related transcription factor 3 [E130320J01] -1.689Signaling molecules- 36 genesAJ539223 mRNA for erythroid differentiation regulator (edr gene). [AJ539223] -3.106NM_009362 trefoil factor 1 (Tff1), mRNA [NM_009362] -2.899NM_007445 anti-Mullerian hormone (Amh), mRNA [NM_007445] -2.469NM_008712 nitric oxide synthase 1, neuronal (Nos1), mRNA [NM_008712] -2.342NAP102169-1 oxytocin (Oxt), mRNA [NM_011025] -2.331NM_009379 thrombopoietin (Thpo), mRNA [NM_009379] -2.3201500036H07 weakly similar to ARF GTPASE-ACTIVATING PROTEIN GIT2 (G PROTEIN-COUPLED RECEPTOR KINASE- INTERACTOR 2) (TYROSINE-PHOSPHORYLATED PROTEIN CAT-2) [] [1500036H07] -2.183U49723 guanylyl cyclase C (Gucy2c) mRNA, partial cds. [U49723] -2.037D930034C02 protein tyrosine phosphatase, non-receptor type 14 [D930034C02] -2.004E130103H07 inferred: ADP-ribosylation factor-directed GTPase activating protein isoform b {} [E130103H07] -1.9926330436O20 hypothetical Sushi domain / SCR repeat / CCP module containing protein [6330436O20] -1.992ENSMUST00000053066 SIMILAR TO QUAKING II. [Source:SPTREMBL;Acc:Q8K4Y1] [ENSMUST00000053066] -1.927NM_010473 histidine rich calcium binding protein (Hrc), mRNA [NM_010473] -1.927NM_011477 small proline-rich protein 2K (Sprr2k), mRNA [NM_011477] -1.912NAP019468-001 hematopoietic cell signal transducer (Hcst), mRNA [NM_011827] -1.883Gene name Description Fold ChangeNAP106185-1 AXU1_ AXIN1 up-regulated gene 1 protein (TGF-beta induced apoptosis protein 3) (TAIP-3). [] {}, partial (33%) [TC1023468] -1.869NM_007522 Bcl-associated death promoter (Bad), mRNA [NM_007522] -1.859NM_016933 protein tyrosine phosphatase, receptor type, C polypeptide-associated protein (Ptprcap), mRNA [NM_016933] -1.848NM_199042 THAP domain containing, apoptosis associated protein 1 (Thap1), mRNA [NM_199042] -1.835NM_031192 renin 1 structural (Ren1), mRNA [NM_031192] -1.835NM_033614 phosphodiesterase 6C, cGMP specific, cone, alpha prime (Pde6c), mRNA [NM_033614] -1.783NM_011108 phospholipase A2, group IIA (platelets, synovial fluid) (Pla2g2a), mRNA [NM_011108] -1.776NM_007828 death-associated kinase 3 (Dapk3), mRNA [NM_007828] -1.770AK053554 0 day neonate eyeball cDNA, RIKEN full-length enriched library, clone:E130107I17 product:INOSITOL POLYPHOSPHATE 4-PHOSPHATASE TYPE II-ALPHA homolog [Rattus norvegicus], full insert sequence. [AK053554] -1.739NAP027959-1 similar to ERF-2 (LOC333473), mRNA [XM_285657] -1.721L33768 (clone 32D5-1) protein tyrosine kinase (JAK3) mRNA. [L33768] -1.7182610524P08 Era (G-protein)-like 1 (E. coli) [2610524P08] -1.701Receptor and adhesion molecules - 29 genesNM_146275 olfactory receptor 1402 (Olfr1402), mRNA [NM_146275] -4.237NM_031499 proline rich protein 2 (Prp2), mRNA [NM_031499] -2.494NAP000791-001 AB065580 seven transmembrane helix receptor {Homo sapiens}, partial (9%) [TC1021675] -2.331AF199614 fibroblast growth factor homologous factor 2 isoform 1U (FHF-2) mRNA, partial cds. [AF199614] -2.208NM_010704 leptin receptor (Lepr), mRNA [NM_010704] -2.174NM_023304 fibroblast growth factor 22 (Fgf22), mRNA [NM_023304] -2.155NM_146649 olfactory receptor 1160 (Olfr1160), mRNA [NM_146649] -2.1052310003H23 LEUCINE-RICH-DOMAIN INTER-ACTING PROTEIN 1 (PPARGAMMA COFACTOR 2) (PEROXISOME PROLIFERATIVE ACTIVATED RECEPTOR, GAMMA, COACTIVATOR 2) homolog [] [2310003H23] -2.062NM_001001999 glycoprotein Ib, beta polypeptide (Gp1bb), transcript variant 1, mRNA [NM_001001999] -2.016NM_015738 galanin receptor 3 (Galr3), mRNA [NM_015738] -1.980S78451 interleukin-3 receptor beta subunit [mice, D35 promyelocytic cells, mRNA Partial Mutant, 331 nt]. [S78451] -1.946NM_020291 olfactory receptor 480 (Olfr480), mRNA [NM_020291] -1.852NAP053944-1 AF093669 peroxisomal biogenesis factor {}, partial (8%) [TC1055468] -1.835BC051445 interleukin 1 receptor-like 1, mRNA (cDNA clone MGC:60556 IMAGE:30073421), complete cds. [BC051445] -1.835AF464177 protocadherin mRNA, partial cds; alternatively spliced. [AF464177] -1.783BC030075 T-cell receptor beta, variable 13, mRNA (cDNA clone MGC:41416 IMAGE:1531980), complete cds. [BC030075] -1.779NM_011174 proline rich protein HaeIII subfamily 1 (Prh1), mRNA [NM_011174] -1.727AK048080 16 days embryo head cDNA, RIKEN full-length enriched library, clone:C130033F22 product:CADHERIN FIB1 (FRAGMENT) homolog [Homo sapiens], full insert sequence [AK048080] -1.727NAP107834-1 AF195661 transmembrane protein I1 {Homo sapiens}, partial (81%) [TC1021639] -1.704cytoskeleton - 9 genes NM_172868 paralemmin 2 (Palm2), mRNA [NM_172868] -2.315NM_009711 artemin (Artn), mRNA [NM_009711] -2.304NM_016887 claudin 7 (Cldn7), mRNA [NM_016887] -2.066AK088011 2 days neonate thymus thymic cells cDNA, RIKEN full-length enriched library, clone:E430002D17 product:UNCONVENTIONAL MYOSIN 1G VALINE FORM (FRAGMENT) homolog [Homo sapiens], full insert sequence. [AK088011] -2.004NP063775 AAA79963.1 synapsin I [NP063775] -1.969ENSMUST00000055959 synaptopodin (Synpo), mRNA [XM_129030] -1.802AF205079 strain ICR 90 kDa actin-associated protein palladin mRNA, partial cds. [AF205079] -1.764Metabolic enzymes -13 genesNM_133657 cytochrome P450, family 2, subfamily a, polypeptide 12 (Cyp2a12), mRNA [NM_133657] -2.012NM_010292 glucokinase (Gck), mRNA [NM_010292] -1.992NM_175140 carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8 (Chst8), mRNA [NM_175140] -1.927ENSMUST00000051542 SEPIAPTERIN REDUCTASE. [Source:SPTREMBL;Acc:Q62218] [ENSMUST00000051542] -1.845NM_021306 endothelin converting enzyme-like 1 (Ecel1), mRNA [NM_021306] -1.832M36289 beta-1,4-galactosyltransferase mRNA, 5' end. [M36289] -1.7838030457O12 cytosolic 5' nucleotidase, type 1A [8030457O12] -1.761NM_008437 napsin A aspartic peptidase (Napsa), mRNA [NM_008437] -1.718NM_011637 three prime repair exonuclease 1 (Trex1), mRNA [NM_011637] -1.692Gene name Description Fold ChangeProteases- 10 genesNM_008950 protease (prosome, macropain) 26S subunit, ATPase 5 (Psmc5), mRNA [NM_008950] -4.098NM_009776 serine (or cysteine) proteinase inhibitor, clade G, member 1 (Serping1), mRNA [NM_009776] -2.151NM_011458 serine (or cysteine) proteinase inhibitor, clade A, member 3K (Serpina3k), mRNA [NM_011458] -1.828NM_008407 inter-alpha trypsin inhibitor, heavy chain 3 (Itih3), mRNA [NM_008407] -1.773NM_009430 protease, serine, 2 (Prss2), mRNA [NM_009430] -1.767AB049453 tessp-1 mRNA for testis serine protease-1, complete cds. [AB049453] -1.761NAP102783-1 similar to SPI3C (LOC193403), mRNA [XM_111405] -1.712Ion channel and Transporters- 16 genesNAP019559-001 AF115505 sodium/calcium exchanger 1 splice variant NaCa10 {Homo sapiens}, partial (7%) [TC1006772] -2.604NAP029777-1 similar to potassium channel, subfamily K, member 9 (Task-3) (LOC223604), mRNA [XM_139425] -2.577NM_011773 solute carrier family 30 (zinc transporter), member 3 (Slc30a3), mRNA [NM_011773] -2.008NM_008172 glutamate receptor, ionotropic, NMDA2D (epsilon 4) (Grin2d), mRNA [NM_008172] -2.000NM_007378 ATP-binding cassette, sub-family A (ABC1), member 4 (Abca4), mRNA [NM_007378] -1.942NM_080466 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 (Kcnn3), mRNA [NM_080466] -1.842BC023117 solute carrier family 6 (neurotransmitter transporter, GABA), member 13, mRNA (cDNA clone MGC:28956 IMAGE:4240641), complete cds. [BC023117] -1.8322210409B01 NG22 PROTEIN homolog [Homo sapiens] [2210409B01] -1.773NM_022017 transient receptor potential cation channel, subfamily V, member 4 (Trpv4), mRNA [NM_022017] -1.770NM_205783 cholinergic receptor, muscarinic 5 (Chrm5), mRNA [NM_205783] -1.751AY255605 gamma-aminobutyric acid type B receptor mRNA, partial cds. [AY255605] -1.701BC039157 glutamate receptor, ionotropic, NMDA1 (zeta 1), mRNA (cDNA clone MGC:25375 IMAGE:4507986), complete cds. [BC039157] -1.678Extracellular matrix proteins - 7 genesNM_015784 periostin, osteoblast specific factor (Postn), mRNA [NM_015784] -1.992NM_053185 procollagen, type IV, alpha 6 (Col4a6), mRNA [NM_053185] -1.812NM_146007 procollagen, type VI, alpha 2 (Col6a2), mRNA [NM_146007] -1.730NM_139001 chondroitin sulfate proteoglycan 4 (Cspg4), mRNA [NM_139001] -1.706Others- 20 genesF830014N06 lymphocyte antigen 108 [F830014N06] -3.300AB032764 halap-X mRNA for haploid specific alanine-rich acidic protein, complete cds. [AB032764] -2.358Z31359 M.musculus (Balb/C) HTx02 mRNA. [Z31359] -2.062NAP100607-001 AE003774 CG31019-PA {Drosophila melanogaster}, partial (16%) [TC1024875] -1.996BC024677 allergen dI chain C2A, mRNA (cDNA clone MGC:19070 IMAGE:4193111), complete cds. [BC024677] -1.953BC051228 heat shock protein, alpha-crystallin-related, B6, mRNA (cDNA clone IMAGE:3471643), partial cds. [BC051228] -1.876U63712 testis-specific HMG-box protein m-tsHMG precursor mRNA, partial cds, and mitochondrial transcription factor m-mtTFA precursor mRNA, nuclear mRNA encoding mitochondrial protein, partial cds. [U63712] -1.859NM_011859 odd-skipped related 1 (Drosophila) (Odd1), mRNA [NM_011859] -1.835NM_012033 tubulointerstitial nephritis antigen (Tinag), mRNA [NM_012033] -1.783NM_013480 bone gamma-carboxyglutamate protein, related sequence 1 (Bglap-rs1), mRNA [NM_013480] -1.779NM_008995 peroxisome biogenesis factor 5 (Pex5), mRNA [NM_008995] -1.767AK046712 4 days neonate male adipose cDNA, RIKEN full-length enriched library, clone:B430320C24 product:weakly similar to HYPOTHETICAL 13.8 KDA PROTEIN [Homo sapiens], full insert sequence [AK046712] -1.761S83543 Cer-1=cerebellar-expressed gene/granule neuron differentiation-associated gene [mice, cerebellum, granule neurons, mRNA Partial, 622 nt]. [S83543] -1.727AK017618 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730435J01 product:hypothetical Serine-rich region containing protein, full insert sequence. [AK017618] -1.712NM_010245 Friend virus susceptibility 4 (Fv4), mRNA [NM_010245] -1.689

Table 3. CCAT versus GFP downregulated genes Page 2

76

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5 (G

jb5)

, mR

NA

[NM

_010

291]

Fmn1

NM

_010

230

1.65

3 (1

.642

to 1

.663

)7.

836

(5.1

04 to

9.7

2)16

.37

(14.

58 to

17.

78)

Mus

mus

culu

s fo

rmin

1 (F

mn1

), m

RN

A [N

M_0

1023

0]M

pv17

NM

_008

622

2.85

3 (0

.32

to 9

.876

)3.

812

(2.9

5 to

5.2

09)

1.06

8 (1

.055

to 1

.08)

Mus

mus

culu

s M

pv17

tran

sgen

e, k

idne

y di

seas

e m

utan

t (M

pv17

), m

RN

A [N

M_0

0862

2]C

x3cl

1N

M_0

0914

21.

821

(1.3

84 to

2.1

82)

3.13

6 (2

.51

to 3

.814

)1.

899

(1.7

29 to

2.0

82)

Mus

mus

culu

s ch

emok

ine

(C-X

3-C

mot

if) li

gand

1 (C

x3cl

1), m

RN

A [N

M_0

0914

2]Az

gp1

AY24

8694

;2.

031

(1.5

35 to

2.6

18)

2.88

(2.5

81 to

3.0

62)

1.84

2 (1

.558

to 2

.042

)M

us m

uscu

lus

zinc

-alp

ha-2

-gly

copr

otei

n 1

(Azg

p1) m

RN

A, c

ompl

ete

cds

[AY2

4869

4]Tr

im30

NM

_009

099

2.07

4 (1

.723

to 2

.416

)2.

611

(1.9

21 to

3.3

36)

2.25

9 (1

.842

to 2

.759

)M

us m

uscu

lus

tripa

rtite

mot

if pr

otei

n 30

(Trim

30),

mR

NA

[NM

_009

099]

Rim

bp2

XM_1

3239

61.

755

(1.7

07 to

1.8

47)

2.50

1 (2

.377

to 2

.619

)2.

543

(2.2

36 to

2.7

69)

Mus

mus

culu

s R

IMS

bind

ing

prot

ein

2, tr

ansc

ript v

aria

nt 1

Cat

nal1

NM

_018

761

3.54

8 (2

.99

to 4

.127

)2.

468

(1.7

58 to

3.0

41)

0.84

4 (0

.804

to 0

.876

)M

us m

uscu

lus

cate

nin

alph

a-lik

e 1

(Cat

nal1

), m

RN

A [N

M_0

1876

1]Se

leN

M_0

1134

51.

752

(1.2

65 to

3.1

88)

2.41

3 (1

.747

to 2

.873

)1.

947

(1.3

44 to

2.9

68)

Mus

mus

culu

s se

lect

in, e

ndot

helia

l cel

l (Se

le),

mR

NA

[NM

_011

345]

Slfn

10N

M_1

8154

21.

731

(1.4

67 to

2.1

49)

2.18

6 (1

.808

to 2

.82)

2.75

3 (1

.902

to 3

.375

)M

us m

uscu

lus

schl

afen

10

(Slfn

10),

mR

NA

[NM

_181

542]

1700

011H

14R

ikNM

_025

956

1.82

2 (1

.552

to 2

.129

)2.

148

(2.0

49 to

2.3

61)

1.11

2 (1

.038

to 1

.167

)M

us m

uscu

lus

RIK

EN c

DN

A 17

0001

1H14

gen

e (1

7000

11H

14R

ik),

mR

NA

[NM

_025

956]

AK08

0352

;1.

803

(1.7

61 to

1.8

31)

2.05

4 (1

.805

to 2

.295

)1.

709

(1.4

05 to

2.0

41)

Mus

mus

culu

s 3

days

neo

nate

thym

us c

DN

A, R

IKEN

full-

leng

th e

nric

hed

libra

ry, c

lone

:A63

0063

H21

pro

duct

:hyp

othe

tical

pro

tein

, ful

l ins

ert s

eque

nce

[AK0

8035

2]V1

rh13

NM

_134

238

3.02

6 (2

.773

to 3

.474

)2.

053

(0.8

03 to

3.3

6)13

.15

(10.

87 to

16.

45)

Mus

mus

culu

s vo

mer

onas

al 1

rece

ptor

, H13

(V1r

h13)

, mR

NA

[NM

_134

238]

Cdo

nN

M_0

2133

92.

173

(2.0

36 to

2.3

17)

2.02

6 (1

.289

to 2

.742

)1.

659

(1.0

52 to

2.2

04)

Mus

mus

culu

s ce

ll ad

hesi

on m

olec

ule-

rela

ted/

dow

n-re

gula

ted

by o

ncog

enes

(Cdo

n), m

RN

A [N

M_0

2133

9]R

gs5

BC00

5656

;3.

106

(2.5

69 to

3.8

)1.

89 (1

.44

to 2

.727

)0.

911

(0.9

02 to

0.9

29)

Mus

mus

culu

s cD

NA

clon

e IM

AGE:

3710

250,

com

plet

e cd

s. [B

C00

5656

]C

ldn1

9N

M_1

5310

51.

988

(1.4

33 to

2.6

66)

1.88

5 (1

.416

to 3

.09)

0.86

6 (0

.86

to 0

.871

)M

us m

uscu

lus

clau

din

19 (C

ldn1

9), m

RN

A [N

M_1

5310

5]

Gen

es d

ownr

egul

ated

by

CC

AT in

all

expe

rimen

ts <

0.5

fold

CC

AT v

rs c

ontr

olC

CAT

vrs

UN

TC

CAT

TA v

rs U

NT

Gck

NM

_010

292

0.19

2 (0

.131

to 0

.32)

0.23

(0.1

67 to

0.2

81)

0.18

3 (0

.138

to 0

.289

)M

us m

uscu

lus

gluc

okin

ase

(Gck

), m

RN

A [N

M_0

1029

2]St

yk1

NM

_172

891

0.25

8 (0

.174

to 0

.357

)0.

466

(0.4

05 to

0.5

36)

0.16

1 (0

.124

to 0

.222

)M

us m

uscu

lus

serin

e/th

reon

ine/

tyro

sine

kin

ase

1 (S

tyk1

), m

RN

A [N

M_1

7289

1]An

krd4

3N

M_1

8317

30.

307

(0.1

71 to

0.8

33)

0.40

4 (0

.317

to 0

.497

)0.

276

(0.2

46 to

0.2

97)

Mus

mus

culu

s an

kyrin

repe

at d

omai

n 43

Mfa

p3BC

0068

28;

0.32

8 (0

.242

to 0

.433

)0.

558

(0.4

29 to

0.6

66)

0.37

3 (0

.221

to 0

.508

)M

us m

uscu

lus

mic

rofib

rilla

r-ass

ocia

ted

prot

ein

3, m

RN

A (c

DN

A cl

one

MG

C:1

1870

IMAG

E:35

9787

8), c

ompl

ete

cds

[BC

0068

28]

Artn

NM

_009

711

0.34

5 (0

.317

to 0

.368

)0.

