regulation of expression and activity of the bhlh-pas ......bacteria to humans (crews and fan 1999;...
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Regulation of Expression and Activity of
the bHLH-PAS Transcription Factor NPAS4
David Christopher Bersten
B.Sc. (Biomedical Science), Honours (Biochemistry)
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
Discipline of Biochemistry
School of Molecular and Biomedical Science
University of Adelaide, Australia
June 2014
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Contents
Abstract ................................................................................................................................................... 3
PhD Thesis Declaration ........................................................................................................................... 5
Acknowledgements ................................................................................................................................. 6
Publications ............................................................................................................................................. 8
Conference oral presentations ........................................................................................................... 9
Additional publications ....................................................................................................................... 9
Chapter 1: .............................................................................................................................................. 10
Introduction ...................................................................................................................................... 10
bHLH-PAS transcription factor structure and mechanism ............................................................ 10
bHLH-PAS dimerization, DNA binding and signal transduction mechanisms ............................... 16
bHLH-PAS transcription factor function ........................................................................................ 19
Neuronal PAS Factor 4 .................................................................................................................. 19
Drosophila Dysfusion .................................................................................................................... 20
NPAS4 Expression and Regulation ................................................................................................ 21
NPAS4–dependent Transcriptional Regulation ............................................................................. 24
NPAS4 Function ............................................................................................................................. 26
Chapter 2: Results ................................................................................................................................. 31
2.1 Elucidation of molecular mechanisms which restrict NPAS4 expression to the brain. .............. 31
2.2 Human variants in the NPAS4/ARNT2 transcriptional complex can disrupt function ................ 32
2.3 Generation and validation of Inducible and reversible systems for lentiviral and ES cell (Col1a1-RMCE) mediated manipulation of NPAS4 levels. ................................................................ 33
Chapter 3: Discussion ............................................................................................................................ 36
References............................................................................................................................................. 45
Appendix 1 .......................................................................................................................................... 103
Appendix 2 .......................................................................................................................................... 104
Appendix 3 .......................................................................................................................................... 105
Appendix 4 .......................................................................................................................................... 106
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Abstract
Development of the Central Nervous System (CNS) relies on complex transcriptional programs
to specify distinct neuronal areas/cell types, and guide the formation of neuronal networks.
Synaptic activity during post-natal brain development dictates the number and strength of
synapses as well as promoting neuronal cell survival through activation of transcriptional
programs. The establishment of synapses during this “critical period” of post-natal neuronal
development and the local rearrangement, fine tuning and maintenance of synaptic
connections into adulthood contributes to synaptic plasticity, memory, learning and cognitive
function, while dysfunction in these processes is thought to contribute to a number of
neuropsychiatric diseases. Studying transcription which underlies these events and disease
states has been technically challenging due to the lack of gain and loss of gene expression
systems to interrogate complex biological questions in primary neurons or the developing
nervous system of rodents. As a result, despite clinical and anatomical data, the molecular
mechanisms underlying neuropsychiatric disease or memory and learning remain poorly
understood.
The basic-Helix-Loop-Helix (bHLH) – Per/Arnt/Sim (PAS) (bHLH-PAS) homology domain
transcription factor Neuronal PAS factor 4 (NPAS4) is tightly coupled to neuron function by
homeostatically regulating neuronal activity via stimulating formation of inhibitory synapses.
NPAS4 expression is brain restricted and highly induced following neuronal depolarisation,
paradigms of learning, seizure or ischemia. NPAS4 null mice are prone to seizures,
hyperactivity, have defects in memory formation, social interaction, cognitive impairments, as
well as age related neurodegeneration.
This thesis shows that NPAS4 expression is highly restricted to the CNS, in particular the
cortex, by repressive activity of RE-1 Silencing Transcription Factor/Neuron-Restrictive Silencer
Factor (REST/NRSF) in non-neuronal cells and stem cells. In addition, we also provide evidence
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that microRNA-224 targets the NPAS4 3’UTR, which may contribute to regionalised NPAS4
expression in the brain. We identify human variants within NPAS4 and ARNT2 which disrupt
NPAS4 function, which may have implications for neuropsychiatric disease. Using structural
modelling and biochemical experiments we show that one of these variants disrupts
dimerisation, providing insight into bHLH-PAS dimerisation mechanisms.
We also describe a new system for knockdown and ectopic expression which is broadly
applicable for reliable, flexible and temporal control of gene expression to facilitate
investigating gene function. This system incorporates single gateway compatible vector
systems for lentiviral infection and Recombination Mediated Cassette Exchance (RMCE), the
latter targeting the Collagen 1a1 (Col1a1) locus in germline competent embryonic stem cells.
Using an optimised reverse tetracycline transactivator (rtTA) system with reduced background
expression and increased sensitivity to doxycycline, we have shown that we can rapidly
generate inducible overexpression and short hairpin RNA (shRNA) mediated knockdown cell
lines with homogenous, inducible expression.
The work encompassed within this thesis investigates the molecular mechanisms underlying
the restricted expression pattern of NPAS4, the contribution of human non-synonymous
variants to NPAS4/ARNT2 transcription factor function, and the development of flexible,
inducible and reversible gene expression systems for studying NPAS4 function in vitro and in
vivo.
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PhD Thesis Declaration
This thesis contains no material which has been accepted for the award of any other degree or
diploma in any university or other tertiary institution to David Christopher Bersten and, to the
best of my knowledge and belief, contains no material previously published or written by
another person, except where due reference has been made in the text. I give consent to this
copy of my thesis when deposited in the University Library, being made available for loan and
photocopying, subject to the provisions of the Copyright Act 1968. The author acknowledges
that copyright of published works contained within this thesis (as listed below*) resides with
the copyright holder(s) of those works. I also give permission for the digital version of my
thesis to be made available on the web, via the University’s digital research repository, the
Library catalogue, and also through web search engines, unless permission has been granted
by the University to restrict access for a period of time.
David Christopher Bersten
June 2014
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Acknowledgements
Without the support, patience, generosity, friendship and love of colleagues, mentors, friends,
and family the PhD journey would not have been as enjoyable or rewarding. For these people I
must acknowledge.
Ass. Prof. Murray Whitelaw, you are truly an inspiring scientist and educator. I am forever
indebted to you for nurturing and supporting my interest in science, and guiding my
development as an independent researcher. I aspire to be as purely principled in scientific
pursuit, as kind, as patient and as enthusiastic as you are.
Dr. Daniel Peet, you have been a fantastic co-supervisor and mentor for me over the years.
Your door has always open for scientific discussion or a beer on a Friday afternoon, the former
being invaluable to me.
The biochemistry discipline – I would like especially thank all the past and present researchers
and educators in the biochemistry discipline for creating and maintaining an environment of
excellence and scientific rigor within the discipline.
For all those who have helped be along the way, colleagues and friends thank you for all the
help and support. There are many to name but I would specifically like to thank Adrienne
Sullivan, Pete McCarthy, Jo Wright, James Hughes, Paul Thomas, Sandy Piltz, Stephen Bent,
and Andrew (Nan) Hao.
Jayne, the love and support you have given me is above and beyond, thank you for being so
understanding. I would also like to thank the McConachy’s and my siblings for their support
and patience.
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Lastly, I must acknowledge my parents Libby and Andrew. This was not possible without your
unfailing support and gentle guidance. Thank you for having confidence in me and allowing me
to pursue my interests.
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Publications
This thesis is based on the following publications and referred to in the text:
I. Bersten DC, Sullivan AE, Peet DJ, Whitelaw ML. bHLH-PAS proteins in cancer. Nat Rev
Cancer. 2013 Dec;13(12):827-41.
II. Bersten DC, Wright JA, McCarthy PJ, Whitelaw ML. Regulation of the neuronal
transcription factor NPAS4 by REST and microRNAs. Biochim Biophys Acta. 2014
Jan;1839(1):13-24.