437

(0.4

16 to

0.4

61)

0.37

8 (0

.359

to 0

.389

)M

us m

uscu

lus

arte

min

(Artn

), m

RN

A [N

M_0

0971

1]G

rin2d

NM

_008

172

0.36

7 (0

.347

to 0

.394

)0.

505

(0.5

03 to

0.5

08)

0.71

7 (0

.693

to 0

.735

)M

us m

uscu

lus

glut

amat

e re

cept

or, i

onot

ropi

c, N

MD

A2D

(eps

ilon

4) (G

rin2d

), m

RN

A [N

M_0

0817

2]Po

stn

NM

_015

784

0.38

4 (0

.359

to 0

.432

)0.

507

(0.4

81 to

0.5

24)

0.33

2 (0

.286

to 0

.409

)M

us m

uscu

lus

perio

stin

, ost

eobl

ast s

peci

fic fa

ctor

(Pos

tn),

mR

NA

[NM

_015

784]

Ltbp

1N

M_0

1991

90.

408

(0.3

22 to

0.4

9)0.

588

(0.4

77 to

0.7

03)

0.32

7 (0

.318

to 0

.344

)M

us m

uscu

lus

late

nt tr

ansf

orm

ing

grow

th fa

ctor

bet

a bi

ndin

g pr

otei

n 1

(Ltb

p1),

trans

crip

t var

iant

1, m

RN

A [N

M_0

1991

9]In

hbc

NM

_010

565

0.41

9 (0

.382

to 0

.443

)0.

468

(0.3

97 to

0.5

21)

0.44

4 (0

.435

to 0

.457

)M

us m

uscu

lus

inhi

bin

beta

-C (I

nhbc

), m

RN

A [N

M_0

1056

5]Pt

prd

AK00

3303

;0.

423

(0.3

82 to

0.4

5)0.

535

(0.5

14 to

0.5

78)

0.43

6 (0

.391

to 0

.462

)M

us m

uscu

lus

18-d

ay e

mbr

yo w

hole

bod

y cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:1

1100

02J0

3 pr

oduc

t:unk

now

n ES

T, fu

ll in

sert

sequ

ence

. [AK

0033

03]

AK03

2063

;0.

424

(0.3

45 to

0.4

96)

0.48

1 (0

.359

to 0

.623

)0.

86 (0

.789

to 0

.997

)M

us m

uscu

lus

adul

t mal

e m

edul

la o

blon

gata

cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:6

3305

68D

16 p

rodu

ct:u

ncla

ssifi

able

, ful

l ins

ert s

eque

nce.

[AK0

3206

3]H

s6st

2N

M_0

1581

90.

426

(0.3

94 to

0.4

44)

0.49

3 (0

.379

to 0

.564

)0.

489

(0.3

03 to

0.7

55)

Mus

mus

culu

s he

para

n su

lfate

6-O

-sul

fotra

nsfe

rase

2 (H

s6st

2), m

RN

A [N

M_0

1581

9]49

3343

9C10

RikAK

0171

15;

0.44

(0.3

85 to

0.5

45)

0.50

1 (0

.37

to 0

.64)

0.29

8 (0

.216

to 0

.352

)M

us m

uscu

lus

adul

t mal

e te

stis

cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:4

9334

39C

10 p

rodu

ct:u

nkno

wn

EST,

full

inse

rt se

quen

ce. [

AK01

7115

]Xr

cc1

AK00

9778

;0.

45 (0

.41

to 0

.479

)0.

591

(0.5

48 to

0.6

24)

0.38

3 (0

.346

to 0

.458

)M

us m

uscu

lus

adul

t mal

e to

ngue

cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:2

3100

43G

11 p

rodu

ct:X

-ray

repa

ir co

mpl

emen

ting

defe

ctiv

e re

pair

in C

hine

se 1

, ful

l ins

ert s

eque

nce.

[AK0

0977

8]Am

hN

M_0

0744

50.

453

(0.4

09 to

0.4

77)

0.41

4 (0

.386

to 0

.468

)0.

473

(0.4

04 to

0.5

21)

Mus

mus

culu

s an

ti-M

ulle

rian

horm

one

(Am

h), m

RN

A [N

M_0

0744

5]So

x3N

M_0

0923

70.

461

(0.3

55 to

0.5

96)

0.50

9 (0

.474

to 0

.55)

0.42

9 (0

.409

to 0

.454

)M

us m

uscu

lus

SRY-

box

cont

aini

ng g

ene

3 (S

ox3)

, mR

NA

[NM

_009

237]

OR

F34

BC02

5887

;0.

461

(0.4

31 to

0.5

19)

0.51

8 (0

.49

to 0

.571

)0.

569

(0.5

56 to

0.5

93)

Mus

mus

culu

s op

en re

adin

g fra

me

34, m

RN

A (c

DN

A cl

one

IMAG

E:52

5296

7), p

artia

l cds

. [BC

0258

87]

Gal

r3N

M_0

1573

80.

469

(0.4

17 to

0.5

75)

0.51

4 (0

.445

to 0

.594

)0.

68 (0

.661

to 0

.706

)M

us m

uscu

lus

gala

nin

rece

ptor

3 (G

alr3

), m

RN

A [N

M_0

1573

8]Si

dt2

AK08

1177

;0.

471

(0.4

59 to

0.4

93)

0.58

3 (0

.56

to 0

.598

)0.

542

(0.5

39 to

0.5

44)

Mus

mus

culu

s 10

day

s ne

onat

e ce

rebe

llum

cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:B

9300

96O

19 p

rodu

ct:C

GI-4

0 PR

OTE

IN h

omol

og [H

omo

sapi

ens]

, ful

l ins

ert s

eque

nce.

[AK0

8117

7]Fi

gla

NM

_012

013

0.48

5 (0

.333

to 0

.84)

0.51

3 (0

.472

to 0

.548

)0.

843

(0.5

08 to

1.4

95)

Mus

mus

culu

s fa

ctor

in th

e ge

rmlin

e al

pha

(Fig

la),

mR

NA

[NM

_012

013]

Serp

ing1

NM

_009

776

0.49

4 (0

.453

to 0

.547

)0.

475

(0.4

45 to

0.5

29)

0.57

(0.4

87 to

0.7

73)

Mus

mus

culu

s se

rine

(or c

yste

ine)

pro

tein

ase

inhi

bito

r, cl

ade

G, m

embe

r 1 (S

erpi

ng1)

, mR

NA

[NM

_009

776]

Myo

1gN

M_1

7844

00.

496

(0.4

32 to

0.6

42)

0.50

9 (0

.481

to 0

.534

)0.

507

(0.4

91 to

0.5

19)

Mus

mus

culu

s m

yosi

n IG

(Myo

1g),

mR

NA

[NM

_178

440]

Kcnn

3N

M_0

8046

60.

504

(0.4

89 to

0.5

31)

0.55

2 (0

.54

to 0

.569

)0.

526

(0.5

2 to

0.5

37)

Mus

mus

culu

s po

tass

ium

inte

rmed

iate

/sm

all c

ondu

ctan

ce c

alci

um-a

ctiv

ated

cha

nnel

, sub

fam

ily N

, mem

ber 3

(Kcn

n3),

mR

NA

[NM

_080

466]

Wds

ub1

NM

_028

118

0.50

7 (0

.435

to 0

.563

)0.

575

(0.5

2 to

0.6

07)

0.48

2 (0

.415

to 0

.58)

Mus

mus

culu

s W

D re

peat

, SAM

and

U-b

ox d

omai

n co

ntai

ning

1Fo

xe3

NM

_015

758

0.50

7 (0

.463

to 0

.546

)0.

513

(0.5

to 0

.521

)0.

558

(0.5

55 to

0.5

64)

Mus

mus

culu

s fo

rkhe

ad b

ox E

3 (F

oxe3

), m

RN

A [N

M_0

1575

8]BI

1510

98;

0.51

(0.4

7 to

0.5

56)

0.51

9 (0

.466

to 0

.556

)0.

707

(0.6

88 to

0.7

28)

BI15

1098

602

9170

37F1

NC

I_C

GAP

_Lu2

9 M

us m

uscu

lus

cDN

A cl

one

IMAG

E:50

6736

8 5'

, mR

NA

sequ

ence

[BI1

5109

8]Tr

pv4

NM

_022

017

0.51

8 (0

.449

to 0

.598

)0.

576

(0.5

08 to

0.6

49)

0.54

4 (0

.512

to 0

.586

)M

us m

uscu

lus

trans

ient

rece

ptor

pot

entia

l cat

ion

chan

nel,

subf

amily

V, m

embe

r 4 (T

rpv4

), m

RN

A [N

M_0

2201

7]N

frkb

NM

_172

766

0.53

5 (0

.47

to 0

.613

)0.

582

(0.4

21 to

0.7

41)

0.46

1 (0

.437

to 0

.508

)M

us m

uscu

lus

nucl

ear f

acto

r rel

ated

to k

appa

B b

indi

ng p

rote

in (N

frkb)

, mR

NA

[NM

_172

766]

Dna

hc1

Z838

15;

0.53

8 (0

.296

to 0

.8)

0.35

5 (0

.276

to 0

.414

)0.

566

(0.3

98 to

0.9

1)M

.mus

culu

s m

RN

A fo

r axo

nem

al d

ynei

n he

avy

chai

n (p

artia

l, ID

mdh

c7).

[Z83

815]

2810

488G

03R

ikAK01

3449

;0.

54 (0

.511

to 0

.598

)0.

532

(0.5

25 to

0.5

37)

0.47

3 (0

.44

to 0

.493

)M

us m

uscu

lus

10, 1

1 da

ys e

mbr

yo w

hole

bod

y cD

NA,

RIK

EN fu

ll-le

ngth

enr

iche

d lib

rary

, clo

ne:2

8104

88G

03 p

rodu

ct:in

ferre

d: R

IKEN

cD

NA

2810

488G

03 g

ene,

full

inse

rt se

quen

ce. [

AK01

3449

]Ba

dN

M_0

0752

20.

546

(0.5

26 to

0.5

82)

0.54

1 (0

.504

to 0

.567

)0.

493

(0.4

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78

FIGURE LEGENDS

Figure 1. The C Terminus of Cav1.2 Is Found in the Nucleus of Neurons

(A) Western blot of HEK 293T cells expressing Cav1.2 probed with anti-II-III loop

antibody (left gel) or anti-CCAT (right gel). The first lane of each gel contains lysate

from untransfected cells (Unt), and the second contains lysate from cells expressing

CaV1.2.

(B) Western blots of membrane (Mem), nuclear (Nuc), and cytoplasmic (Cyt) extracts

from the cortex of P7 rats analyzed with the anti-CCAT (upper gel), anti-CREB (second

gel), anti-Cav1.2 II-III loop (third gel), and anti-GAPDH (bottom gel) antibodies. The

first two lanes contain extracts from HEK 293T cells expressing Cav1.2. CREB, and

CCAT immunoreactivity are detected only in the nuclear fraction, Cav1.2 probed with

anti-II-III loop antibody and GAPDH are found in the membrane and cytoplasmic

fractions confirming the efficacy of the fractionation.

(C) Immunocytochemistry of cortical neurons grown 6 days in vitro. Anti-CCAT

staining is shown in green and nuclei is shown in blue.

(D) High-power image shows strong anti-CCAT staining (green) of nuclei (blue) and

lighter staining of dendrites.

(E) Staining with anti-CaV1.2 II-III loop antibody (green) reveals strong membrane and

ER staining but little nuclear staining (blue).

(F) Costaining with anti-CCAT antibody (green) and anti-GAD65 antibody (red)

reveals that CCAT is strongly nuclear in a subpopulation of GAD65-positive neurons.

(G and H) Immunohistochemistry of P30 rat-brain sagittal sections reveals strong

nuclear staining with the anti-CCAT antibody (green) in the inferior colliculus (G:IC)

and inferior olivary nucleus (H:IO). The cerebellum is labeled C and the brain stem is

labeled BS.

(I) High-power images of anti-CCAT (green), anti-GAD65 (red), and nuclear (blue)

staining of rat olfactory lobe neurons shows that only a subpopulation of neurons have

nuclear CCAT and that many of the cells are positive for GAD65.

79

Figure 2. Ectopically Expressed CCAT Localizes to the Nucleus of Neurons via a

Nuclear Retention Domain

(A) Neurons expressing Cav1.2 tagged at the C terminus with YFP (Cav1.2-YFP) show

pronounced nuclear and cytoplasmic fluorescence. Nuclei are shown in red in all panels.

(B) Neurons expressing the Cav1.2 tagged at the N terminus with YFP (YFP-Cav1.2)

show membrane and ER fluorescence but little nuclear fluorescence.

(C) Full-length CCAT-YFP (c503-YFP) is nuclear when expressed in neurons, cardiac

myocytes, and HEK 293T cells and forms prominent punctae.

(D) Epifluorescence (left panel) and confocal (right panel) image of neurons stained

with anti-CCAT antibody showing endogenous nuclear punctae.

(E) Schematic representation of YFP-tagged CCAT-deletion mutants (top). Top panel

and second panels show nuclear punctae in HEK 293T cells expressing CCAT

containing a deletion of 133 aa from the carboxyl terminus (c503–133) and a truncated

CCAT lacking aa 1642–1814 of Cav1.2 (c330) respectively. The third panel shows cells

expressing a truncated CCAT lacking aa 1814–1864 (c280). The domain important for

nuclear localization is highlighted in red in the schematic.

(F) Mean nuclear to cytoplasmic fluorescence ratios for all the CCAT deletions

constructs schematized in part (E) (means ± SEM; n > 75). ** p < 0.001 versus c503.

(G) Time-lapse images showing fluorescence recovery after photobleaching (FRAP) of

Cav1.2-YFP in the nucleus of Neuro2A cells. The area outlined in the red circle was

bleached for 300 ms with a high-intensity pulsed 488 nM laser beam. Images were

collected every 300 ms.

(H) Time course of recovery of nuclear fluorescence (solid symbols) and time course of

loss of cytoplasmic fluorescence (empty symbols) following bleaching. Nuclear

fluorescence recovered with a time course faster than 300 ms in cells expressing YFP

alone. Shown is a representative example of eleven experiments.

Figure 3. The Nuclear Localization of CCAT Is Regulated by Intracellular

Calcium and by Developmental Processes in the Brain

80

(A) Cortical neurons stained with anti-CCAT antibody (green) and Hoechst (red)

treated with 5 mM K+ (1 hr), 65 mM K+ (1 hr), 100 µM glutamate (30 min), or 2.5 mM

EGTA (1 hr).

(B) Higher magnification image of neurons treated with 2.5 mM EGTA reveals strong

and punctate anti-CCAT staining.

(C) Histograms of the ratio of nuclear to cytoplasmic anti-CCAT fluorescence in

neurons treated with 65 mM K+, glutamate, or EGTA. Treatment with 100 µM

glutamate (blue) and 65 mM K+ (green) reduce the amount of CCAT in the nucleus of

neurons, while EGTA (red) increases the amount of CCAT in the nucleus. Control cells

in 5 mM K+ are shown in black (n = 375 per condition).

(D) Time course of the decrease in anti-CCAT nuclear fluorescence following

stimulation with 65 mM K+ (green line; n = 200) or 100 µM glutamate (blue line; n =

200).

(E) Mean nuclear to cytoplasmic fluorescence ratio of CCAT in neurons treated for 1 hr

with 2.5 mM EGTA (bar 2), 65 mM K+ (bar 3), and 65 mM K+ in the presence of 2.5

mM EGTA, 10 µM nimodipine, 10 µM MK-801 or 10 µM NBQX (bars 5–8; n = 200).

(means ± SEM) * p < 0.001

(F) Mean anti-CCAT cell body fluorescence in neurons treated with 5mM or 65mM K+

(n=50).

(G) Mean nuclear to cytoplasmic fluorescence ratio of CCAT in neurons treated with

5mM and 65mM K+ in the presence of 5µM lactacystin (n=50).

(H) Western blot analysis of membrane (top) and nuclear fractions (middle) obtained

from E18, P1, P21, and adult-brain cortex probed with the anti-CCAT antibody. CREB

was used as a loading control (lower gel).

(I) Western blot analysis of cortical membrane extracts obtained from E18, P1, P8, P21,

and adult rats analyzed with anti-CaV1.2 II-III loop antibody (top gel) and anti-β-actin

(bottom) as a loading control. Adult lane exposed 4X relative to other lanes.

Figure 4. The C Terminus of Cav1.2 Binds to Nuclear Proteins and Activates

Transcription

81

(A) CCAT immunoprecipitates endogenous p54(nrb)/NonO. Upper panel shows

p54nrb/NonO levels in the lysates. The middle panel shows immunoprecipitated c503-

Gal4 and c280-Gal4 that lacks a nuclear localization domain. The lower panel shows

endogenous p54(nrb)/NonO coimmunoprecipitated by c503-Gal4 but not by c280-Gal4.

(B) Schematic representation of the CaV1.2-Gal4 fusion and CCAT deletion proteins.

c503 CCAT is the full-length C terminus of CaV1.2 downstream of the IQ motif. c330

CCAT lacks the N-terminal transcriptional activation domain (shown in red). c280 lacks

the nuclear retention domain of CCAT and c503Δ133 lacks the C-terminal

transactivation domain (shown in blue).

(C) Reporter gene activity of Neuro2A cells expressing a UAS-luciferase-reporter

plasmid along with either the Gal4-DNA binding domain, full-length CaV1.2, full-

length CaV1.2-Gal4, or CaV1.2-Gal4 channel lacking 133 amino acids from the

carboxyl terminus. Cells were cotransfected with a Renilla driven by the thymidine

kinase promoter to control for cell number and transfection efficiency. The results are

given as the ratio of Firefly to Renilla-luciferase activity. (means ± SD) ** p < 0.0001

versus Gal4.

(D) Luciferase activity of neurons transfected with the UAS-luciferase-reporter gene

and either Gal4 alone, CREB-Gal4, or four different CaV1.2 C-terminal fragments

c503, c330, or c280 and C503Δ133 fused to Gal4. The two domains identified as

important for transcriptional activation are highlighted in B. (means ± SD; ** p < 0.005)

(E) Luciferase activity of neurons transfected with Gal4-DNA binding domain alone or

Gal4 fused to the C terminus of CaV1.2, CaV1.3, or CaV2.1. (means ± SD) ** p <

0.0001 versus Gal4.

(F) Luciferase activity of Neuro2A cells expressing CaV1.2-Gal4 treated with 5 mM, 65

mM, and 65 mM K+/2.5 mM EGTA. (means ± SD; ** p < 0.005 versus untreated).

Figure 5. CCAT Regulates Endogenous Genes

(A) A subset of mRNAs identified in microarray experiments that were upregulated (red

bars) or downregulated (green bars) by overexpression of CCAT relative to CCATΔTA

or GFP. For a full list of genes, see Tables S1–S4.

82

(B) RT-PCR analysis of mRNA levels from Neuro2A cells overexpressing CCAT

confirming changes in gene expression for a subset of these genes identified in A. Bars

represent mean fold changes relative to CCATΔTA or GFP and were normalized to β-

actin and GAPDH levels. Data are means ± SD of three independent experiments

performed in triplicate.

(C) Luciferase activity of neurons expressing a Cx31.1-luciferase-reporter gene along

with empty vector, full-length CCAT, or CCAT lacking the C-terminal transactivation

domain (CCATΔTA) (means ± SD; ** p < 0.0001).

(D) 4OHT-induced nuclear translocation of a CCAT-ER fusion. Immunocytochemistry

of Neuro2A cells expressing myc-CCAT-ER (green) before (top panels) and after

(bottom panels) addition of 5 µM 4OHT for 1 hr. Nuclei are shown in blue.

(E) Transcription of a Cx31.1-luciferase-reporter gene in Neuro2A cells expressing a

Cx31.1-luciferase reporter along with ER alone, CCAT-ER, or CCATΔTA-ER before

and after nuclear translocation by addition of 5 µM 4OHT for 6 hr.

(F) Mean luciferase activity (mean ± SEM; n = 3) of Neuro2A cells expressing CCAT

and deleted forms of the Cx31.1 promoter. The 125 bp 3′ region of the promoter that

binds CCAT is shown in red.

(G) Representative ChIP assay showing that CCAT immunoprecipitates the endogenous

Cx31.1 promoter. Agarose gel electrophoresis of PCR products amplified from either

input DNA (I) or from DNA that was immunoprecipitated by GST-CCAT (IP) or by

GST alone (C). The upper gel shows the PCR products using primers that recognize two

regions in the Cx31.1 promoter (5′ promoter and 3′ promoter). The lower gel shows

PCR products using primers that recognize the 3′ region of the Cx31.1 gene several KB

from the start site (n = 3).

Figure 6. Endogenous CCAT Regulates Transcription Driven by the Cx31.1

Promoter

(A) Depolarization of neurons with 65 mM KCl decreases the activity of the Cx31.1

reporter relative to unstimulated cells (5 mM KCl; mean ± SD; n > 3; ** p < 0.0001).

(B) RT-PCR analysis of endogenous Cx31.1 mRNA in Neuro2A cells and cortical and

thalamic neurons treated with 65 mM KCl or 0 calcium showing that depolarization

83

causes a pronounced decrease in Cx31.1 expression. mRNA levels are normalized to

mRNA levels in unstimulated cells (bars represent mean ± SEM; n = 5). (C) Western

blots showing reduced expression of FLAG-tagged rat CaV1.2 expressed in Neuro2A

cells by expression of two rat shRNAs (RCaV1.2 sh6410 and RCaV1.2 sh6500; lanes 1

and 2, top panel) but not by a mouse shRNA (MCaV1.2 sh6203) that differs by two

base pairs (lane 3, top panel). CaV1.2ΔTA is resistant to RCaV1.2 sh6500 (lane 4),

which targets the TA domain of CaV1.2. Ds-Red, expressed from the shRNA vector,

was used as a loading control (bottom panel). (D) Mean luciferase activity (± SD) of rat

neurons transfected with the Cx31.1-luciferase-reporter gene and either a scrambled

control shRNA (sh-scr), shRNA constructs targeting the mouse CaV1.2 (MCaV1.2 sh-

6203), or the rat CaV1.2 mRNAS (shRNA RCaV1.2 sh-6500, RCaV1.2 sh-6410; ** p <

0.001 versus scrambled (sh-scr) or MCav1.2 sh-6203).

(E) CCAT* is resistant to knockdown by an shRNA targeting CaV1.2 (CaV1.2 sh6500).

Western blot analysis of lysates from Neuro2A cells expressing CCAT or an RNAi-

resistant CCAT* along with the RCaV1.2 sh-6500 shRNA vector that targets rat

CaV1.2 (upper gel). Ds-Red, expressed from the shRNA vector, was used as a loading

control (lower gel). (F) Expression of RNAi-resistant CCAT* reversed the effect of

CaV1.2 sh6500 on Cx31.1 expression (bar 3), but CCATΔTA, which lacks the C-

terminal transactivation domain, does not (bar 4; means ± SD; n = 3; ** p < 0.001

versus sh-scr).

(G) CaV1.2-FLAG* is resistant to knockdown by RCav1.2 sh6500. Western blot

analysis of lysates from Neuro2A cells expressing CaV1.2-FLAG or RNAi-resistant

CaV1.2-FLAG*, and RCaV1.2 sh6500 or MCaV1.2 sh-6203 (upper gel). Ds-Red,

expressed from the shRNA vector, was used as a loading control (lower). (H)

Expression of RNAi-resistant CaV1.2-Flag* partially reversed the effect of RCaV1.2

sh6500 on Cx31.1 expression (bar 3), but CaV1.2-ΔTA*, which lacks the C-terminal

transactivation domain, did not (bar 4; means ± SD; n = 3). ** p < 0.0001 versus sh-scr.

Figure 7. CCAT Regulates Neurite Growth in Primary Neurons

84

(A and B) Representative low- and high-magnification images of cerebellar granule

cells grown in vitro for 5 days transfected with a vector expressing GFP and either

CCATΔTA (A) or CCAT (B).

(C) Histograms of mean neurite length for granule neurons expressing CCATΔTA (top

graph), a vector with GFP alone (middle graph), or CCAT (bottom graph).

(D) Average neurite length for cells expressing CCATΔTA, GFP alone, or CCAT

(means ± SEM, n = 200; ** p < 0.005 versus vector control).

(E) Average number of primary dendrites for granule neurons expressing CCATΔTA,

empty vector, or CCAT.

Figure S1. CCAT’s nuclear localization and transcription activation domains are

conserved among CaV1.2 channels in vertebrates

(A-B) and (D) Multiple alignment of the nuclear localization domains (A), N-terminal

transactivation domains (B) and C-terminal transactivation domains of homologous

CaV1.2 channels (CAC1C) from different species. Accession numbers: CAC1C_RAT

AAA18905, CAC1C_MOUSE, Q01815, CAC1C_RABBIT P15381,

CAC1C_HUMAN Q13936, CAC1C_ZEBRAFISH: NM_131900.1. (B) Multiple

alignment of the N-terminal transcriptional activation domain of rat CCAT with

GATA4 sequences from different species. N-terminal transcriptional activation domain

is 42% homologous and 27% identical to a conserved C-terminal domain of the

transcription factor GATA4. Consensus symbols indicate identity (*) and similarity (:).

All alignments were generated using the ClustalW program. Black and gray shading

show identical and similar amino acid residues respectively.

Figure S1. CCAT’s nuclear localization and transcription activation domains are

conserved among CaV1.2 channels in vertebrates

(A-B) and (D) Multiple alignment of the nuclear localization domains (A), N-terminal

transactivation domains (B) and C-terminal transactivation domains of homologous

CaV1.2 channels (CAC1C) from different species. Accession numbers: CAC1C_RAT

AAA18905, CAC1C_MOUSE, Q01815, CAC1C_RABBIT P15381,

CAC1C_HUMAN Q13936, CAC1C_ZEBRAFISH: NM_131900.1. (B) Multiple

alignment of the N-terminal transcriptional activation domain of rat CCAT with

85

GATA4 sequences from different species. N-terminal transcriptional activation domain

is 42% homologous and 27% identical to a conserved C-terminal domain of the

transcription factor GATA4. Consensus symbols indicate identity (*) and similarity (:).

All alignments were generated using the ClustalW program. Black and gray shading

show identical and similar amino acid residues respectively.

Figure S2. CCAT derived from CaV1.2-YFP channel is regulated by depolarization

(A) Neuro2A cells expressing the C-terminally tagged CaV1.2-YFP treated with either

5mM K+ (upper panels) or 65mM K+ (lower panels).

(B) Histogram of mean nuclear fluorescence of cells in part A treated with 5mM K+

(gray; n=50) and 65mM K+ (black; n= 50).

Supplemental Figure 3. CCAT regulates expression of endogenous genes

(A) Graph of the expression level of mRNAs isolated from cells expressing either the

intact CCAT (X axis) or the CCAT∆TA (Y axis) that lacks the transcriptional activation

domain. Points below the diagonal green lines line correspond to genes that are down-

regulated in the CCAT relative to the CCAT∆TA by more than three standard

deviations. Data is derived from Agilent mouse two color arrays and the expression of

each gene was normalized to the mRNA levels in untransfected Neuro2A cells. The

color of each spot represents the expression level of each mRNA relative to

untransfected cells.

(B) Graph of the expression level of mRNAs isolated from cells expressing the CCAT

(Y axis) or GFP (X axis). Points that are above the diagonal green lines correspond to

genes that are expressed at least three standard deviations higher in CCAT expressing

cells than in GFP control cells. Points that are below the diagonal (blue) correspond to

genes that are repressed by the CCAT. Only the mRNAs whose expression was more

that 100 intensity units (three SD over the background) were analyzed. Genes are color

coded according to the ratio of expression in CCAT over GFP containing cells.

(C-D) Pie charts showing the functional distribution of 66 genes that are up-regulated

(C) and 206 genes that were down-regulated (D) by CCAT relative to GFP. See

Supplemental tables 2 and 3 for additional analysis and for a complete list of genes.

86

Figure S4.

(A) RCaV1.2 sh-6500 knockdown of endogenous CaV1.2 in neurons decreases CREB

activation induced by K+. The luciferase activity of rat neurons expressing a CREB

reporter gene is reduced by coexpression of an shRNA that targets rat CaV1.2 (RCaV1.2

sh6500; black) relative to coexpression of a control shRNA (MCaV1.2 sh6203; gray).

Cortical neurons were stimulated with 65mM K+ or 65mM K+ in the presence of 10µM

nimodipine. RCaV1.2 sh6500 reduces the activation of CREB in response to KCl in a

manner that is similar to the effect of the LTC blocker nimodipine, suggesting that it is

reducing the expression of endogenous CaV1.2 in neurons. Data represents means ± SD

of at least three independent experiments performed in quadruplicate; ** p<0.0001.

(B) Blockade of CaV1.2 channel pore by nimodipine does not affect expression from the

Cx31.1 reporter in the presence or absence of CCAT. Data represents means ± SD of at

least three independent experiments performed in quadruplicate; ** p<0.0001.

87

CHAPTER 3:

An independent promoter in the CACNA1C channel gene generates short CCAT

88

SUMMARY

Thus far we have discussed how the c-terminus of the voltage-gated calcium

channel Cav1.2 encodes a transcription factor within its c-terminal domain. This

protein, which we termed CCAT, is found in the nucleus of neurons, has a potent

transcription activation domain and its intracellular localization is regulated by

intracellular calcium levels. Here we show that a short CCAT is generated from a

second transcript from both exogenous channel cDNA and the endogenous channel

gene. Consistent with this, we find that CCAT expression is independent of full-length

channel protein, suggesting that this protein is not released from proteolysis of full-

length channel. Transcription of the CCAT message is driven by an exonic promoter

whose transcriptional activity is demonstrated in the channel’s cDNA. Activity at this

promoter, and consequently CCAT expression, is regulated spatially and temporally in

the brain having highest expression during embryonic stages and predominantly in

regions of the brain rich in inhibitory neurons. We also provide evidence for two other

endogenous transcripts containing the C-terminus. Analysis of 5’ ends from

CACNA1C derived transcripts showed two additional transcriptional start sites one of

which we propose is CCAT’s transcriptional start site in vivo. The second transcript is

predicted to encode a membrane bound CCAT containing a voltage sensor. These

findings uncover another unexpected detail of CCAT’s biology and provide a unique

example in which two proteins with distinct biologic functions can be derived from a

single gene. Furthermore, we provide an example in which exonic promoters can be

used to contribute to transcriptional and protein complexity.

89

INTRODUCTION

Voltage-gated calcium channels are large complexes composed of an α1C

subunit which forms the pore, and accessory subunits β and α2δ which play a role in

targeting, tethering and regulation of the biophysical properties of these channels

(Catterall et al., 2005). Calcium influx through L-type calcium channels, encoded by

the Cav1.2, Cav1.3 α1C subunits, has been shown to be particularly effective at

regulating gene expression in response to depolarization (Morgan and Curran, 1986;

Murphy et al., 1991). This has been thought to occur mainly via the activations of

calcium-regulated transcription factors such as CREB, NFAT and MEF (Graef et al.,

1999; Hardingham et al., 1999; Mao et al., 1999; Sheng et al., 1990; Zafra et al.,

1990). Specifically for Cav1.2 and Cav1.3 signaling to CREB has been shown to be

dependent on calcium influx through the channels, calmodulin-binding motifs in the

channel’s sequence and activation of the MAP kinase pathway (Deisseroth et al.,

1998; Dolmetsch et al., 2001; Weick et al., 2003). We have recently described a

calcium channel influx-independent mechanism in which Cav1.2 containing channels

can regulate gene expression (Gomez-Ospina et al., 2006). In this model the carboxyl-

terminal domain of the channel translocates to the nucleus and directly influences

transcription. In our previous study we showed this Calcium Channel Associated

Transcription regulator or CCAT is able to bind to nuclear proteins, associate with

endogenous promoters and both augment and repress the transcription of a wide

variety of endogenous genes. In the adult brain, CCAT nuclear localization is

90

restricted to inhibitory neurons in the thalamus, inferior colliculus and several brain

stem nuclei.

One of the most important questions remaining regarding CCAT’s biology is

how this C-terminal fragment is generated. We know based on its restricted nuclear

localization that the mechanism involved would be cell-type and stimulus specific.

One possibility is that CCAT is a c-terminal fragment released after a proteolytic event

from channels at the membrane. Such a mechanism would directly link electrical

activity, i.e., activation of voltage-gated channels, to the liberation of a transcriptional

regulator during periods of increased activity. Early biochemical studies of the pore-

forming unit of L-type channels including Cav1.1, Cav1.2, and Cav1.3 found that, in

most native tissues, these channels exist as truncated proteins lacking the c-terminus

(De Jongh et al., 1994; De Jongh et al., 1991; Gerhardstein et al., 2000). It was also

reported that NMDA stimulation increased the appearance of the Cav1.2 truncated

channel in hippocampal slices (Hell et al., 1996). Calpain protease inhibitors blocked

this effect, suggesting that increased neuronal activity could lead to the activation of

calcium-regulated proteases, which could then release CCAT.

A second possibility is that CCAT is made as an independent protein expressed

either from an alternative promoter hidden within the channel’s gene, as an

independent transcript, or from an internal ribosomal entry site in the channel’s

mRNA. Regardless of the exact mechanism, this would be the first example in which

alternative transcription or translation initiation leads to the expression of two proteins

with completely separate biological functions: a calcium channel and a transcription

factor. Alternative first promoters are commonly used for tissue specific expression.

91

In fact, alternative selection of P1 promoters in the CACNA1C gene results in Exon1a

being predominantly expressed in the heart and Exon1b in smooth muscle and brain

(Saada et al., 2005). Hitherto, most alternative promoters described include 5’

promoters that lead to alternative 5’ UTRs and identical proteins or different N-termini

resulting in protein isoforms with the same general biologic function (Davuluri et al.,

2008). Examples of this alternative promoter usage include the p53, TCF/LEF and

CREM/ICER families of isoforms (Arce et al., 2006; Mioduszewska et al., 2003;

Murray-Zmijewski et al., 2006). An alternative transcript based mechanism would be

especially plausible given our most recent understanding of the eukaryotic

transcriptome. Genome-wide analyses of the mouse and human transcriptomes have

revealed that almost one half the protein-coding genes contain alternative promoters

and, not uncommonly, genes have additional internal transcriptional start sites

(Carninci et al., 2006; Frith et al., 2008; Gustincich et al., 2006).

In these study we investigated how CCAT is generated and have identified that

a short CCAT is expressed from an alternative promoter within the channel’s coding

sequence. Consistent with this, we report that in vivo and in vitro, CCAT is made

independently of existing channel protein. Using Northern blots and 5’ RACE

experiments we demonstrate that other 3’ transcripts are present in cells containing

exogenous channel and in brain mRNA extracts. In addition, we have found that an

exonic promoter residing within the penultimate exon of the rat and mouse genes is

responsible for CCAT message expression and is sufficient to promote transcription of

a reporter gene. Using minigenes we show that this exonic promoter activity is

preserved in the context of large intron sequences. As predicted by an alternative

92

transcript mechanism, a single methionine mutation can abolish CCAT expression

from both exogenous cDNA and from minigenes. We find that the level of expression

of the endogenous transcript as well as the abundance of CCAT nuclear protein is

highest in embryonic stages and decreases in post-natal life. Early in development

both CCAT transcript and CCAT protein have highest expression in the thalamus and

lowest in the cortex. Accordingly, we find that reporter gene expression driven by the

exonic promoter is higher in thalamic than cortical neuronal cultures. Finally, our 5’

RACE and northern blot experiments suggest the existence of an additional transcript.

This message would encode for the C-terminus of the channel as well as 7

transmembrane helices including S6 of domain III and S1-S6 of domain IV, predicting

a membrane bound CCAT with a voltage sensor.

These data identify an unexpected alternative transcription initiation mechanism

responsible for CCAT expression and a novel promoter residing within the coding

sequence of the channel.

RESULTS

CCAT is not generated by Proteolytic Cleavage of Exogenously Expressed

Channels

We have previously described that Cav1.2 channels tagged with the Gal4 DNA

binding domain at its c-terminus led to constitutive and strong luciferase expression

from a UAS-luciferase reporter gene when expressed in N2A cells and myocytes

93

(Gomez-Ospina et al., 2006). These data demonstrated to us that a c-terminal fragment

was produced which was free from the membrane and able to translocate to the

nucleus, and that this fragment contained a potent transactivation domain. We used

this assay to examine how CCAT was released from Cav1.2 channels at the membrane

by measuring the generation of CCAT protein from mutant channels expressed in cells

(Figure 1A). We first expressed Cav1.2 -Gal4 channels in Neuro2A cells (N2As) along

with accessory subunits and a UAS-luciferase reporter gene. We followed the

appearance of CCAT in two ways: we monitored protein expression using

immunoprecipitation and western blots and we measured its transcriptional activity

and abundance using transcription assays. When expressed in N2As cells wild-type

Cav1.2–Gal4 channels (WT) generated two Gal4 tagged proteins: a full-length channel

and a smaller protein of approximately 250 KDa and 40 KDa respectively (Figure 1b).

This 40 KDa band is a fusion of part of the c-terminus and Gal4 DBD thus predicting

a molecular weight for CCAT of approximately 20 KDa. In the transcription assays

WT channels evoked strong luciferase expression, one hundred fold better than Gal4

alone (Figure 1C). In the previous study, we determined that the transactivation

domain (TA) resides within the last 133 amino acids of the channel and consistent

with this, deletion of TA completely abolished transcriptional activation and the

CCAT band (Figure 1B and 1C).

We then examined whether Cav1.2 channels needed to be at the plasma

membrane to produce CCAT. We deleted a region of 150 amino acids between the last

transmembrane domain (TM) and the IQ domain. This region has been previously

shown to be required for expression of functional channels at the membrane (Wei et

94

al., 1994). We observed that this deletion had no effect in the size or abundance of

CCAT protein. Additionally, incubation of the cells in brefeldinA, which inhibits

anterograde protein transport from the endoplasmic reticulum (ER) to the Golgi

apparatus, yielded similar results (Sup Fig1). These data could only be reconciled with

a mechanism where cleavage occurs co-translationally or within other compartments

of the secretory pathway such as the endoplasmic reticulum or the Golgi apparatus.

We next tested whether a reported cleavage site of Cav1.1 was necessary for the

production of CCAT protein. Mass spectrometry analysis of Cav1.1 (the skeletal

isoform of Cav1.2) purified from skeletal muscle identified the site of proteolytic

processing as Alanine 1664 (Hulme et al., 2005). The surrounding sequence,

including the Alanine is conserved in Cav1.2, corresponding to Alanine 1773,

suggesting that this could constitute a cleavage site in these channels as well. We

found that deletion of 50 amino acids surrounding the proposed cleavage site had no

effect on the size or abundance of CCAT protein (Figure 1B and 1C) indicating that

for Cav1.2 this sequence was not necessary for CCAT production. Because it seemed

difficult to explain how cleavage of channel within intracellular membrane

compartments would be employed as a way to link channel activity to CCAT

production we asked whether channel protein was at all necessary for the generation of

CCAT. We introduced a stop codon at amino acid 1910, upstream of the TA domain.