III. Bersten DC, Bruning JB, Peet DJ, Whitelaw ML. Human Variants in the Neuronal Basic
Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex
NPAS4/ARNT2 Disrupt Function. PLOS one. 2014 Jan, DOI:
10.1371/journal.pone.0085768
IV. Bersten DC, Sullivan AE, Bhakti V, Li D, Thomas PQ, Bent S, Whitelaw ML, Inducible
and reversible lentiviral and recombination mediated cassette exchange (RMCE)
systems for controlling gene expression. Manuscript submitted Nucleic Acids Research
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Conference oral presentations
REGULATION OF EXPRESSION AND ACTIVITY OF THE NEURONAL TRANSCRIPTION FACTOR
NPAS4
Bersten D.C., Peet D.J. and Whitelaw M.L. Australian Society of for Biochemistry and
Molecular Biology annual conference (ComBio 2012)
Additional publications
Bonnefond A, Raimondo A, Stutzmann F, Ghoussaini M, Ramachandrappa S, Bersten DC,
Durand E, Vatin V, Balkau B, Lantieri O, Raverdy V, Pattou F, Van Hul W, Van Gaal L, Peet DJ,
Weill J, Miller JL, Horber F, Goldstone AP, Driscoll DJ, Bruning JB, Meyre D, Whitelaw ML,
Froguel P.
Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features.
J Clin Invest. 2013 Jul 1;123(7):3037-41.
All publications have been reproduced with the permission from the copyright holders
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Chapter 1:
Introduction
NPAS4 is a member of the basic-Helix-Loop-Helix (bHLH) – Per/Arnt/Sim (PAS) (bHLH-PAS)
transcription factor family which is almost exclusively expressed within the central nervous
system (CNS) and is tightly regulated by neuronal activity. Like other members of this family it
must heterodimerise with a class II bHLH-PAS transcription factor (Aryl hydrocarbon receptor
nuclear translocator (Arnt) or Arnt2) to bind DNA and activate transcription. The heterodimer
binds to asymmetric E-BOX like elements (NNCGTG) within the promoters and enhancers of
target genes to modulate gene expression. The function of NPAS4 is primarily to
homeostatically regulate the activity state of excitatory neurons to keep a balance between
excitatory and inhibitory inputs within the neuron.
bHLH-PAS transcription factor structure and mechanism
bHLH-PAS proteins are a conserved family of proteins which contain two major functional
domain structures, the bHLH domain and the PAS domain (or PAS repeats)(Crews 1998; Yun,
Maecker et al. 2002; Kewley, Whitelaw et al. 2004; McIntosh, Hogenesch et al. 2010).
Transcription factors containing basic Helix-Loop-Helix (bHLH) motifs contribute to gene
expression profiles critical for many development processes such as myogenesis,
haematopoiesis, neurogenesis, heart and pancreatic development (Massari and Murre 2000).
This motif mediates dimerisation and DNA binding to a degenerate E-box CANNTG to regulate
transcription (Blackwell, Kretzner et al. 1990; Blackwell and Weintraub 1990; Baxevanis and
Vinson 1993). The N-terminal basic region of this motif contacts bases in the major groove of
DNA, while the HLH domain primarily contributes to dimmer stability (Ma, Rould et al. 1994).
The interaction between the two amphipathic helices of two proteins allows the formation
of homo- or heterodimers, which pairs the basic regions to facilitate binding to their cognate
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half sites (Ferre-D'Amare, Prendergast et al. 1993; Ma, Rould et al. 1994). Over 240 HLH
proteins have been described in almost all eukaryotes ranging from Saccharomyces cerevisiae
to Caenorhabditis elegans, Drosophila melanogaster to mammals (Atchley and Fitch 1997;
Massari and Murre 2000).
There are two sub-families of bHLH transcription factors which utilize secondary dimerisation
domains adjacent to the bHLH domain; these are the bHLH leucine zipper (Zip) transcription
factor family and the bHLH-Per/Arnt/Sim (bHLH-PAS) homology domain family of
transcriptional regulators (Massari and Murre 2000). The bHLH-PAS proteins are characterized
by two N-terminal PAS domains denoted PAS A and PAS B. The PAS domain or the PAS repeats
are also ancient sensor and protein interaction domain structures found in organisms from
bacteria to humans (Crews and Fan 1999; McIntosh, Hogenesch et al. 2010; Henry and Crosson
2011). The PAS domain was initially defined as a ~275 amino acid motif with shared sequence
homology between the Drosophila melanogaster clock protein Period (Per), the midline
expressed neurogenesis factor Single Minded (Sim) and vertebrate ARNT (Reddy, Zehring et al.
1984; Crews, Thomas et al. 1988; Hoffman, Reyes et al. 1991). However, recent structural
insights have indicated that isolated PAS domains encompass an ~110 amino acid generic PAS
fold which is structurally highly conserved but poorly conserved in amino acid sequence
(Figure 1)(Huang, Chelliah et al. 2012; Bersten, Sullivan et al. 2013).
The PAS domains are present in thousands of proteins and are most highly represented in
bacterial proteins (Moglich, Ayers et al. 2009). A general mechanism of PAS domain sensing
coupled to transduction effector output domains, such as protein kinase, catalytic,
dimerisation, DNA binding or ion channel domains, has been proposed for PAS containing
proteins. Indeed PAS domain containing proteins are involved in sensing and responding to
light, voltage, oxygen, xenobiotics, redox potential and biological time keeping (McIntosh,
Hogenesch et al. 2010; Henry and Crosson 2011). A classic signal sensing mechanism exists in
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Photoactive Yellow Protein (PYP), providing an example of a blue light sensing PAS protein
which acts negatively on phototaxis (Genick, Borgstahl et al. 1997; Henry and Crosson 2011).
In contrast to prokaryotes, few ligands or stimuli directly targeting the PAS domains have been
described for the mammalian PAS containing proteins. The best example lies with planar
aromatic hydrocarbons and dioxins, ligands which have been shown to directly bind to the PAS
B repeat in the Aryl hydrocarbon Receptor (AhR), thereby stimulating translocation to the
nucleus and activation of genes encoding xenobiotic metabolising enzymes (Whitelaw,
Gottlicher et al. 1993; Antonsson, Whitelaw et al. 1995; Whitelaw, McGuire et al. 1995). It has
also been reported that the NPAS2 PAS A domain can bind heme and carbon monoxide,
resulting in inhibition of an interaction with its bHLH/PAS partner, BMAL1 (Dioum, Rutter et al.
2002). Per2 has also been shown to bind heme, which mediates degradation of the protein.
As Per is an inhibitor of CLOCK/BMAL1, reduced Per levels result in increased CLOCK/BMAL1
dimer formation and function (Yang, Kim et al. 2008). CLOCK and NPAS2 have overlapping
function and can both bind nicotinamide adenine dinucleotide (NAD), which inhibits DNA
binding in vitro (Rutter, Reick et al. 2001). Recent structural resolution of the PAS B domain of
the hypoxia inducible factor HIF2 reveal its ability to bind small synthetic ligands and X-ray
crystallography of the bHLH-PAS A-PAS B regions of the CLOCK-BMAL1 dimer show a CLOCK
cavity which could potentially be targeted by small molecules (Scheuermann, Tomchick et al.
2009; Huang, Chelliah et al. 2012). While ligands or stimuli that directly act on the PAS
domains have not been described for most bHLH-PAS proteins there is generally a signal
regulated role for most PAS containing proteins.
Across all phyla, the PAS domain is frequently used as a protein-protein interaction interface
and ligand binding domain and forms a conserved structural module despite poor primary
sequence homology (Taylor and Zhulin 1999; Moglich, Ayers et al. 2009). Conformity of the
PAS domain structure has been elucidated over the last decade, mostly from crystallisation of
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bacterial PAS containing proteins. The overall PAS fold and structure comprises five central
stranded anti-parallel -sheets (denoted A , B , G , H and I ) which are flanked by alpha
helices (denoted C , D , E , and F ) and connected by flexible linkers ( Figure 1). This forms
the PAS core or PAS fold which presents an ideal pocket for binding natural or synthetic
ligands (exploited by antagonist discovery for HIF2 (Scheuermann, Tomchick et al. 2009)).