As expected, this construct when expressed in cells could no longer make full-length

Gal4-tagged channel but surprisingly Gal4 tagged CCAT protein was intact. This

result implied that channel protein was not necessary for CCAT production and that

CCAT was made as an independent protein.

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We next designed a series of experiments to determine how this channel-

independent protein was made. One possibility is that CCAT is translated from the

channel mRNA via an IRES or as a distinct message transcribed from the same cDNA.

Regardless of which model is at play, it can be predicted that an initiating methionine

would be necessary for the production of CCAT. To test this prediction, we mutated

two methionine residues in the channel sequence selected based on predicted CCAT

size of ~20 KDa and the predicted strength of the initiating AUG (Algire and Lorsch,

2006). Mutation of Methionine 2011 to Isoleucine and not Methionine 2078

completely prevented the expression of the 40KDa CCAT band and abolished

transcriptional activation. Instead of the 40KDa protein a smaller 32KD protein was

made (Figure 1D and 1E). The appearance of this smaller band can be explained by

the scanning mechanism by which the ribosome determines the starting ATG. The

translational start site in the M2011I mutant then likely becomes M2073, 62 amino

acids downstream. Additionally, a significant increase in the amount of the smaller c-

terminal protein could be seen in the western blot and is better quantified in the

luciferase assay (Fig 1E). This effect can be explained by the competitive inhibitory

effect that surrounding Methionines have on translation initiation (Kozak, 2000).

Cav1.2 Channel Protein is not necessary for CCAT Expression In Vivo

To test whether channel protein is necessary for the production of CCAT in

vivo. We looked for endogenous CCAT in Cav1.2 knockout mice (kindly provided by

Dr Jean Pierre Kinet at Harvard). The Cav1.2 conditional null allele was generated by

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homologous recombination with a construct designed to delete exons 14 and 15

(Supplementary Figure 1B and 1C). CMV-CRE-transgenic mice were used to produce

ubiquitous deletion of the floxed exon and create the Null allele. Cav1.2 deletion is

lethal and mouse embryos do not survive past 13.5 days post conception (dpc).

Consequently, we examined the embryos from heterozygous Null crosses at 11.5 and

12.5 dpc for the presence and localization of CCAT. At the 11.5 and 12.5 dpc Cav1.2

knockout embryos were anatomically and functionally normal. We first used a c-

terminal antibody (anti-CCAT) to probe for the presence of full-length channel in

heterozygous and homozygous Null 11.5 dpc embryos from the same litter. We used

biochemical fractionation to separate microsomal and nuclear fractions from N/+ and

N/N and embryos. In membrane fractions, anti-CCAT antibody recognized a 240KDa

band corresponding to the full-length channel in wild type and heterozygote embryos.

This band was absent in the knockout embryos confirming the efficacy of the

knockout strategy (Figure 1F). We have previously reported that this antibody shows

nuclear staining in a subset of neurons in the brain as well as in N2A cells. This

nuclear localization of CCAT is further supported by the fact that exogenous c-

terminus tagged with YGP localizes to the nucleus in cells (Gomez-Ospina et al.,

2006). To examine whether nuclear CCAT staining was preserved in the knockout

embryos, we probed sections of wild type and Cav1.2 KO 11.5 dpc embryos with anti-

CCAT antibody. At this age, in wild type animals, cytoplasmic and membranous anti-

CCAT immunoreactivity was seen in the developing neural tissue, heart muscle and

major blood vessels. Strong nuclear staining was noted in somites and mesenchymal

tissue. No nuclear CCAT staining was seen in the developing brain at this stage (Sup

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Fig1D). Cav1.2 null embryos showed intact nuclear staining in somites suggesting that

anti-CCAT reactive nuclear protein was still present in cells lacking full-length

channel (Figure 1G).

CCAT is translated from a Separate Transcript from the cDNA

Our data thus far suggested that an alternative mechanism independent of

channel protein was giving rise to CCAT protein. CCAT could be translated from the

Cav1.2 mRNA via an IRES or from a separate transcript. Given our observations using

channel constructs, it was clear that such mechanism is preserved in the context of the

cDNA, which supported the idea of an IRES within the channel’s message. We used

northern blots to probe for the last exon, i.e. Exon47 (Acc# AAA18905), of the

channel gene to examine transcripts from N2A cells expressing Cav1.2 -Gal4 channels.

We observed two transcripts, one of approximately 8 Kb corresponding to full-length

channel and a second transcript of approximately 1.4 Kb (Figure 2A). Even though

this strongly suggested that a second transcript was involved, it did not rule out the

possibility of an IRES within the full-length channel transcript. To test this, we deleted

the CMV promoter in the pcDNA4 Cav1.2 -Gal4 construct and checked for CCAT

expression. As expected, deletion of the CMV promoter led to the loss of the

channel’s transcript and protein. However, neither the 1.4 Kb transcript nor CCAT

protein are affected by the deletion of the promoter (Figure 2A and 2B). Luciferase

expression from the UAS reporter in the presence of the promoterless Cav1.2 -Gal4

plasmid was not only preserved but was significantly increased compared to wild-type

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plasmid. This increase, we speculate, is due to decreased transcriptional quenching in

the absence of the potent CMV promoter (Figure 2C). Together these data

demonstrated that, in the cDNA, CCAT is translated from a separate transcript and

suggested that the sequences required to drive the transcription of this message reside

within the coding sequence of the Cav1.2 gene.

An Exonic Promoter Drives CCAT Expression

To find the sequences involved in transcriptional activity, we cloned fragments

of the coding sequence of Cav1.2 in front of luciferase and tested for luciferase

expression (Figure 2D). The full coding sequence of the channel can robustly drive

expression of luciferase when taking the position of the promoter (Figure 2E). This

activity can only be observed when the channels sequence is placed in frame of the

luciferase sequence and any intervening stop codon is mutated to code for amino acid,

thus maintaining the open reading frame. Consistent with our hypothesis, this finding

suggested that translation and consequently transcription was initiated within the

channel’s coding sequence. Using truncation analysis, we found that a substantial part

of the promoter activity resided within the penultimate exon of the channel, i.e. Exon

46. Specifically, the 238 base pair region within Exon 46 upstream of M2011 was

sufficient for promoter function (Figure 2E).

We next examined whether this promoter activity was present in the context of

the gene. For this purpose we constructed several CCAT minigenes containing 4 Kb of

genomic sequence comprising the last two introns and last two exons of the

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CACNA1C gene fused to Gal4 (Figure 2F). In the control minigene, both the 238 bp

promoter and the M2011 reside within Exon 46 more than 2 Kb upstream of Gal4

(4Kb-0). TA and, more precisely, the sequence between M2011 and M2073, which is

required for transcriptional activation, straddle Exon 46 and Exon 47 (Figure 1D and

Sup Fig 2). We predicted that, if such promoter activity was present and could be

recapitulated in the minigene, it should lead to a transcript whose appropriate splicing

would create a fusion of CCAT’s transcriptional activation domain to Gal4. The

abundance of this protein could be sensitively measured by its ability to promote

luciferase expression from the UAS-luciferase reporter. As a negative control we built

a minigene where the reading frame between CCAT and Gal4 was frameshifted by

introducing a single deoxyguanosione (4Kb-1). When expressed in N2A and 293

cells, 4Kb-0 had 10 fold more activity than 4Kb-1. Deletion of the 238 bp promoter

(∆238 bp) decreased protein expression by 75% confirming that this sequence is

necessary to drive full expression of CCAT-Gal4. The M2011I mutation led to a

similar decrease in protein expression. There are three possible methionines within

this putative transcript: M2011, M2073 and M2078. To test the possibility that

translation was initiated at the methionines downstream, we built three more

minigenes where only one of the afore mentioned methionines was intact in the

context of the control construct (4Kb-0). In these experiments, only M2011 was able

to produce CCAT-Gal4 protein (Figure 2H).

We next compared the strength of the identified promoter to that of the full-

length channel’s promoter. To this end we cloned a 4 Kb genomic fragment upstream

of methionine M2011 in front of the firefly luciferase coding sequence.

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Concomitantly, a 4 Kb genomic fragment upstream of the putative methionine for

neuronal Cav1.2 transcript (rbcII) was cloned in the same manner. We introduced

neuronal/ Cav1.2 –luciferase and CCAT/Cav1.2-luciferase reporter genes into N2As

and HEK cells and measured luciferase expression. In both cell types, the genomic

region upstream of M2011 showed 2.5X higher protein expression than promoterless

vector alone. In N2As cells the promoter activity of this region was comparable to the

neuronal promoter/enhancer region (Sup Fig2A). Together, these results confirm our

hypothesis that the 238 bp region within Exon 46 of CACNA1C gene has promoter

activity. The resulting transcript is spliced as endogenous full length channel transcript

giving rise to a protein that encodes the transcription activation domain of CCAT. Our

data also strongly points at M2011 as the starting methionine for the transcript in vivo.

We compared sequences from Cav1.2 channels from Zebrafish to Human to look at

the conservation of the promoter region and M2011. This amino acid sequence and the

exon-exon borders within the c-terminus are highly conserved (Sup Fig 2B).

Alignment of these sequences reveals that the M2011 is conserved in primates,

rodents: including rat mouse and guinea pig and in Zebrafish (Sup Fig 2C).

Interestingly, M2011 is not conserved in all species even if the amino acid sequence

and exon organization is preserved. However the downstream Methionine 2073 is

conserved in all species and if alternative transcripts were made, translation would

initiate at this methionine. The sequence upstream, which includes the 238 bp

promoter region, is also highly conserved at the amino acid level. This would suggest

that Exon n-1 in other channels could function as a promoter. However, it is likely that

key differences at the nucleotide level dictate promoter activity and so without a better

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understanding of the DNA elements required for transcription, it is difficult to

speculate regarding promoter activity in other channels.

CCAT is Translated from a Separate Transcript In Vivo whose Expression is

Spatiotemporally Regulated in the Brain

To investigate the presence of independent transcripts in vivo we extracted

mRNA from the cerebral cortex, midbrain and cerebellum of rats at different

developmental stages and analyzed them using northern blots. Because our

experiments predict a transcript whose transcriptional start site (TSS) is in the coding

sequence of the channel no distinct sequence could be used for precise quantification

with RT-PCR and other hybridization methods. Thus, the main distinction is in

transcript size. Hybridization of a probe designed to recognize CCAT-TA domain

revealed two additional transcripts of approximately 4 Kb and 2.2 Kb (Figure 3A).

These transcripts were absent when using a probe that recognized S3 of domain 3 of

the full-length channel (Figure 3B). For the three transcripts we plotted the normalized

signals of the 18S ribosomal RNA from three independent experiments (Figure 3C).

The Full-length channel message was expressed at higher levels in the adult brain.

Conversely, the 2.2 Kb message was most abundant in cerebellum and midbrain of P1

rats and in E18 whole brain compared to these structures in the adult brain. The

normalized signal of 4.0 Kb band was most abundant in E18 whole brain. These data

suggests that subpopulations of neurons express at least two other CCAT-TA

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containing transcripts and their expression level is inversely correlated to the full-

length channel’s.

In parallel experiments we looked at the amount of CCAT nuclear staining in

brains during development using immunohistochemistry with anti-CCAT antibody. In

the brain, the number of cells showing CCAT nuclear staining also decreased with

age. In E18 embryos most nuclear CCAT reactive cells were found in the developing

striatum, thalamus, brain stem and cerebellar bud (Figure 3D and 3E). Nuclear

staining was almost nil in the cortex (Figure 3D and Sup Figure 3A and B). At P1 the

nuclear staining became more restricted to subpopulations of cells in the cerebellum to

the developing Purkeji layer, the thalamus and to 20-30% of the cells in the striatum.

In 3wk brains, most anti-CCAT reactivity came from membranous full length Cav1.2

channels which localized to the cell bodies and dendrites as has been reported

previously (Hell et al., 1993). This was particularly evident in the hippocampus and

cortex. Nuclear staining at this age was sparse and found mostly in the inferior

colliculus, and brain stem nuclei with scattered cells in the thalamus (Figure 3E and

(Gomez-Ospina et al., 2006)). Together, these results showed that the distribution and

abundance of CCAT nuclear protein was consistent with the distribution of the 2.2 Kb

transcript. Both transcript and protein coincide with highest expression in E18 brain,

P1 cerebellum and thalamus and decreasing levels in the adult.

The observed distribution of CCAT expression implied tissue specific CCAT

promoter usage. Specifically, we learned that the level of endogenous transcript and

nuclear staining was higher in the thalamus than in the cortex in E18 brains. Hence,

we predicted that there would be a higher level of promoter activity in thalamic

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neurons compared to cortical neurons. We tested this by comparing UAS-luciferase

expression from cultured thalamic and cortical neurons transfected with Cav1.2-Gal4

channels knowing that sufficient part of the promoter is preserved in the cDNA. As

Figure 3F shows, in thalamic cultures, 40 ± 10% of neurons have robust nuclear

CCAT compared to less than 5± 5% in cortical neuron cultures. Furthermore, in the

transcription assays thalamic neurons replicated what we have reported in N2As:

Cav1.2 -Gal4 channels upregulate the UAS promoter independently of channel protein

and rely on M2011 for expression. In cortical neurons, Cav1.2-Gal4 channels did not

lead to quantifiable differences in luciferase expression compared to untagged Cav1. 2

channels, suggesting this promoter is not transcriptionally active in these cells. This

would predict less nuclear CCAT in these neurons consistently with what we have

observed.

To better understand the expression of CCAT protein the developing brain, we

stained for CCAT at earlier embryonic stages. Our data indicated that early in the

developing mouse brain, nuclear CCAT expression was largely confined to cells in the

ventral telencephalon. We have observed expression of nuclear CCAT as early as

embryonic day 12 in parts of the ventral telencephalon corresponding to the

developing caudate and putamen (striatum), as well as in the developing thalamus,

distinct domains of periventricular nuclei in the hypothalamus, and in the developing

cerebellum (Supplementary Figure 3B and 3C). Expression of nuclear CCAT in these

regions persisted through embryonic neural development but began to decline early in

postnatal life and subsequently disappears into adulthood. Notably, in the developing

cortical plate and ventricular zones immunofluorescence staining with the C-terminal

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Cav1.2 antibody revealed primarily cytoplasmic expression, corresponding to the

presence of the full length Cav1.2 calcium channel but no CCAT expression.

CACNA1C has Multiple TSS Predicting Multiple Proteins

To provide independent evidence for the existence of an alternative transcript

encoding for CCAT in vivo we employed Rapid Amplification of cDNA Ends (5’

RACE) analysis of CACNA1C derived transcripts. Moreover, 5’ end analysis could

help us identify the TSS, corroborate the role of methionine 2011 and locate the cis-

acting element, which we predict include the sequence herein identified. We used the

RNA ligase-mediated (RLM) and oligo-capping approaches to selectively ligate an

RNA oligo adapter to previously capped full-length mRNAs. cDNA synthesis was

performed with a primer complementary to a region of Exon 47 as a gene specific

primer or an oligo dT primer. cDNA’s were PCR amplified using a primer within the

5’ oligo adapter sequence as a forward primer and a nested primer in Exon 47 as the

reverse primer. The PCR products were cloned, sequenced and the 5’ 20 bp sequences

mapped onto the gene. First, to test our approach and to determine the transcriptional

start site from the exonic promoter within the channels cDNA, we used 5’ RACE on

mRNA extracted from cell expressing Cav1.2 -Gal4 channels. For this experiments a

Gal4 specific primer was used for cDNA synthesis to select only transcripts from

exogenous construct. Cells transfected with Cav1.2-Gal4 and Cav1.2-gal4

promoterless constructs expressed a second transcript whose TSS mapped 50 bp

upstream of M2011 (Figure 4A and 4B).

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We next isolated transcripts from cortex and thalamus of E18 rats. We focused

on this stage given the greatest difference in abundance of nuclear protein between

these two brain regions and the greatest abundance of short transcript as suggested by

the northern blots. We performed five independent RACE experiments to really

convince ourselves that the 5’ ends we found corresponded to true ends of capped

RNA's and not to truncated transcripts that had escaped dephosphorylation during the

chemical selection step. Transcriptional start sites were considered equivalent if they

mapped within 20bp. The most reproducible 5’ starts and the corresponding

chromosomal location of the 20 upstream nucleotides on the mouse genome are listed

in Table 1. Three additional TSS’s were expressed from the CACNA1C gene in

addition to the already established TSS’s for full-length channel transcripts. All three

sites were within exons. For all three possible transcripts we used three available

translation initiation algorithms to predict whether and what proteins could be

translated from them (Nadershahi et al., 2004). The start site for transcript variant 4

was found to reside within Exon 46, 119 bp upstream of M2011 and 60 bp upstream

of the TSS found expressed from the channel cDNA. In both cases, the UAG codon

for M2011 is predicted to be the initiating codon and the predicted molecular weight

for CCAT is 15KDa. Thus, transcript variant 4 would presumable encode for CCAT in

vivo. Furthermore the promoter is localized at least in part to the upstream sequence in

Exon 46. In order to predict its full-length size, we thought it would be reasonable to

expect that all additional transcripts share the same 3’ termination signals and

therefore the 3’UTR. For the majority of CACNA1C-derived transcripts found in the

databases the 3’ UTR is roughly 1.6 Kb. Consequently, the estimated size for

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transcript variant 2a is 2.1 Kb. Notably, this approximates the size of the 2.2 Kb seen

our northern blot assays (Figure 3A).

Our 5’ RACE experiments also discovered two other TSS’s. Transcript 2a

resides within Exon 27 of CACNA1C. For this transcript the first and strongest

starting AUG codons were in frame with the channel’s sequence. This predicted a C-

terminal channel protein of approximately 110KDa. This predicted protein would

encompass Cav1.2’s sixth transmembrane helix of ion pore domain III, all of domain

IV and the cytoplasmic c-terminus. Hence, this protein was predicted to be membrane

bound and would have a voltage sensor. The predicted full-length transcript size for

his message was roughly 4.4 Kb, which also corresponded to the size of the higher

molecular weight transcript found in the Northern blot experiments. Lastly, transcript

3 has a start site within Exon 42 of the channel. Sequence analysis of the 5’ UTR

predicts it to be non-coding.

Systematic and genome-wide 5’end analysis of the mouse and human

transcriptomes has been carried out using cap analysis of gene expression (CAGE)

technology (Carninci et al., 2006; Kawaji et al., 2006; Kodzius et al., 2006). The basis

of this approach is the chemical targeting of 5’-capped RNA's and the formation of

short, 20-21 bp sequences of the 5’ ends of the cDNA's. These tags have been mapped

onto the mouse genomic sequences. To look for independent evidence for these

transcription start sites, we searched the CAGE tag library looking for co-clustering

with our TSS’s (http://fantom3.gsc.riken.jp/). We found 2 tags that mapped within 100

bp of the TSS’s found (Table 1). In support of transcript variant 4, we found one tag

that maps 77 bp downstream of the TSS found. The predicted starting AUG was also

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M2011. The second tag and its expected TSS mapped 92 bp downstream of the one

we identified for transcript variant 3. No protein was predicted to be translated from

this transcript. We found no CAGE tags in the vicinity of the 5’ end of transcript

variant 2a on Exon 27. Interestingly, in the CAGE database the biggest tag cluster for

CACNA1C was not the for the full-length channel but for a TSS located between

Exons 28 and 29 of the channel

(http://gerg01.gsc.riken.jp/cage/mm5/SummaryTss.php?tss_id=T06R07183F39 and

Table 1, transcript 2b). Without additional splicing of the intron sequence, this

transcript is not predicted to be protein coding. However the best, though weak,

initiator AUG is in frame with the channel and would generate a 100KDa protein with

similar localization and properties of transcript variant 2b.