When dual PAS repeats are present they are usually separated by a variable, unstructured
linker domain. Initially overlaying the structures from the Light, Oxygen and Voltages (LOV)
PAS domain containing sensors PYP, FixL and HERG demonstrated the conservation of the
overall PAS fold structure. As additional structures have been solved the overall conservation
of the PAS fold has held true even between kingdoms of life. However, the notion of signal
regulated domains and specificity of protein-protein interactions between PAS domains or PAS
domain contain proteins suggests a significant degree of conformation flexibility or surface
specific interactions. Recently the concept of surface specific interactions which confer
specificity has been described for the ARNT/AhR transcriptional complex in a screen for PAS A
domain residues critical for dimerisation (Hao, Whitelaw et al. 2011). Furthermore, recent
structures of the bHLH-PAS (PAS A and PAS B) of CLOCK and BMAL1 support the notion of
conserved PAS fold structure but divergent conformation. For example, while PAS A domains
of CLOCK and BMAL interact through reciprocal -helical and -sheet interactions, which is
also observed for NifL and Per/CLOCK PAS interactions, the CLOCK/BMAL1 PAS B domain
interactions fundamentally differ from those seen in HIF2 /ARNT PASB interactions (Huang,
Chelliah et al. 2012).
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Figure 1 | Roles and structures of class I and class II bHLH–PAS family members. a | Examples of heterodimeric basic
HLH (helix-loop-helix)–PER–ARNT–SIM (bHLH–PAS) transcription factors are shown. Dimers are formed between a class I
factor and a class II factor (AHR nuclear translocator (ARNT), ARNT2, brain muscle ARNT-like 1 (BMAL)). A class I factor may
be tissue-restricted in expression (such as single-minded homologue (SIM) and neuronal PAS domain-containing protein
(NPAS)) or active in response to a stimulus (such as a ligand for aryl hydrocarbon receptor (AHR) and hypoxia for hypoxia-
inducible factor (HIF)). The heterodimers bind to class I-specific variations of the canonical E-box sequence and regulate
specific target genes, thereby mediating various developmental or homeostatic processes and/or responses to
environmental or physiological stresses. Class II factors are capable of forming dimers with more than one class I factor,
although some combinations may be limited in vivo; for example, ARNT2 expression is mostly restricted to neurons, and it
functions as the obligate partner in vivo for SIM1 and probably for NPAS4. HIF1 can form active dimers with either ARNT
or ARNT2 (REF. 37), but the HIF–ARNT dimer is more abundant overall owing to both proteins being ubiquitously
expressed. Conversely, ligand-bound AHR can dimerize with either ARNT or ARNT2 in vitro, but only AHR–ARNT dimers can
activate target genes171. b | The HLH and PAS folds constitute the interface of dimerization between class I and class II
bHLH–PAS factors. Comparison of the limited available structural information indicates that the tertiary structure of the
PAS fold is well conserved. However, this does not preclude different orientations and surfaces from being involved in
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PAS–PAS interactions throughout the family. The PASB regions of BMAL1 and circadian locomotor output cycles kaput
(CLOCK) associate in roughly parallel orientations, whereas isolated PASB folds of HIF2 and ARNT show antiparallel
orientation and a -sheet interface8 65. These different interfaces may contribute to the specificity of binding and partner
selection, and they present different surfaces for specific interactions with accessory signalling proteins or transcription
co-regulators. The period proteins, which lack a bHLH, show promiscuous binding with other family members and, similar
to ARNT, can homodimerize172. Nuclear receptor co-activator 1 (NCOA1) is one of a small class of co-activator proteins that
contain typical PAS folds but that do not seem to function as obligate heterodimers. Structures shown: HIF2 PASB, ARNT
PASB (Protein Data Bank (PDB) ID: 4GHI); CLOCK PASB, BMAL1 PASB (PDB ID: 4F3L); PER2 PASB (PDB ID: 3GDI); NCOA1
PASB (PDB ID: 1OJ5). ARC, activity-regulated cytoskeleton-associated protein; BDNF, brain-derived neurotrophic factor;
CME, central midline element; CRY, cryptochrome; CYP1A1, cytochrome P4501A1; EGR1, early growth response 1; EPO,
erythropoietin; GLUT1, glucose transporter 1; GST, glutathione S-transferase; HRE, hypoxia response element; PER,
period circadian protein homologue; ROR, retinoic acid-related orphan receptor; VEGF, vascular endothelial growth factor;
XRE, xenobiotic response element.
Reproduced from Paper I Bersten et al, Nat Rev Cancer. 2013.
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bHLH-PAS dimerization, DNA binding and signal transduction mechanisms
The bHLH-PAS class of proteins can be generally separated into transcription factors or
coactivator proteins which contain a shared domain structure (Kewley, Whitelaw et al. 2004;
Partch and Gardner 2010). bHLH-PAS proteins are characterised by an invariant N-terminal
bHLH domain which is involved in primary dimerisation and DNA binding (Reisz-Porszasz,
Probst et al. 1994; Fukunaga, Probst et al. 1995) followed by a PAS domain which
encompasses two PAS repeats denoted PAS A and PAS B, and a PAC domain (Ponting and
Aravind 1997) or S2 boxes (Zhulin, Taylor et al. 1997) which are structurally associated with the
PAS domain (Hefti, Francoijs et al. 2004). The bHLH-PAS transcription factors are further
divided into class I or class II factors, to form functional transcriptional complexes (Figure 1).
Class I factors (AhR, HIF1-3 , Sim1-2, NPAS1-4, and CLOCK) must heterodimerise with class II
factors (ARNT, ARNT2, BMAL1, BMAL2) to enable DNA binding and transcriptional activation.
Unlike the bHLH and bHLH/Zip transcription factors which bind the classic CANNTG E-box, the
bHLH-PAS transcription factors utilize an atypical E-box which has variable 5’ sequence
(Massari and Murre 2000; Kewley, Whitelaw et al. 2004). It has been shown in vitro and in
cultured cells that class II factors such as ARNT can homodimerise to activate canonical E-box
elements, although the biological significance of this has not been addressed (Sogawa, Nakano
et al. 1995; Swanson and Yang 1999).
The PAS A domain of bHLH-PAS transcription factors also contributes to dimerisation strength
(Erbel, Card et al. 2003; Chapman-Smith, Lutwyche et al. 2004) as wells as mediating
dimerisation specificity between bHLH-PAS family members (Pongratz, Antonsson et al. 1998).
Interestingly, the PAS A domain has also been implicated in direct DNA contact and DNA
bending, downstream of the primary bHLH-major groove contact (Chapman-Smith and
Whitelaw 2006). While PAS B also provides a dimerisation interface between bHLH-PAS
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proteins (Card, Erbel et al. 2005; Huang, Chelliah et al. 2012), in selected cases it also directly
binds ligands (Whitelaw, Pongratz et al. 1993; Scheuermann, Tomchick et al. 2009),
coactivator proteins (Partch, Card et al. 2009; Partch and Gardner 2010; Partch and Gardner
2011) and chaperone proteins to control signal transduction (Pongratz, Mason et al. 1992;
Coumailleau, Poellinger et al. 1995).
Importantly, the PAS domains can also contribute to target gene specificity, for example, in
Drosophila the bHLH/PAS members Trachealess (Trh) and Single Minded (SIM) are able to bind
to the same DNA recognition sequence in collaboration with the obligate bHLH-PAS partner
factor, Tango (Tgo; equivalent to Aryl Hydrocarbon Nuclear Translocator (ARNT) in mammals).
While the DNA response elements of these two bHLH/PAS heterodimers are identical, target
gene induction can be switched by swapping the PAS domains between Trh and SIM,
suggesting that in this case target gene specificity is conferred by the PAS domain (Zelzer,
Wappner et al. 1997). How they achieve this is unclear, but may involve ancillary factors
recruited by the PAS domains or direct contact between the PAS domain and DNA elements
adjacent to the primary bHLH contact (Kimura, Weisz et al. 2001; Pearson, Watson et al. 2012).
This is somewhat supported by the ability of some bHLH-PAS transcription factors to bind to
the same DNA binding motifs yet have distinct target gene profiles or the capacity to bind to
several variants of the asymmetric E-box core (Swanson, Chan et al. 1995; Woods and
Whitelaw 2002; Ooe, Saito et al. 2004; Jiang and Crews 2007; Farrall and Whitelaw 2009;
Bersten, Sullivan et al. 2013).