Our experiments thus far suggested that alternative promoter sequences within

CACNA1C promote expression of alternative transcripts some of which could encode

for non-channel proteins including CCAT and a 110KDa, membrane bound channel

fragment or mem-CCAT (Figure 4C). To characterize the subcellular localization of

these proteins we cloned their predicted coding sequence in front of an N-terminal

GFP tag. When expressed in N2As, mem-CCAT was localized to the endoplasmic

reticulum and to a smaller extent to the plasma membrane. CCAT appeared to be

soluble, distributed between the cytoplasm and nucleus, with increased nuclear signal

(Figure 4D). Analysis of the untagged proteins using SDS-Page reveal a running

molecular weight of 120KDa for mem-CCAT and 20KDa for CCAT (data not shown).

We next sought to determine whether proteins of predicted molecular weights

could be found in brain protein extracts. First, we assumed that mem-CCAT and

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CCAT share their C-terminal sequences and therefore both proteins should be detected

using the anti-CCAT antibody. Second, both proteins were predicted to be expressed

from independent transcripts and consequently their expression should remain in our

Cav1.2 KO mice. As previously described, we fractionated membrane and nuclear

proteins from wild type and Cav1.2 null 11.5 dpc embryos separated them using SDS-

page and probed with the anti-CCAT antibody. In membrane fractions two other

additional bands in addition to the full-length channel could be detected corresponding

to MW 160 and 120KDa (Figure 1F). In nuclear fractions of WT and KO embryos,

anti-CCAT antibody recognized several bands including two small 22K and 15 KDa

bands. Thus, proteins of predicted molecular weights can be detected in the

appropriate subcellular fraction. The presence of these CCAT reactive bands in protein

extracts from embryos lacking full-length cav1.2 would be consistent with the idea

that the endogenous CCAT fragment and mem-CCAT can be generated in the absence

of Cav1.2 channel. However, the possibility that some of these bands are the result of

cross-reactivity of anti-CCAT with other nuclear proteins must also be considered.

DISCUSSION

The results of the present study demonstrate that a short CCAT is translated

from an alternative transcript whose expression is driven by an exonic promoter.

These findings provide a unique example in which two proteins with distinct biologic

functions can be derived from a single gene. They also highlight what has been

recently discovered about the eukaryotic transcriptome: many more start sites, many

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more RNAs, much more complexity than initially predicted. Here we provide an

example in which an exonic promoter can be used to contribute to transcriptional as

well as protein complexity. Such transcriptional phenomena may be at play in many

other genes throughout the genome and has far reaching implications for prediction of

gene products and interpretation of phenotypes in gene mutations and knockout

studies.

Functional Consequences of Exonic Promoters

Genome-wide analyses of the mouse and human transcriptomes have revealed

that our initial predictions greatly underestimated the number of transcripts expressed

in cells (Yasuda and Hayashizaki, 2008). The genomic sequences transcribed are more

extensive than we originally thought. The distribution of CAGE tags clearly shows

many additional promoters and common exonic transcription start sites, especially in

3’ UTR sequences (Carninci et al., 2006). Many of these exonic promoters are

conserved between species. These additional start sites can give rise to new proteins or

variations in protein sequence, thereby increasing proteome complexity. More than

half of these start sites are predicted to generate a wealth of non-coding RNAs for

which we have no function, thereby increasing RNA and regulatory complexity.

Furthermore, these additional exonic promoters will contribute to cell-type, tissue-type

and developmental gene regulation. Thus, mutations thought to be in coding

sequences may actually impact gene transcription. It can be predicted that aberrant use

of these promoters could be implicated in disease as it is known for many alternative

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promoters such as abnormal expression of c-myc in Burkitt’s lymphoma and

expression of short isoform p53 in several human cancers (Davuluri et al., 2008).

There are few examples of exons as promoter sequences in the literature. For

instance, in D. Melanogaster, the gene encoding for the sperm specific axonemal

dynein subunit Sdic, has a promoter derived from a protein-coding region from the

gene encoding the cell adhesion protein annexin (AnnX, (Nurminsky et al., 1998)).

The gene is posited to have evolved from the fusion AnnX exon 4 with Cdic intron 3

(cytoplasmic dynein intermediate chain). Transcription is initiated from promoter

elements within the AnnX Exon 4 region, and translation is initiated within the

sequence derived from Cdic Intron 3. Another example, also in flies, is NonA, a gene

important for vision, courtship song and viability in flies. NonA has upstream

regulatory regions embedded within the coding regions of adjacent gene dGpi1

(Sandrelli et al., 2001). While these two examples share the motif of coding

sequences having dual roles as promoters, the transcription of CCAT is unique in that

both promoter and coding sequences reside within a that also happens to code for a

calcium channel. Furthermore, by maintaining the reading frame CCAT is in fact a

channel fragment and has, as we have previously shown, a separate function in the

nucleus.

Our findings also beget the question of how expression from channel and

CCAT promoters is regulated. From our studies in N2A cells we surmise expression

of both proteins is not mutually exclusive. In the gene, these promoters are separated

by millions of nucleotides and could represent two very different chromatin

environments. Furthermore, the cell and tissue type specific expression of CCAT and

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the different patterns in mRNA expression during development argue that regulation

of each promoter occurs independently. However, given the evidence that the c-

terminus may also inhibit channel function, controlling their promoter elements would

be a necessary mechanism to establish an adequate expression ratio between channel

and putative negative regulator (Dzhura et al., 2003; Gao et al., 2001; Hulme et al.,

2006a). It is also conceivable that the transcript generated from the upstream channel

promoter impedes the formation of the transcription complex at the downstream

CCAT promoter. This type of relationship between these adjacent promoters is

exemplified by the human dihydrofolate reductase gene (DHR) (Martianov et al.,

2007). This mechanism would be consistent with the observed reverse expression

patterns of full-length channel and CCAT transcripts. Future experiments aimed at

understanding the DNA elements responsible for regulation of both promoters will

help understand how signaling cascades impinge on expression at both promoters.

CCAT Promoter Architecture

Our understanding of mammalian promoter architecture and evolution is

increasing rapidly. Still, present algorithms aimed at promoter or TSS prediction have

proven unsatisfactory even with the addition of phylogenetic conservation. Promoter

prediction algorithms such as Promoter 2.0 and Promoter inspector failed to predict

the CCAT promoter. Mammalian promoters can be separated in two classes,

conserved TATA-box rich promoters and CpG rich promoters. Several CpG islands

are found within the 3’end of the mouse CACNA1C gene. Two reside downstream at

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1.3 Kb and 2 Kb from the TSS and a third CpG island is 2.5 Kb upstream. However, it

is unclear whether any of these would influence transcription at the CCAT promoter

given than transcription driven by CpG islands usually occurs within 1.0 Kb (Saxonov

et al., 2006). The PolII core eukaryotic promoter is frequently observed to contain

TATA, TFII recognition, downstream core and initiator elements as well as CCAAT

and GC boxes (Carey et al., 2009). The 238 bp sequence identified here lacks a TATA

box but has a CCCAT and 2 Sp1 GC boxes at -140 bp, -170 bp and -40 bp

respectively from the TSS's found using 5’RACE (Sup Fig 4A). Promoters with

similar architecture have been reported in several brain specific genes (Christensen et

al., 2004; Ross et al., 2002; Schmitt et al., 2003; Schwarzmayr et al., 2008; Skak and

Michelsen, 1999). TATA-less promoters have broader distributions in the TSS's and

can vary by 100 bp. This can explain the range observed in our experimentally found

TSS's and CAGE tags. Future analysis of these sequences may help narrow down the

DNA elements required for CCAT promoter activity.

CCAT Promoter Evolution

Voltage-gated channels are multidomain proteins whose domains evolved separately

and consequently individual domains can be found as independent proteins in lower

organisms. It is reasonable to hypothesize that CCAT and its promoter constitute an

independent transcriptional unit that was acquired as a module during the channels’

evolution. One way to explore this is to look for sequences similar to the last two

exons of the channel while preserving the exon-exon boundary in the genome of

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ancient organisms using Blast. We found no independent proteins in lower organisms

with significant similarity to CCAT suggesting the CCAT promoter may have evolved

from sequential modifications to an ancestral channel and that the CCAT

transcriptional unit was not added to the channel gene in single step.

The evolutionary history of the C-terminus of Cav1.2 can be glimpsed by

looking at the evolution of the voltage gated channel family. The Cav 1 families of

channels diverged early during evolution from other Cav channels and have as earliest

ancestor the L-type channel (LTC) in worms and flies (http://www.treefam.org/cgi-

bin/TFinfo.pl?ac=TF312805#description). We selected channel proteins derived from

transcripts containing the longest c-terminus from human, mouse, Zebrafish and

compared to the ancestral worm and fly channels for all L-type families. Predictably,

the alignments show that the regions of greatest variation are the N and C-terminal

cytoplasmic regions. All channels in the LTC family are conserved to 20-30 amino

acid segment beyond the calmodulin binding motif, highlighting the importance of this

domain in LTC function (Sup Fig 4C and D). Multiple sequence alignment of the

mouse Cav 1.1, 1.2, 1.3 and 1.4 and the worm and fly LTC sequences highlight a

second region of conservation, a modified leucine zipper domain (LZ) (Figure). The

LZ is entirely contained in the last exon of the channel and moreover the E n-1/E n

boundary is also conserved in all channels including C. elegans. This suggests that the

last exon and therefore a significant part of the CCAT sequence was present early in

the evolution LTC genes. The sequence for E n-1, which contains the 238 bp promoter,

is on the other hand highly variable at the amino acid and nucleotide level. This

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suggests that the new regulatory sequence might have evolved around the divergence

of Cav1.2 channels.

How does a sequence that has no prior function in regulating gene expression

can become a fortuitous promoter? One hypothesis is that new regulatory sequences

can be inserted through transposition and many specific examples of mammalian

genes regulated by promoters donated by endogenous transposable elements have

been reported (Cohen et al., 2009; Ferrigno et al., 2001; Oei et al., 2004). A search for

interspersed repeats in the Mouse CACNA1C gene using RepeatMaster failed to

revealed any repetitive sequences near CCAT’s promoter. However, these sequences

can be hard to recognize, especially if the integration occurred in a distant past.

Alternatively, de novo regulatory sequences can be created through, small-scale, local

mutations which modify and create transcription binding sites.

Recent evidence has shown that LZ is required for the targeting of PKA to

Cav1.2 channels and functional regulation of the channels by PKA signaling which is

an important mechanism of cardiomyocyte regulation in response to β-adrenergic

stimulation (Hulme et al., 2003; Hulme et al., 2004). Interestingly, we found that

mutation of the I-F-I-L residues within the rat or mouse CCAT sequence is sufficient

to abolish transcriptional activation (Sup Fig 4B). This highlights the importance of

this domain in channel and CCAT function.

Alternative Transcript vs. Channel Cleavage

115

Several laboratories have reported that LTCs are cleaved at their c-terminus

and the site of cleavage of Cav1.1, the skeletal isoform of Cav1.2, has been recently

identified (De Jongh et al., 1994; De Jongh et al., 1989; De Jongh et al., 1991; Hulme

et al., 2005). Thus far, our experiments on heterologously expressed Cav1.2 channels

have failed to show cleavage under conditions of low and high electrical activity and

in different cell types. We have used sensitive methods including luciferase assays and

immunoprecipitation assays to demonstrate that, in the absence of this alternative

protein, or conditions where the channel is degraded such as in 293t cells, no

additional c-terminal fragments can be observed (Figure 1). Nevertheless, our results

do not preclude the possibility that endogenous full-length channels proteins are

proteolytically processed and that we have yet to determine the exact combination of

cell type and stimulus that lead to cleavage of channels. The phenomenon of channel

proteolysis and its mechanism, as it is described until now, does necessitate further

experimental validation. For Cav1.2, the exonic promoter precludes cleavage studies in

heterologous channels. Future experiments would require mutation of all Methionines

downstream of the TSS. Perhaps studies in Cav1.1 where a cleavage site has been

identified can help elucidate the conditions that trigger cleavage of channels, the

channel sequences involved, and the exact nature of the fragment.

Other CCAT Proteins

Our results also put forward the possibility that other proteins are expressed

from the CACNA1C gene. Transcript 2a has its TSS within Exon 27 of the Mouse

116

gene. The existence of this protein is supported by the presence of a transcript of

appropriate size in mRNA extracts from brain detected with a c-terminal probe and a

protein of predicted molecular weight in tissue extracts detected with a c-terminal

antibody. The predicted protein encodes for Cav1.2’s sixth transmembrane helix of

ion pore domain III, all of domain IV and the cytoplasmic c-terminus. Thus, this

membrane protein would have a plausible voltage sensor. There are several intriguing

possibilities for the role of such a protein in excitable cells. Such protein may affect

the function of individual channels or channel complexes. It could also influence

channel transport along the secretory pathway and provide a regulatory step for

channels at the membrane. This protein could also function independently to release

the transcription factor CCAT with the appropriate voltage stimulus or as an oligomer

to form functional channels or signaling complexes.

We provide strong evidence that CACNA1C encodes in addition to a calcium

channel a transcription factor within its 3’ region. We show that at least in mice, the

expression of the transcription factor CCAT is driven by a promoter nestled within the

penultimate Exon of the gene. Transcription at this promoter is regulated in a cell-type

and developmental specific ways. In the brain, regulated expression from this

promoter results in highest level of expression in subpopulations of inhibitory neuron

before postnatal life. These findings reveal another unexpected chapter of CCAT’s and

CACNA1C’s biology. The gene products from the many exonic transcriptional start

sites found in vivo will be a rich area of study in all fields of biology.

117

EXPERIMENTAL PROCEDURES

Materials

Brefeldin A was purchased from Calbiochem and used at 10µM.

The generation of Anti-CCAT was described earlier (Gomez-Ospina et al., 2006). The

antibody was used 1:500-1:1000 for western blots. Anti-gal4 SC-510 was used at

1:200 for western blots and 4 µg for immunoprecipitations and was purchased from

Santa Cruz Biotechnology. Anti-Map2 was used at 1:1000 for IHC and was obtained

from Chemicon/Millipore.

Cell culture and Transfection

Neuro2A cells were cultured in Dulbecco's Minimal Essential Media (DMEM)

containing 10% fetal bovine serum (FBS; 15% for PC12s), penicillin, streptomycin

(P/S), and L-glutamine (LQ).

Cortical neurons were dissociated from E17-19 Sprague Dawley rats as described (Xia

et al., 1996) and maintained for 6–14 days in culture in Basal Medium Eagle with 5%

FBS, P/S, LQ, and 1% glucose or in Neurobasal medium containing B27 supplement

(Invitrogen).

Thalamic neurons were dissected from E17-19 Sprague Dawley rats in ice-cold Hank's

Balanced Salt Solution without Ca++ and Mg++ (HBSS, Gibco). Thalami were

enzymatically digested using trypsin (Worthington, 10mg/ml), DNase (Sigma, 200

U/ml) in HBSS at room temperature for 10 min. Thalami were washed 3x in Basal

Medium Eagle with 5% FBS, P/S, LQ and 1% glucose and gently triturated in the

118

same media. Neurons were plated at 25,000/cm2. Arabinosylcytosine was added 24 h

after plating to inhibit glial cell growth.

Neuro2As (24 hr), cortical and thalamic neurons (96 hr) were transfected using

lipofectamine 2000 according to manufacturer's instructions.

Plasmid Construction

Note: All mutations were generated using the Quick Change XL mutagenesis

kit according to manufacturer’s instructions. All amino acid and base pair positions in

Cav1.2 refer to Acc# AAA18905.1

Construction of the dihydropyridine resistant Cav1.2 -Gal4 and ∆TA channels

has been previously described (Dolmetsch et al., 2001; Gomez-Ospina et al., 2006).

Construction of ∆TM-IQ was achieved in a three-step process. First, we inserted a

KpnI site immediately downstream of the IQ at position 4929 of the Cav1.2 -Gal4

channel’s coding sequence. The was accomplished by PCR amplification with the

following primers: 4930-4965 Fwd 5’-

GGCAAGCCCTCGCAGAGGAATGCACTGTCTCTGCAG-3’ and IQ reverse

(4893-4929)

5’-GGTACCGACCAGCCCCTGCTCTTTTCGCTTCTTGAATTTCCTG-3’. The

amplicon was a linearized channel vector with the KpnI site added to the reverse

primer. The PCR product was then DpnI digested, blunt ligated and transformed.

The second step was to insert another KpnI site at 4477-4482 nucleotide position

taking care of maintaining the channel’s reading frame. The primers used were: (4477-

4482) KpnI fwd 5’-

119

GGATTGGTCTATCCTTGGTACCCATCACCTGGATGAATTCAAGAG-3’

(4477-4482) KpnI rev 5’-

CTCTTGAATTCATCCAGGTGATGGGTACCAAGGATAGACCAATCC-3’

Finally to obtain ∆TM-IQ, the mutant plasmid was subjected to KpnI digestion, gel

extraction, ligation and transformation.

The delta cleavage site channel was generated by deleting 150 a.a. downstream of the

IQ motif. The Cav.12-Gal4 channels was amplified using the following primers:

5371-5406 Fwd GTCAGCACTGTGGAGGGCCATGGGCCTCCCTTGTCC

IQ reverse (4893-4929)

GGTACCGACCAGCCCCTGCTCTTTTCGCTTCTTGAATTTCCTG. This linear

construct was then DpnI treated, blunt ligated and transformed.

The translational stop channel was created after deletion of 193 bp between

nucleotides 5588 and 5781 using the double KpnI site strategy using the following

primers:

5588 KpnI fwd 5’-

GCTCTCCACAGATATACTCTGGTACCAGGACGATGAAAACCG-3’

5588 KpnI rev 5’-

CGGTTTTCATCGTCCTGGTACCAGAGTATATCTGTGGAGAGC-3’ and

5781 KpnI fwd 5’-GCCTTGCCCTTGCATCTGGTACCTCACCAGGCATTGG-3’

5781 KpnI rev 5’-CCAATGCCTGGTGAGGTACCAGATGCAAGGGCAAGGC-3’

After KpnI digestion and excision of the intervening sequence there was a frameshift

in the sequences creating an early stop codon at a.a. 1910.

120

Methionine mutations of Cav1.2-Gal4 channels and minigenes and CCAT

minigenes were generated using the primers:

M2011I: F 5’-TTGGCAGTGGCAGGGATCCCCCGGAGAGCCCGG-3’

R 5’-CCGGGCTCTCCGGGGGATCCCTGCCACTGCCAA-3’

(Primers add a silent Cspc site used for screening mutants).

M2073I: F 5’-CTGGCTGACGCCTGCGATATCACAATAGAGGAGATGGAG-3’

R 5’-CTCCATCTCCTCTATTGTGATATCGCAGGCGTCAGCCAG-3’

(Primers add a silent EcorV site used for screening mutants).

M2078I:

F: 5’CGACATGACAATAGAGGATATCGAGAACGCCGCAGACAACATC-3’

R: 5’-GATGTTGTCTGCGGCGTTCTCGATATCCTCTATTGTCATGTCG-3’

(Primers add a silent EcorV site used for screening mutants).

Promoterless constructs were generated by MfeI digestion and self-ligation of

the pcDNA4 Cav1.2 -Gal4 plasmid. This removed additional 407 base pairs from the

channel’s 5’ end.

The Cav1.2 CDS as promoter constructs except for the full length channel

sequence were built by PCR amplification of : Ion Pore: 4 – 445 nt, E46-E47: 5794 –

6429 nt, E46: 5794-6129 and E47: 6130-6429 and insertion via KpnI digestion into

pGL3 basic luciferase reporter vector (Promega). The full length channel sequence

was subcloned from PA1-Cav1.2 plasmid which contains the full length channel’s

sequence in between two KpnI sites in the PA1 expression vector originally obtained

from Dr. Michael Lin and described earlier (Gomez-Ospina et al., 2006). The

channel’s termination codon and on additional stop codon in between the channel’s

121

and luciferase’s CDS were mutated using the following primers:

Pgl3- Cav1.2 6490 stop

F 5’-GAGATCTGCGATCTAAGGAAGCTTGGCATTCCGGTACTG-3’

R 5’-CAGTACCGGAATGCCAAGCTTCCTTAGATCGCAGATCTC-3’

Pgl3- Cav1.2 6436 stop

F 5’-CTATGTCAGCAACCTGTACGGTACCGAGCTCTTACGC-3’

F 5’-GCGTAAGAGCTCGGTACCGTACAGGTTGCTGACATAG-3’

Minigenes were generated using a multistep cloning process: 1. An NcoI site

was mutated to a MluI site in the pGL4.10 vector (Promega) with the purpose of

creating a compatible site where the genomic sequence could be inserted into. 2. To

make the construct usable for stable cell line creation, we inserted an amplified Zeocin

resistance cassette from pCDNA4 into the BamI/Sal1 sites of pGL4.10. 3. To create

4Kb-0/luciferase, a 4 Kb genomic region was PCR amplified using NcoI/MluI sites

and the BAC clone RP23-158O9 3’ as template which contains the 3’ end of

CACNA1C. 5. The Gal4 based minigene was finally generated by removing the

luciferase and subcloning a PCR amplified sequence of Gal4-DBD using the

restriction sites MluI and Xba1. 4Kb-1 was created by using the mutagenesis kit to

insert a G between the channel’s and luciferase’s coding sequence in the 4Kb-0

construct. ∆238 was created by inserting a CspcI site at 1579 bp of the 4Kb construct

within Exon 46. The sequence already had a CspcI site at the 5’ end of Exon 47.

Subsequent digestion with CspcI removed 238 bp sequence.

The neuronal channel 4Kb promoter was cloned by PCR amplification of 4Kb

122

upstream of the initiating methionine for the neuronal channel from a BAC clone

RP23-117C19 that contained the 5’ end of CACNA1C. The PCR product had

EcoRV/HindIII sites added which were used for cloning into the pGL4.10 vector.

CCAT TA promoter was generated by PCR amplification of 4Kb upstream of the

2011 methionine from BAC clone RP23-158O9. This was inserted into the XhoI/MluI

sites in pGL4.10 .

The plasmids encoding the N-terminal tagged YFP-mem-CCAT and YFP-

CCAT were generated using Gateway technology (Invitrogen) by first cloning coding

the sequences from a.a. 1160-2144 for mem-CCAT and 2011-2144 for CCAT

amplified from a pCDNA4.0 Cav1.2 plasmid into the TOPO sites of the pCR8 entry

vector and subsequently transferring the Cav1.2 coding sequence into a destination

vector called pDEST-pGWYFP that contains a CMV promoter and an N-terminal YFP

in frame with the ATTR (Cox and Emili, 2006) acceptor sequences.

Subcellular Fractionation

Note: For biochemical experiments all protein samples were kept at or bellow -4°C.

Complete protease inhibitor tablets were added to all solutions fresh before each

experiment.

For Cav1.2 knockout mice and control pubs, pregnant females at 11 days post plugging

were anesthetized using CO2 and embryos were dissected in cold PBS. The tails were

cut and separated for DNA genotyping. Tissue subcellular fractionation and protein

extraction was performed as described (Cox and Emili, 2006). Briefly, after dissection

embryos were cut and washed in cold 250-STMPBS, homogenized and centrifuged at

123

800g for 15 min. The nuclear pellet was resuspended and homogenized again.

Supernatants were pooled to form the cytosolic fraction I and the pellet constituted

nuclear fraction I. The cytosolic fraction was centrifuged 1h at 100,000 g to pellet the

microsomal fraction which was solubilized in ME buffer for 1h and finally centrifuged

for 30min at 9000g. This supernatant constituted the solubilized membrane fraction.

The nuclear fraction I was resuspeded in 2M-STMDPS buffer and layered on a 2M

STM cushion in a centrifuge tube. Samples were spun at 80,000g for 30-45 min. The

pelleted nuclei were solubilized in NE buffer, then passaged through an 18 gauge

needle and spun at 9000g for 30 min. Supernatant was used as salt soluble nuclear

proteins.

Immunoprecipitation and Western Blotting

N2A cells were lysed 24 hrs post-transfection using lysis buffer containing 1.5%

TritonX-100, 50 mM TRIS-HCL pH 7.5, 150mM NaCl amd 10mM EDTA and

protease inhibitor tablets (Roche). Immunoprecipitations were carried out using 4 µg

of Gal4 antibody and Protein A/G beads (Santa Cruz).

Western blotting was conducted using standard protocols. Antibodies and dilutions are

included in Supplemental Experimental Procedures. Protein concentration was

measured by the BCA method (Pierce).

Immunoflurescence

Cortical and thalamic neurons were fixed in 4% paraformaldehyde/2% sucrose and

10mM EDTA in PBS for 10 minutes followed by permeabilization with 0.025%

124

TritonX-100 and blocking with 3% bovine serum albumin (BSA) in phosphate-

buffered saline (PBS). Cultured neurons were stained primarily with rabbit anti-CCAT

(1:500) and secondarily with 1:500 dilutions of Alexa 594-conjugated or Alexa 488-

conjugated anti-rabbit antibodies (Molecular Probes). Nuclei were stained using

Hoechst 33258 or DAPI (Molecular Probes).

Brains from embryonic and postnatal rats and wildtype C57/bl6 mice were

dissected and fixed in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB, pH

7.4). Brains from animals younger than embryonic day 18 (E18) were fixed in PFA for

30 minutes, whereas brains from postnatal mice were fixed overnight at 4°C.

Subsequently brains were transferred to a 30% sucrose solution overnight for

cryoprotection, embedded in Tissue-Tek OCT compound, and cut (10µm for

embryonic brains, 25-30µm for postnatal brains) on a freezing cryostat (Leica,

CM3050). All tissue was stored at -80°C until further use. For immunofluorescence

analysis of CCAT expression, slides were washed in PBS containing 0.1% bovine

serum albumin (BSA) to remove excess OCT. Sections were blocked in 10% normal

goat serum (NGS; Gibco) containing 0.25-3% Triton X-100 for 1 hour at 25°C.

Primary antibody (anti-CCAT, 1:150) was applied overnight in 10% NGS with 0.1%

Triton X-100 at 4°C followed by the appropriate fluorochrome conjugated secondary

antibody (Alexa conjugates; Molecular Probes) for 1 hour at 25°C. Slides were then

washed in PBS with 0.1% BSA, counterstained with Hoechst or DAPI, and mounted

in Aqua Poly/Mount (Polysciences, Inc.) for fluorescence microscopy.

125

Slides were visualized by conventional epifluorescence microcopy using a cooled

CCD camera (Hamamatsu) coupled to an inverted Nikon Eclipse E2000-U

microscope.

Luciferase Assay Transfections

Cav1.2-Gal4 channels were transfected in a ratio of 2:1:1:0.5 Cav1.2, β1b subunit,

firefly luciferase and Renilla luciferase reporters.

Minigenes were transfected as a 1:1:0.25 ratio of Minigen to UAS-firefly luciferase to

Renilla luciferase.

Channel coding sequence as promoter constructs were transfected at a ratio of 2:1

pGL3 based vector to Renilla luciferase.

Most luciferase assays were performed 24 hr after transfection using the Dual-Glo

luciferase assay kit from Promega. A Veritas 96-well luminometer (Turner

Biosystems) was used to measure light emission. PFA-CMV (Gal4 alone) and UAS-

luciferase constructs and were obtained as part of the PathDetect transreporting system

(Strategene). Data sets were analyzed using Igor Pro and Prism4 software. Two-paired

t tests were performed between relevant conditions.

Northern Blots

RNA isolation: mRNA was extracted from rat brains and N2A cells using the Fast-

Track mRNA isolation kit according to manufacturer’s instructions (Invitrogen).

Probe: Exon 47-pGL3 plasmid was used as template to PCR amplify Exon 47’s

sequence. After gel extraction, 25ng of PCR template were used for labeling using

126

prime-it II random primers labeling kit (Stratagene) and 5µl of {α-32P]dCTP at 3000

Ci/mmol (Amersham) by following the manufacturer’s protocol. For loading control a

900bp region of the 18S RNA was amplified using the following primers:

18S RNA L 5’-GAGGGAGCCTGAGAAACGGCTA-3’

18S RNA R 5’-AACTAAGAACGGCCATGCACCA-3’ and used as template for

random labeling as described above. The full length channel probe used contained

sequence nt 2659 to 2966 in domain III of the channel’s coding sequence and was

amplified using the following primers:

IIIS1 F 5’-GCGAAGCTTagcccaaacaacaggttc-3’

IIIS3 R 5’-gcgAAGCTTatgccaaaggagatgagg-3’

Blots: Northern blots were carried out using the NorthernMax Kit solutions and

followed the protocol as recommended by the Manufacturer (Ambion). Briefly, 5µg

of mRNA were loaded into 1% RNAse free agarose gel. Electrophoresis was carried

out at ~5 V/cm. RNA was then transferred to Ambion’s BrightStar-Plus membranes

by downward capillary transfer. The RNA was crosslinked to the membrane using a

commercial crosslinker. Membranes were prehybridized at 68°C for 5-6h in

prehybridization solution plus 100µg of salmon sperm DNA. Labeled probe was

denatured before adding to hybridization solution and incubated at 55-65°C overnight.

Membranes were washed using low and high stringency washes as outlined in

Northern Max kit. Films were exposed form 5-48h at -80°C.

5’ RACE experiments

127

5’ RACE was carried out using Invitrogen’s GeneRacer kit according to

manufacturer’s instructions with the following specifications:

1) 250ng of each mRNA template were dephosphorylated using calf intestinal

phosphatase (CIP) at 50°C for 1h. 2) After precipitation the cap structure was removed

using Tobacco acid pyrophosphatase (TAP) at 37° C for 1h. 3) After precipitation the

RNA oligo :

5’-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3’

was ligated to the 5’ end using T4 RNA ligase by incubating at 37C for 1 h. 4)

Reverse transcription followed using RACE outer primer: 5’-

CTACAGGTTGCTGACATAGGACCTGCT-3’ encompassing sequence including

the channel’s termination codon or olido-dT primer. 5) PCR amplification was carried

out using the primers: GeneRacerTM 5’-CGACTGGAGCACGAGGACACTGA-3’

primer and 5’RACE reverse inner primer 5’-

CACAAAAGGTAAGAGGGTGCCGTTG-3’. To increase abundance of longer

transcripts a reverse primer closer to 5’ end (5970nt of cDNA) was used for CDNA

synthesis: 5’-GAAGCTGCTGTTGAGTTTCTCACTGGACTC-3’ and the nested 5'-

CTGGTGATGAACCAGATGCAAGGGCA-3’ (5793 nt) was used for amplification.

GeneRacerTM 5′ Nested Primer 5’-GGACACTGACATGGACTGAAGGAGTA-3’

5’ RACE of the second transcript from the Cav1.2-Gal4 channel was accomplished

using the following primers: for cDNA synthesis Cav1.2-Gal4 6865 (up to stop) R:

5’-TGACCGGCGATACAGTCAACTGTCTTTG-3’ and for PCR the Cav1.2-Gal4

6565 R 5’-TCAGCGGAGACCTTTTGGTTTTGGG-3’ and GeneRacerTM 5’ as

forward primer.

128

Generation of conditional knockout mice for the Cav1.2 calcium channel gene

Mice lacking the L-type a1c (Cav1.2) calcium channel gene were generated by

homologous recombination mediated gene targeting (Supplementary Figure 1B and

1C). The targeting construct was designed to delete exons 14 and 15 of the a1c gene

under the control of CRE recombinase. To facilitate Southern screening, a new BamHI

site was generated 5’ to Exon 16, and the original BamHI site 5’ to Exon 14 was

eliminated in the targeting construct (Fig X). The a1c targeted mice were maintained

in 129/sv-C57BL/6 mixed background. The a1c floxed (Neo deleted) mice were

generated by crossing a1c targeted mice with FLP transgenic mice (kindly provided by

Dr.Dymecki). Ubiquitous deletion of the floxed exon was achieved using CMV-CRE-

transgenic mice.

Sequence Analysis and Multiple Sequence Alignments

The following accession numbers were used for alignments:

Cav1: Drosophila Melanogaster NP_602305.1, Caenorhabditis elegans

NP_001023079.1

Cav1.1: Human NP_000060.2, Mouse NP_055008.2, Zebrafish NP_999891.1

Cav1.2: Human NP_001123312.1 Mouse NP_001153006.1 Zebrafish NP_571975.1

Rat P22002.1, Chimpanzee XP_522315.2, Rhesus monkey XP_001117926.1, Guinea

pig dbj|BAA34185.2, Rabbit NP_001129994.1, Horse XP_001490707.1, Dog

XP_534932.2,

Bovine XP_001255123.2, Chicken XP_416388.2

Cav1.3: Human NP_001122312.1, Mouse NP_001077085.1, Zebrafish NP_982351.1

129

Cav1.4: Human NP_005174.2, Mouse NP_062528.2

Sequences were aligned with ClustalW and MAFFT multiple sequence alignment

programs. The alignments were edited using Jalview and colored using percentage

identity with a conservation color increment set to 20.

130

FUTURE EXPERIMENTS

Experiments over the last five years have been aimed at characterizing basic

properties of CCAT. We first showed that such a fragment was present in cells. We

then were able to demonstrate that this channel fragment played a role in nucleus and

shared characteristics of many transcription factors. Later, we were able to discern the

way in which CCAT was made in cells. Now, we are finally in a position to ask what

is perhaps the biggest question remaining regarding CCAT’s biology: what is CCAT’s

function in vivo? In the following paragraphs I’ll be discussing several experiments

that are underway that will help us understand CCAT’s function in vivo.

The best way to ask whether CCAT is necessary for the function or

development of cells is to use loss of function studies. In order to be able to design

experiments to investigate CCAT’s role in cells using a “knockout” approach we

needed to understand how CCAT was being produced. Loss of function experiments

with CCAT are complicated by the fact CCAT is a fragment of a channel who is vital

to survival and the function of excitable cells. Consequently, any perturbations to the

CACNA1C gene sequence should be carefully designed so that they disrupt CCAT

production/function while maintaining the properties of the channel intact. We have

shown that CCAT is expressed from an alternative message and we predict that, as we

have shown in vitro, a single methionine mutation should be able to knockout the

production of the 15 KDa CCAT protein in vivo. One future experiment is to generate

mice using homologous recombination, where only M2011 has been disrupted. Our

preliminary studies suggest this mutation has no effect on the calcium conduit or

131

signaling to CREB properties of the full-length channel. We are particularly interested

in the role that CCAT has in development of the brain. Our data regarding the very

specific and temporally regulated expression of CCAT in inhibitory neurons, suggest a

role for this protein in their development. We plan to study the number, migration and

behavior of inhibitory neurons in these mice, using immunohistochemical and

electrophysiology techniques as well as genetic mouse reporters.

Understanding the precise timing and location of CCAT expression will help

understand CCAT’s role in development. Toward this end, we plan to create a BAC

transgenic mouse that reports on expression from the exonic promoter identified in

chapter 3. We have obtained a BAC containing 120 Kb of the mouse CACNA1C gene.

This BAC contains the last 37 exons and the 3’ UTR of the channel thus lacking the

full-length channel’s promoter including the first ten exons. We have introduced a

GFP reporter at the 3’ end of the channel’s coding sequence immediately before the

termination codon using bacterial recombineering. Expression of GFP tagged protein

from this BAC would strongly support the presence of internal promoter elements

within the gene. Transgenic mice generated using this construct can be used to study

the activity of this promoter in vivo by analyzing the tissue and cellular distribution of

GFP at different developmental stages.

Another approach towards understanding CCAT’s function is to perform gain

of function studies. In these experiments, CCAT can be added exogenously using

transfection, electroporation or even expression from a transgene. These experiments,

especially under controlled conditions, can be used to ask very exact questions about

the effect of CCAT on specific cellular processes. One confounding factor as it

132

pertains to CCAT specifically is that CCAT overexpression may perturb the channel’s

function by inhibiting the channel or by competing for interacting proteins that may be

important for targeting or function of the channel. We have already reported the effect

of CCAT on neurite growth in cultured neurons. In the same manner, this approach

can be used to study CCAT’s role in neuronal differentiation by expressing CCAT in

precursor neuron cultures and quantifying the number of differentiated neurons and

the percentages of specific subpopulations. A version of CCAT with a mutated leucine

zipper differs by four amino acids from the wild type protein but completely lacks

transcriptional activation, making it an optimal negative control for these studies.

Finally, one can also envision more in vivo approaches where the effect of CCAT

expression is studied in developing brains of mouse embryos or newborn pubs after

introduction of the CCAT transgene using in vivo electroporation.

B C

250

40

Cav1.2

CCAT

anti-Gal4

WT∆TM-IQ

∆C.S.STOP

∆TA

UAS

Tra

nscr

iptio

n (

Fire

fly/R

enilll

a)

Gal4 WT ∆TM-IQ ∆C.S. STOP ∆TA

0.00

0.25

0.50

0.75

1.00

1.25

Cav1.2 channels

ED

WT M2011I M2078I

Anti Gal4

Cav1.2

CCAT

250

40

0.00

0.25

0.50

0.75

1.00

1.25

UAS

Tra

nscr

iptio

n (

Fire

fly/R

enilll

a)

WT M2011I M2078IGal4

Cav1.2-Gal4 channels

A

IQ TACleavage Site

Translational

Stop

Gal4TM

DAPI MERGED

100X

600X

CCATN/+ N/+ N/+ N/N N/N N/N

250

60

F G

32

150

100

*

*

*

*

133

0.0

0.5

1.0

1.5

UAS Transcription (Firefly/Renillla)

Gal

4

Cav

1.2-

Gal

4

∆Pro

mot

er+P

rom

oter

01234567

Luciferase expression (Firefly/Renillla)

Full

leng

thIo

n Po

reE4

6-47

E46

E47

M20

11AAGCTA...STOP

TTCGAT ...STOP

AA

CC

TG

TT

GG

AC

..

..

..

..

..

..

AT

GT

AC

Gal

4

AAGCTA...STOP

TTCGAT ...STOP

AA

CC

TGG

TT

GG

AC

..

..

..

..

..

..

AT

GT

AC

C AAGCTA...STOP

TTCGAT ...STOP

AA

CC

TG

TT

GG

AC

..

..

..

..

..

..

AT

GT

AC

AAGCTA...STOP

TTCGAT ...STOP

AA

CC

TG

TT

GG

AC

..

..

..

..

..

..

ATC

TAG

E47

4Kb-

0

4Kb-

1

∆238

bp

M20

11I

0.00

0.01

0.02

0.03

0.04

UAS Transcription (Firefly/Renillla)

4Kb-0

4Kb-1

∆238b

p

M2011

I

M20

11IQ

Ion

Pore

Luci

fera

se

TACGGT...ACC

GGTGGC ...GTA

...

...

ATG

TAC

AAC

CTG

TTG

GAC

..

..

..

ATG

TAC

..

..

..

Full

leng

th

TACGGT...ACC

GGTGGC ...GTA

...

...

ATG

TAC

Ion

Pore

AAC

CTG

TTG

GAC

..

..

..

ATG

TAC

..

.TACGGT...ACC

GGTGGC ...GTA

...

...

ATG

TAC

E46-

47

ATG

TAC

TACGGT...ACC

GGTGGC ...GTA

...

...

ATG

TAC

E46

AGC

CTG

TCG

GAC

..

..

..

TACGGT...ACC

GGTGGC ...GTA

...

...

ATG

TAC

E47

A

7.6

Kb

2.4

Kb

1.4

Kb

Cav

1.2-

Gal

4

Unt

+Pro

mot

er ∆Pro

mot

er

9.5

Kb

Anti-

Gal

4

Cav

1.2

CC

AT

250

Prom

oter

+

40

BC

DE

FG

H

4Kb-0

4Kb∆

MM20

11I

M2073

IM20

78I

0.00

0.01

0.02

0.03

0.04

0.05

UAS Transcription*

*

**

***

*

134

E18 P1 AdultCtxMid

9.57.5

4.4

2.2

1.8

18S

A C

).U. A( S81/ noi sser pxe dezil a

mr oN

CB Ctx MidCB

Cav1.2 E47

18S

1234

0

Full length channel5

123456

0

2.2 Kb

1

2

0E18

P1 AdultCtxCBMid CtxCB Mid

4 .0 Kb

E

3

Coronal Sagital

CbS

BS V

Th

C

V

S

CV

VC

S

0.5

1.5

2.5

3.5

0.5

1.5

2.5

3.5

U

AS T

rans

crip

tion

(N

orm

alilz

ed to

Gal

4)

UAS

Tra

nscr

iptio

n (

Nor

mal

ilzed

to G

al4)

Cav1.2

Gal4 Stop

Gal4 M2011

I

Gal4

Cav1.2

Gal4 Stop

Gal4 M2011

I

Gal4

Cor

tical

Neu

rons

Thal

amic

Neu

rons

CCAT

CCAT

DAPI

DAPI

F

***

Cerebellum Thalamus

P1

D

E18 E18

P1

3W 3W

CCAT CCAT

CCATMap2

18S

E18 P1 AdultCtxMidCB Ctx MidCB

9.57.5

4.4

2.2

1.8

Cav1.2 IIIS3

B

135

5’ MetG AAAAAA3’

AAAAAA3’

cDNA synthesis

cDNA

Exon 46 Exon 47 Gal4

M2011

RNA oligo

PCRF R

A B

Predicted Proteins

CACNA1Cgene

Chr6 119200000 119100000 119000000 118900000 118800000 118700000 118600000 118500000

CalciumChannelCaV1.2

Membrane-AnchoredC-term with Voltage Sensor

CCAT

Mem-CCAT CCAT

Cav1.2-Gal4Promoter+ -

2.01.61.0

0.5

5983 nt 6432 nt 6874 nt

N/N N/N N/+ N/+40

35

25

15

C

D

E

136

C

A CCAT DAPI Merged

Brain

Heart

Bloodvessels

Somites

HeadMesenchyme

Liver

0.00

0.25

0.50

0.75

1.00

Brefeldin AGal4Cav1.2 Gal4

+_

UA

S Tr

ansc

rip

tio

n

(Fir

efly

/Ren

illa)

C

V

HM

C

M

Cav1.2 Targeting Strategy

Chr6

Targeting Construct

Targeting Allele

NULL

FLOXED

B

Null/+

FLox/+

+/+ Targe

ted

12.1 Kb9.5 Kb8.3 Kb

D

CRE

FLP

Supplentary Figure 1

137

0.0

0.1

0.2

0.3

0.4

EmptyVector

Neuronal 4kb

CCAT 4Kb

A B

Cav1.2_HumanCav1.2_ChimpCav1.2_RhesusCav1.2_MouseCav1.2_RatCav1.2_Guinea pigCav1.2_RabbittCav1.2_HorseCav1.2_DogCav1.2_BovineCav1.2_ChickenCav1.2_Zebrafish

Consensus

305305303304305305305305305306306300

Q A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R V R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q L V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C G S WA - E T T P G G G D S N T T R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P S T F P R P R P T P P V T P G S R - - - G R P L Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G G S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T A I P R P C A T P P A T P G S R - - - GWP P K P I P T L R L E G A E S C E K L N S S F P S I H C S S W S E E P S P C G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L A E AQ A L A V A G L S P L L Q R S H S P T S L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A D S S E K L N S S F P S I H C G S W S G E N S P C R G D S S A A R R A R P V S L T V P S Q A G AQ G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S R A P T T C P Q P W- - - - A T P S S Q - - - GWP P R P I P T L R L E G A E S S E K L N S S F P S I H C G S W S G E P T A C G G G S S A L R R A R P V S L T V P S R A G A P G R Q L - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P G T L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S N E K L N S S F P S I H C S S W S E E P T P C G G G D S T I R R A R P V S L T V P S Q A G A R G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H P P G T L P P P R L T P P A T P G P - - - - AWP P R P V P T L R L E G A E S S DK L T S S F P S I H C DP H I G E P T P C - G V V G T P R R A R P V S L T V P S P A G P Q G R P F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F S R L C A T P P A T P C S R - - - GWP QQ P I P T L R L E G A E S S E K L N S S F P S V H C S S R F P D S S DC G - - - - S P R R A R P V S L T V P S P T A G S S R Q F - H G S A S S L V E AQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N- - - - G N H S G R AQ R P V S L T V P P V T R R D S I S L A H G S A G S L V E A

Q A L A V A G L S P L L Q R S H S P T T F P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T P C G G G S S A A R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E A

417417415417418418418414418417415413

V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - P S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C A R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V A P E - D E S C A Y A L G R - G R S E E A L A D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V V P E - D E S C V Y A L G R - G R S E E A L P D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I G EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G D E - D E G C A C A L G R - GW S E E E L A D S R V H - - - - - - - - - - - - - - - V R S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C D L T I E EM E N A A DD I L S G G A R Q S P N G T L L P F V N R R DP G R DR A GQ N EQ DA S G A C A P G C - GQ S E E A L A DR R A G - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G T QQ S A N G T L F P F V N C R DP GQ DR A G G E E - N E T C A P A L E R - G K S E G E P Q D S R A C - - - - - - - - - - - - - - - G G S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DK A G G H V - G DA C T A A L A C - Q K S E E E L Q D S R A H - - - - - - - - - - - - - - - T G S LV L I S E G L GQ F AQ DP R F L E A T T Q E L A DA C DMT I E EM E S A A DD I L S G G A GQ S P N G T L L P C A N C R DP G P DR A G G V E - DA AWA P S A E P - R Q G A E E P R D S R A F - - - - - - - - - - - - - - - A S G LV L I S E G L MQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L N G N S KQ S P N G N L L P F V N C R DA GQ D S A G E E E - E E VQ N P - - DC - X K S Q E E L K D S R I Y - - - - - - - - - - - - - - - I S S LV L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N G N L L P F I Q C R DT G S Q E S R C S L - S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L

V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G E E - D+ G C A P A L G R - G K S E E E L Q D S R + Y - - - - - - - - - - - - - - - V S S LConsensus

MM

M

M

M

MMM

Cav1.2_HumanCav1.2_ChimpCav1.2_RhesusCav1.2_MouseCav1.2_RatCav1.2_Guinea pigCav1.2_RabbitCav1.2_HorseCav1.2_DogCav1.2_BovineCav1.2_ChickenCav1.2_Zebrafish

C

IQ

LZ

Exon n-1

Exon n

Supplementary Figure 2

138

CCAT

CCAT

CCAT

DAPI

DAPI

DAPI

CCAT/DAPI

E18

LV

VZ/SVZ,GE

CP

striatum(cp)

E18 Striatum

E18 Cortical plate

CCAT/DAPI

E14

IIIV

IIIV

striatum(cp)

hypothalamicnuclei

thalamus(DM, CM,rhomboid)

CP

VZ/SVZ,GE

LV

CCAT/DAPI

E12.5

LV

VZ/SVZ,GE

striatum(cp)

P1

CCAT/DAPI

striatum(cp)

cortex CCLV

septalnuclei

B C

A

Supplementary Figure 3

139

Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster

I Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V S Y Y Q - S DG R S A F P Q T F T T Q R P L H I N K A G S S - - - - - - - - - - - - - - - - - - Q G DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V T Y Y Q - S D S R G N F P Q T F A T Q R P L H I N K T G N N - - - - - - - - - - - - - - - - - - Q A DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V A K I P P K T - - A L S L Q A G L R T L H DMG P E I R R A I S G D L T V E E E L E R AMK E T V C A A S E DD I F R R S G G L F G N H V N Y Y HQ S DG H V S F P Q S F T T Q R P L H I S K S G S - - - - - - - - - - - - - - - - - - - P G E A E S P S HQ K L V D S TI Q DY F R K F R R R K E K G L L G N DA A P S - T S S A L Q A G L R S L Q D L G P EMR Q A L T C DT E E E E E E - - - - - - - - - - GQ E G V E E E D E K D L E T N K A T MV S Q P S A R R G S G I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F R R R K E K G L L G R E A P T S - T S S A L Q A G L R S L Q D L G P E I R Q A L T Y DT E E E E E E E - - - - - - E A V GQ E A E E E E A E N N P E P Y K D S I D S Q P Q S R WN S R I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E E - - - - - - T K R E E E DDV F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S I P P A S DT E K P L F P P A G N S V C H N H H NHI Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E D- - - - - - S K P E E E D- V F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S MP P A S DT E K P L F P P A G N S G C H N H H NHI Q DY F R K F K K R K E E G L V G V H P AQ N N T A I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E L V D- - - - - - F I P E E D E E I Y R R N G G L F G N H I N H I N G DP R R S S G HQ T N A T Q R P L Q VQ P P P H Y V HM EQ P V G R L G R A N AMAQQ N H H R H H H H H H H H H H HI Q E H F R K F MK R Q E E - Y Y G Y R P - K K D I VQ I Q A G L R T I E E E A A P E I C R T V S G D L A A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MK R Q E E - Y Y G Y R P - K K DT VQ I Q A G L R T I E E E A A P E I H R A I S G D L T A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MQ R Q E E - L Y G Y R P T K K N A D E I K A G L R S I E E E A A P E L H R A I S G D L I A E D EM E R - - - AM E S G - - E E G I Y R R T G G L F G L N A DP F S S E P S S P L S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EM E A K G V L P AQ T P Q AMA L Q A G L R T L H E I G P E L K R A I S G N L E T D F N F D E - - - - - - - - - - P E P Q H R R P H S L F N N L V H R L S G A G S K S P T E H E R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EQ E G K E G H P D S N - - T V T L Q A G L R T L H E V S P A L K R A I S G N L D E L DQ E P E - - - - - - - - - - - - P MH R R H H T L F G S VW S S I R R H G N G T F R R S A K A T A S Q S N G A L A I G G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

F T P S S Y S S T G S N A N I N N A N N T A L G R L P R P A G Y P S T V S T V E G H G P P L S P A I R VQ E V AWK L S S N R C H S R E SQ A AMA GQ E E T S Q D E T Y E V KMN H DT E A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T EM L S Y Q DD E N R Q L T - - - -F T P S S Y S S T G S N A N I N N A N N T A L G R F P H P A G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E SQ G A T V N - Q E I F P D E T R S V R M S E E A E Y C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T DM F S Y Q E D E HR Q L T - - - -F T P S S Y S S S G S N A N I N N A N N T A I G H R Y P K P - - - - T V S T V DGQ T G P P L T T I P L P R P T WC F P N K S S D S S D S R L P I I R R E E A S T D E T Y D E T F L D E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R DQ AM L S MDM L E F Q D E E S KQ L A P M- - V G DR L P D S L S F G P S DDDR - - - - - - - - - - - - - - - G T P T S SQ P S V P Q A G S N T H R R G S G A L I F T I P E E G N S Q P K G T K GQ N KQ D E D E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DR L S Y L D EQ A G T P P C S V L L P P- - V K E K L P D S L S T G P S DDDG - - - - - - - - - - - - - - - L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ DNQ N E EQ E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DWT P D L D EQ A G T P S N P V L L P PN S I G KQ V P T S T N A N L N N A NM S K A A H G K R P S I G N L E H V S E N G H H S S H K H DR E P Q R R S S V K R T R Y Y E T Y I R S D S G D EQ L P T I C R E DP E I H G Y F R DP H C L G EQ E Y F S S E E C Y E DD S S P T W S R Q N Y G Y Y S R Y P G R N I D S E R P R G Y H H P Q G F LN S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S - C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LN N S Y N K S P K S T N I N L N N A N V S S X P N G G H N - - R Y Y E H A P A N G Y P G S Y Y G E Y DK P R T P H GQ R R R Y Y E T Y I R S Q G S DR R R P T I R R E E E Y E E DR Y S G - - - - - - - E Y Y S G E E F Y E DD S M L S G - - - - - - - - DR Y P N S DQ E Y E T P R G Y H H P D S Y Y- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E EM E S P - - - V F L E D F P Q DP R T N P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S N H S N S H V F S S V H- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E E L E S P - - - V F L E D F P Q N P G T H P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S S H R N N P V F S S I C- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T Q V T S Q R P L Q F A E N R P E DA G S P P D S V F L P N T E F F P DNMP T T S N T N N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N F I E E F T F E S E S - - - L S A S R N- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I E K G S T L L P F Q P R S F S P T H S L A G A E G S P V P S QMH R G A P I NQ S I N L P P V N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G S A R R L P A L P P Y A N H I H D E T DDG P- - - - - - - - - - - - - - - - - - - - - - - - - - S A S A A L G V G G S S L V L G S S DP A G G DY L Y DT L N R S V A DG V N N I T R N I MQ A R L A A A G K L Q D E L Q G A G S G G E L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T F G E S I S MR P L A K N G G G A A T V A G T

- L P E E DK R D I R Q S P K R G F L R S A S L G R R A S F H L E C L K R Q K DR G G D I S Q K - - - - - T V L P L H L V H HQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA E T T P G G G G S S - A A R R- C P E E DK R E I Q P S P K R S F L R S A S L G R R A S F H L E C L K R Q K DQ G G D I S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RA E V G E E R R P WQ S P R R R A F L C P T A L G R R S S F H L E C L R K H N R P - - DV S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N G - - - - N H S G R AH R AQ R Y MDG H L V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R N S G P N R AQ G S WA T P P - - - - - Q R G - - - R L L Y A P L L L V E E G A A G E G Y L G R S S G P L R - - - - - - - - - - - - - - - - - - -HW S QQ H V N G H H V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R T S G P S R AQ G S WA A P P - - - - - Q K G - - - R L L Y A P L L L V E E S T V G E G Y L G K L G G P L R - - - - - - - - - - - - - - - - - - -DD S P V C Y D S R R S P R R R L L P P T P A S H R R S S F N F E C L R R Q S S Q E E V P S S P I F P H R T A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - T P C Y T P L I Q V EQ S E A L DQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TD EQ P L Y H D S H R S P K R R L L P P T P Q G N R R P S F N F E C L R R Q S S Q DD L P - - - - - HQ R T A L P L H L MQ HQ VMA V A G L D S S R A H R L S P T R S T R S WA S P P P T P A S K DR - - - T P Y Y T P L I R V DR - P L R D S A S S S H S S I R K S S WY T - - - - - DDP E Y QQ R NR E F P E E T E T P A T R G R A L GQ P C R V L G P H S K P C V EM L K G L L T Q R AMP R GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A P P A P CQ C P R V E S S MP E DR K S S T P G S L H E E T P - - - - - - - - - - - - - - - -R E F L G E A DMP V T R E G P L S Q P C R A S G P H S R S H V DK L K R P MT Q R GMP E GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P P S P CQ L S Q A E H P VQ K E G K G P T S R F L E T P N S R - - - - - - - - - - - - - - -Y E D I R D S S L Y V G G - - - - - - - - - - - - - - - - - - - - - - A S N V N DR R L S D F N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V K T N S T Q F P Y N P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -R Y R D T G DR A G Y DQ S S R MV V A N R N L P V DP D E E EQWMR S G G P S N R S DR R N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P H L R E P M L V A R G A A L A L A GM S S E A Y E G T Y R P V G - - - - - - - - - - - - - -L P P E A N A I N Y DN R N R G I L L H P Y N N V Y A P N G A L P G H E R M I Q S T P A S P Y D- - - - - - - - - - - - - - - - - - - - - - - - - - - Q R R L P T S S DMN G L A E S L I G G V L A A E G L G K Y C D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N

V R P V S L MV P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N - G A L L P F V N C R DA GQ DR A G G E E DA G C V R A R G R - P S E E E L Q D S R V Y V S S L - - - - - - - - - - - - - - - -A R P V S L T V P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N - G T L L P F V N C R DP GQ DR A V V P E D E S C A Y A L G R G R S E E A L A D S R S Y V S N L - - - - - - - - - - - - - - - -Q R P V S L T V P P V T R R D S I S L A H G S A G S L V E A V L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N - G N L L P F I Q C R DT G S Q E S R C S L S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L- T F T C L H V P G T H S - DP S H G K R G S A D S L V E A V L I S E G L G L F A R DP R F V A L A KQ E I A DA C R L T L D EMDN A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q G T S S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -- T F T C L Q V P G A H P - N P S H R K R G S A D S L V E A V L I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G N V R P R A N - G DV G P L S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R D E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N - G DMG P I S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R E E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -X S P V H L Q V P P E Y R - NQ Y L Q K R G S A T S L V E A V L I S E G L G R Y A K DP K F V A A X K H E I A DA C EMT I D EM E S A A S H X L N G G I T P V V N G V N V F P I L G H R E Y E L Q DV S A S Y S D E E P E P E P R P R Y E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -- - - - - - - H S R S T R E N T S R C S A P A T A L L I Q K A L V R G G L G T L A A DA N F I MA T GQ A L A DA CQM E P E E V E I MA T E L L K G - - - - - - - - - - - - - - - - - - - - - - R E A P E GMA S S L G C L N L G S S L G S L DQ HQ G S Q E T L I P P R L - - - - - - - - - - - -- - - - - N F E E H V P R N S A H R C T A P A T AM L I Q E A L V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L KQ - - - - - - - - - - - - - - - - - - - - - - E S P E G G A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - C E E S K NQ R S A A E A S P A T DK L I QQ A L R DG G L E S L A E DP Q F V S V T R K E L A E A V N I G L Q D I E S V AQ G I V N G - - - - - - - - - - - - - - - - - - - - - - Q S G K V T K R K R R P I P V P P S K T K E A T S A V - - - - - - - - - - - - - - - - - - - - - - -- - - E G K S V R L P F S S R P V L R P A E D S R P V DR L I GQ S L G L G R Y A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L A P L MQ H V GMH - - - - - - - - - D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G S Q E D L L L V T T L - - - - - - - - - - - - - - - -G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN R L R R V A T V T T T N N N N K S Q V S Q - - - - - - - - N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S - - - - - - - - - - - - - - - -

V E

E DE DE DY EY E

I Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N

C

Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster

Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster

Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster

IQ

Leucine Zipper

A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster

I Q E Y F R K F K K R K EQ G L V G K - P S Q R - N A L S L Q A G L R T L - H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V T Y Y Q S D S R G N F P Q T F A T Q R P L H I N K T G N NQ A - DT E S P S H E K L V D S - - - T F T P S S Y S S - - - - -I Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L - H D I G P E I R R A I S C D L Q DD E P E D- - - - - - - S K P E E E DV F K R N G A L L G N H V N H V N S DR R D S L QQ T N T T H R P L H VQ R P S MP P A S DT E K P - - - - - - - - - - - L F P P A G N S G C H N H HI Q DY F R K F R R R K E K G L L G R E A P T S - T S S A L Q A G L R S L - Q D L G P E I R Q A L T Y DT E E E E E - - - - - - - - - - - - E E E A V GQ E A E E E E A E N N P E P Y K D S I D S Q P Q S R WN S R I - - - - - - - - - - - - S V S L P V K E K L - - - - - - - - P D S L S T G P S DDDI Q E H F R K F MK R Q E E - Y Y G Y R P - K K - DT VQ I Q A G L R T I E E E A A P E I H R A I S G D L T A E E E L E R AM- - - V E A AM E E G I F R R T G G L F GQ V DN F L E R T N - - S L P P VMA NQ R P L Q F A E I EM E - - - E L E S P V F L E D F P Q N P G T H P L A R A N T - - - - -I Q DY F R R F K K R K EM E A K G V L P AQ T P Q AMA L Q A G L R T L - H E I G P E L K R A I S G N L E T D F N F D - - - - - - - - - - E P E P Q H R R P H S L F N N L V H R L S G A G - - - - S K S P T E H E R I E K G S T L L P F Q P R S F S P - - - - - - - - - - - T H S L A G A E G - - - S PI Q DY F R R F K K R K EQ E G K E G H P - D S - N T V T L Q A G L R T L - H E V S P A L K R A I S G N L - - - D E L DQ - - - - - - - - - E P E P MH R R H H T L F G S VW S S I R R H G N G T F R R S A K A T A S - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q S N G A L A I G - - - - -

- - - - - - - - - - T G S N A N I N N A N N T A L G R F P H P A - - - - - G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E S Q G A T V - - - - - - - - - - - - - - - - - - - - - - - - NQ E I F P D E T R S V R M S E E A E Y C S E P S L L S T DM F S Y Q E D E H R Q L T CN H N S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LG L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DNQ N - - - - - - - - - - - - - - - - - - - - - - - - - - - E EQ E V P DWT P D- - L D EQ A G T P S N P V L L P P HW S QQ H V N G H H - - - -- - - - - - - - - - N N A N A N V A Y G N S S H R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N N P V F S S I C Y E - - - - - - - - - - - - - - - - - - - - - R E F LV P S QMH R G A P I NQ S I N L P P V N G S A R R L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A L P P Y A N H I H D E T DDG P R Y R D T G DR A G Y DQ S S R MV V- - - - - - - - - - G S A S A A L G V G G S S L V L G S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S DP A G G DY L Y DT L N R S V - - - - - - - A DG V N N I T R N I M

P E E DK R - - - E I Q P S P K R S F L R S A S L G - R R A S F H L E C L K R Q K DQ G G - - - - - D I S Q K T A L P L H L V Q AL A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RE DDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQ Q QI M A V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S S WY T D E P D I S - - - - - Y R T- - - - - - - - - - - - - V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D- - - - - - - - - - - - L P I P G T Y H R - - - - - - - - - - - G R T S G P S R A QG S WA A P P - - - - Q K G R - L L Y A P L L L V E E S T V G E G Y L G K L G G P - - - - - - - - - - - - - - - - - - L R TG E A DMP - - - V T R E G P L S Q P C R A S G P H - S R S - - H V DK L K R P MT Q R G - - - - - - - - - - - - - - - - - - - - - - - MP E GQ V P - - - - - - - P S P C QL S Q A E H P VQ K E G K G - - - - - - P T S R F L E T P N S R N F E E H V P - - - - - - - - - - - - - - - - - - - - - - -A NRNL P - - - - V DP D E E E QWMR S G G P S - N R S DR R N P H L R E - - - - - - - - - - - - - - - - - - - P M L V A R G A A L A L A GM- - - - - - - - - - S S E A Y E G T Y R P V G E G K S V R - - - - - - - L P F S S R P V L R P A E D S R P - - - - - - - - - - - - - - - - - - - - - - -

Q A R L A A - - - - A G K L Q D E L Q G A G S G G E - L R T F G E S I S MR - - - - - - - - - - - - - - - - - - - - P L A K N G G G A A T V A G T L P P E A N A I N Y DN R N R G I L L H P Y N N VY A P N G A L P G H E R M I Q S T P A S P Y DQ R R L P - - - - - - - - - - - - - - - - - - - - - - -

A R P V S L T V P S Q A G A P G R Q F H G S A S S - - - L V I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T - - - - - - - - - - - - - - - - - - - - L L P F V N C R DP G - - - Q DR A V V P E D E S C A Y A L G R G R - S E E A L A D SF T P A S L T V P S S F R N K N S DKQ R S A D S - - - L V I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N G D- - - - - - - - - - - - - - - - - - - - MG P I S H R Q DY E L - - Q D F G P G Y S D E E - - - - P DP G R - E E E D L A D EF T - - C L Q V P G A H P N P S H R K R G S A D S - - - L V I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E - - - - S I L S R F D E E D L G D E- - - - - - - - - - - - - - R N S A H R C T A P A T AM L I V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L K - - - Q E S P E G G - - - - - - - - - - - - - - - - - - - - A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - V DR - - - L I GQ S L - - - G A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L - - - - - - - A P L MQ - - - - - - - - - - - - - - - - - - - - H V GMH D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G - S Q E D- - - -- - - - - - - - - - - - - - T S S DMN G L A E S - - - L I A E G L G K Y C - D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN

R S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y V S N LM I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C I T T LMA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C V H A L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L V T T LR L R R V A T V T T T N N N N K S Q V S Q N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S

A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster

A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster

A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster

A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster

E

G

E

A V LE A V LE A V LQ A L

GL R YG V A A

HH

CAGGCATTGGCAGTGGCAGGCTTGAGCCCCCTCCTGCAGAGAAG

CCATTCTCCTACCACATTCCCCAGGCCGTGCCCCACACCCCCTG

TCACTCCAGGCAGCCGGGGCAGACCCCTACGGCCCATCCCTACC

CTACGGCTGGAGGGGGCAGAGTCCAGCGAGAAACTCAACAGCAG

CTTCCCATCCATCCACTGCAGCTCCTGGTCTGAGGAGACGACAG

CCTGTAGTGGGAGCAGCAGC

1

45

91

136

180

225

CCATT Box

Sp1/GC Box SRY

GAPGRQFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQELADACDMTIEEMGAPGRQFHGSASSLVEAVLISEGLGQFAQDPKFAEVTTQEAADACDMTIEEM

GAPGRQFHGSASSLVEAVLASEGLGQAAQDPKFAEVTTQEAADACDMTIEEM

0

1

2

3

4

5

6

7

8

UA

S T

ran

scri

pti

on

(F

ire

fly/

Re

nil

la)

C C A T ∆T A

C C A T I F I L

C C A T A A I L

C C A TA A A A

A B

D

140

Transcript variant

Tissue 5’RACE# of TSS

5’RACE StartSite*

CAGEDatabase# of tags$

CAGE StartSite

PredictedProtein Size

1abrain, heart,

smooth muscle,liver, lung

NT NT 76Ch6:119,058,607-

119,058,62613240KDa

1b brain NT NT 53Ch6:119,036,852-

119,036,871240KDa

2a thalamus 2Ch6:118,604,374-

118,604,396Not found 110KDa

2b liver, lung, brain Not found 87Ch6:118,588,084-

118,588,103? /95KDa

3 thalamus, cortex 6Ch6:118,552,082-

118,552,1031

Ch6:118,552,184-118,5

52,205NC

4 thalamus. cortex 2Ch6:

118,545,992-118,546,013

1Ch6:

118,546,069-118,546,090

15KDa

Table 1. Summary of Transcriptional Start Sites and Nearby CAGE TagsChromosomal addresses for experimental TSS are given as the corresponding location in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding).

141

142

FIGULE LEGENDS

Figure 1. CCAT is Not Generated by Proteolytic Cleavage of Exogenously

Expressed or Endogenous Cav1.2 Channels.

(A) Schematic representation of the Cav1.2 -Gal4 fusion and channel mutants. Four

mutations are depicted: deletion from the TM to IQ motif renders the channel unable to

traffic to he membrane, deletion of conserved cleavage site for Cav1.1, a translational

stop at 1910 a.a. and deletion of TA.

(B) Western blot of N2As expressing Cav1.2 -Gal4 channels depicted in A (upper panel)

and Gal4-tagged C-terminal fragments (bottom panel) probed with an antibody to Gal4.

(C) Reporter gene activity of N2As expressing a UAS-luciferase reporter plasmid along

with Cav1.2 - Gal4 channels depicted in A or Gal4 alone as a control. Cells were co-

transfected with a Renilla luciferase construct driven by the thymidine kinase promoter

to control for cell number and transfection efficiency. Results are given as a ratio of

Firefly to Renilla luciferase activity. (Means ± SD; * < 0.0001 vs. Gal4).

(D) Western blot of N2As expressing WT, Methionine 2011 to Isoleucine and

Methionine 2078 to Isoleucine Cav1.2 -Gal4 channels (Upper Panel). Bottom panel

shows Gal4-tagged C-terminal fragments. Proteins were detected with a Gal4 antibody.

Large molecular protein in channel western represents unsolubilized, multimeric

channel proteins.

(E) Luciferase activity of N2As expressing either Gal4 alone, WT, M2011I and M2078

Gal4 tagged channels. (Means ± SD; * < 0.0001 vs. Gal4).

143

(F) Western blot analysis of membrane fractions obtained from 11.5 dpc heterozygous

(N/+) or homozygous (N/N) Cav1.2 knockout embryos probed with the anti-CCAT

antibody. Bottom panel shows loading control.

(G) Immunohistochemistry of 11.5 dpc Cav1.2 Null embryos reveals strong nuclear

staining with the anti-CCAT antibody (red) in the developing somites. Nuclei are shown

in blue.

Figure 2. CCAT is Translated from a Separate Transcript Driven by an Exonic

Promoter

(A) Northern blot analysis of mRNA extracted from N2A cells expressing Cav1.2 -gal4

channel constructs with or without CMV promoter. The first lane contains mRNA

extracted from untransfected cells. The membranes were hybridized with a

radioactively labeled RNA probe to Exon 47 of the channel.

(B) Western blot of N2As expressing Cav1.2 -gal4 channel constructs with or without

CMV promoter. Upper Panel shows full-length channels. Bottom panels shows Gal4

tagged CCAT. Membranes were immunoblotted with a Gal4 antibody.

(C) UAS reporter activity of N2As expressing Cav1.2 -gal4 channel constructs with or

without CMV promoter. (Means ± SD; * < 0.0001 vs. Gal4).

(D) Schematic representation of Firefly reporter constructs design to map the region

within the coding sequence of the channel responsible for the promoter activity. Full-

length construct has the complete sequence of the channel in place of any upstream

promoter. Ion pore construct includes all the transmembrane domains up to the IQ

motif. E46-47 contains exon 46 and 47 upstream of luciferase.

144

(E) Luciferase activity as a surrogate measure of luciferase expression levels from

constructs depicted in D. Graph compares expression level in N2As transfected with

either full-length, Ion pore, E46-47, E46 or E47 constructs. (Means ± SD).

(F) Schematic representation of the minigenes. A 4 Kb genomic segment containing the

last two exons and introns of CACNA1C was fused to the CDS of Gal4. 4Kb-0 is the

wild type minigene. 4Kb-1 is a negative control where a G has been inserted between

exon47 and Gal4. ∆238bp is the 4K-0 minigene where 238bp identified in E was

removed. M2011I is the 4Kb-0 where Met 2011 has been mutated to isoleucine.

(G) Mean luciferase activity (± SD) in N2A expressing 4Kb-0, 4Kb-1, ∆238bp and

M2011I Gal4 minigenes along with a UAS-luciferase reporter. (* < 0.0001 vs. 4Kb-0).

(H) Mean luciferase activity (± SD) in N2A expressing minigenes where either all or

two of three possible methionines were mutated. 4Kb-0 is the wild type sequence.

4Kb∆M has all three methionines mutated to isoleucines. M2011I has only this

methionine intact where M2073 and M2078 have been mutated to isoleucines. M2073I

and M2078I have only these respective methionine intact.

Figure 3. CCAT is Translated from a Separate Transcript whose Expression is

Cell-type and Developmentally Regulated In Vivo

(A) and (B) Northern blot analysis of mRNA extracted from Cortex, Midbrain and

cerebellum from E18, P1 and Adult rats. The membranes were hybridized with a

radioactively labeled DNA probe to Exon 47 of the channel (A) or to domain III

transmembrane S3 of the channel. Bottom panel shows the same membrane labeled

with an DNA probe to the 18S ribosomal RNA as loading control.

145

(C) Graphs showing the normalized expression of the full-length, 4.0Kb and 2.2Kb

band in the cortex, midbrain and cerebellum at 3 developmental stages: E18, P1 and

Adult. Each line represents an independent experiment. The northern blot shown in A

corresponds to the blue tracing. Signals were normalized to the 18S RNA signal.

(D) Imunohistochemistry of E18 coronal and sagital brain sections labeled with rabbit

anti-CCAT (red) and mouse Map2 antibodies (green). Secondary antibodies were

conjugated to Alexa fluorescent dyes. C (developing cortex), V (ventricle), S

(striatum), Th (thalamus), BS (brain stem), Cb (cerebellum).

(E) Imunohistochemistry of E18, P1 and 3-week-old rat cerebellum and thalamus

showing developmental variation in the amount and distribution of CCAT nuclear

staining. Anti-CCAT is shown in red and nuclei in blue.

(F) Immunocytochemistry of cortical and thalamic neuron grown 5 days in vitro stained

with anti-CCAT. Transcriptional assays of cortical (top) and thalamic (bottom)

neuronal cultures transfected with the UAS-luciferase reporter along with the Gal4

tagged channels described in Figure 1. Bars represent normalized transcription to Gal4

alone. (Means ± SD; * < 0.005 and ** <0.0001 vs. M2011I-Gal4).

Figure 4. CACNA1C has Multiple Transcriptional Start Sites Predicting Multiple

Proteins Including CCAT

(A) Schematic representation of the 5’RACE approach to determine the TSS for the

CCAT transcript generated from Cav1.2 -Gal4. Briefly, two sequential phosphatase

treatments are used to inactivate truncated or non-mRNAs and prepare intact, originally

capped mRNAs for ligation of an RNA oligo to the uncapped 5’ end. In this experiment

146

reverse transcription was performed with a Gal4 reverse primer. Nested primers within

the 5’ tag and the Gal4 coding sequence were used for PCR. The bands were then

cloned and sequenced.

(B) Agarose gel of PCR products amplified after performing 5’ RACE as described in A

of N2As expressing Cav1.2-Gal4 channels with and without CMV promoter. Predicted

PCR size band was 805 bp.

(C) Schematic representation of CACNA1C showing the location of TSS found and

depictions of the proteins predicted to be expressed from these transcripts.

(D) Epifluorescence images of N2A cells expressing YFP-mem-CCAT (left panels) or

YFP-CCAT (right panels).

(E) Western blot of nuclear extracts from 11.5 dpc Cav1.2 null and heterozygote

embryos probed with anti-CCAT.

Table 1. Summary of Transcriptional Start Sites and Nearby CAGE Tags

Chromosomal addresses for experimental TSS are given as the corresponding location

in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding).

Supplementary Figure 1

(A) Mean luciferase expression in N2A cells transfected with the UAS-luciferase

reporter and either Gal4 alone or Cav1.2-Gal4 channels. Channel expressing cells were

treated with 10µM Brefeldin A for 3-6 h.

(B) Schematic of the Cav1.2 knockout strategy.

147

(C) Southern blot showing the efficacy of recombination and the expected molecular

weight of the BamHI digested genomic fragments after recombination.

(D) Immunohistochemistry of heterozygote 11.5 dpc embryos stained with anti CCAT

antibody. Membranous staining is seen in the developing cortex and heart muscle wall

(HM). Staining is noticeably nuclear in somites and mesenchymal cells (M). CCAT

staining of the liver is not detected.

Supplementary Figure 2

(A) Normalized luciferase activity of N2As expressing empty pgl4 vector or constructs

containing 4Kb of genomic sequence upstream of the neuronal Cav1.2 channel

transcript starting AUG or upstream of M2011 AUG in the 3’ end of the gene. Cells

were co-transfected with Renilla luciferase construct driven by the thymidine kinase

promoter to control for cell number and transfection efficiency. Results are given as a

ratio of Firefly to Renilla luciferase activity. (Means ± SD; * < 0.005 vs. empty vector).

(B) Schematic representation of the alignment of the c-termini of Cav1.2 channels from

various species. Color was assigned based on similarity, darker colors being identical.

The exon-exon boundaries are depicted by double arrows.

(C) Alignment of the last two exons of Cav1.2 channels from various species. Possible

initiator methionines are labeled in red.

Supplementary Figure 3

(A) Northern blot analysis of mRNA extracted from Cortex, Midbrain and cerebellum

from E18, P1 and Adult rats. The membranes were hybridized with a radioactively

148

labeled DNA probe to Domain III transmembrane S3 of the channel. Bottom panel

shows the same membrane labeled with a DNA probe to the 18S ribosomal RNA as

loading control.

(B) Imunohistochemistry of E18, P1 and 3 week old rat cortex showing mostly cell

body and dendritic staining of CCAT in the three developmental stages. Anti-CCAT is

shown in red and nuclei in blue.

(C) Nuclear CCAT is highly expressed in subpallial regions during early embryonic

neural development and is not found in all cells expressing Cav1.2.

Immunofluorescence staining with CCAT antibody green) in the developing mouse at

embryonic days 12.5, 14 and 18 and postnatal day 1 (P1) demonstrates strong nuclear

expression of CCAT in subpallial regions corresponding to the developing caudate and

putamen (striatum), in addition to cells of the rhomboid, dorsomedial, posterior and

midline nuclear groups of the thalamus, the ventral pallidum, and periventricular

hypothalamic nuclei. CCAT expression becomes progressively restricted over the

course of embryonic development and is abolished several weeks into postnatal life.

(D) Nuclear expression of CCAT is restricted to the striatum (lower panel), thalamus

and developing cerebellum by E18 and postnatal ages. In contrast, cortical neurons

display mainly cytoplasmic expression when stained with the Cav1.2 C-terminus

antibody (upper panel), indicating the presence of the full-length channel but little or no

CCAT expression.

Supplementary Figure 4

149

(A) A schematic of the 238 bp promoter sequence indicating the location of

transcription factor binding sites. Sites were searched using TFSearch, MatInspector

and rVista and only common binding sites are reported. Default thresholds were used in

all instances.

(B) Mean luciferase activity (± SD) in N2A expressing CCAT-Gal constructs along

with the UAS-luciferase reporter and TK-Renilla luciferase construct as control. CCAT-

∆TA serves as negative control. CCAT-IFIL corresponds to WT sequence. Sequences

are included to show the mutations used. CCAT lacking two of the conserved LZ

residues has <50% activity. Mutation of all four a.a to Alanines completely abrogates

transcriptional activity.

(C) Sequence alignment of the c-termini of L-type calcium family. Sequences for Cav1

channels from Zebrafish, Mouse and human were aligned to the ancestral C. elegans

and D. Melanogaster L-type channels. Colors represent similarity based on percentage

identity. Two regions of conservation are identified the IQ with extended a.a and the

modified Leucine Zipper domain.

(D) Sequence alignment focused on Mouse Cav1 channels and C. elegans and D.

Melanogaster L-type channels.

150

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