The DNA binding specificity of bHLH-PAS transcription factors is conferred by the basic region
within the bHLH (Davis and Weintraub 1992). As with other bHLH containing transcription
factors, basic residues make direct contact with nucleotides to guide DNA binding specificity
(Ma, Rould et al. 1994). bHLH-PAS transcription factors utilising ARNT or ARNT2 bind to a
asymmetric E-Box like element where Class I and Class II factors contribute to three nucleotide
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half site specificity. For example AhR/ARNT heterodimers bind to a Xenoboitic Response
Element (XRE) (TNGCGTG) where the AhR binds to NGC half site and ARNT binds to GTG half
site. In this context the ARNT DNA binding specificity is fixed to GTG whereas the class I
partner protein sequence specificity can vary. Although ARNT2 can also bind to many of the
class I factors the DNA binding specificity appears to be the same as for ARNT. This is in
contrast to various heterodimers formed with either BMAL1 or BMAL2, or BMAL homodimers
or ARNT homodimers, all of which bind to canonical E-Box CACGTG (Hogenesch, Gu et al.
1998; Reppert and Weaver 2002).
Many of the class I bHLH-PAS transcription factors dimerise with a common class II factor,
which can create competition for that Class II factor within the cell (Chan, Yao et al. 1999;
Woods and Whitelaw 2002). In addition, overlapping DNA binding specificities can allow for
further competition between heterodimeric complexes (Woods and Whitelaw 2002; Farrall
and Whitelaw 2009). Transcriptionally inactive or DNA binding defective forms of Class I bHLH-
PAS transcription factors can also compete for Class II factors to regulate the output
transcriptional pathways (Mimura, Ema et al. 1999; Makino, Cao et al. 2001; Makino, Kanopka
et al. 2002). The extent to which cross talk occurs in vivo or in pathological scenarios is yet to
be explored, however it is expected that ARNT levels will be limiting, supporting the concept of
competition for ARNT by class I bHLH-PAS factors (Semenza, Jiang et al. 1996; Holmes and
Pollenz 1997).
While the bHLH-PAS transcription factors primarily act to modulate gene expression by direct
DNA binding, alternate functions for the bHLH-PAS transcription factors have also been
described. The most notable are the direct control of hypoxic induced translation by HIF2 by
incorporation into the translational machinery (Uniacke, Holterman et al. 2012) and the
regulation of ubiquitin ligase protein degradation mediated by ligand activated AhR (Ohtake,
Baba et al. 2007; Kawajiri, Kobayashi et al. 2009).
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bHLH-PAS transcription factor function
bHLH/PAS proteins are involved in a diverse array of physiological and pathological processes,
such as the cellular response to hypoxia (Hypoxia Inducible Factors
(HIF1 /HIF2 /HIF3 IPAS)), the maintenance of circadian rhythms (circadian locomotor
output cycles kaput (CLOCK), Period (PER1/PER2/PER3),Neuronal PAS domain protein 2
(NPAS2)), the response to environmental pollutants (Aryl hydrocarbon Receptor (AhR)/ Dioxin
receptor (DR), AhR repressor AhRR), neurogenesis and lung development (NPAS1, and NPAS3)
and appetite control (Single minded 1 (Sim1))(Reppert and Weaver 2002; Kewley, Whitelaw et
al. 2004; Semenza 2012).
In addition to canonical roles in normal physiology, emerging roles in T cell immunity, cancer
progression and metastasis, diabetes, and behaviour for bHLH-PAS transcription factors are
being discovered (Gunton, Kulkarni et al. 2005; Marcheva, Ramsey et al. 2010; Dang, Barbi et
al. 2011; Semenza 2012; Bersten, Sullivan et al. 2013; Hao and Whitelaw 2013). Normal and
cancer related functions of the bHLH-PAS transcription factors was reviewed recently in Paper
I (Bersten, Sullivan et al. 2013).
Neuronal PAS Factor 4
The mammalian NPAS4 gene (also known as Limbic enhanced-PAS (lePAS and NXF), was
discovered from screening a human fetal brain cDNA library with a PAS domain oligonuceotide
probe (Ooe et al, 2004). Human, rat and mouse NPAS4 were cloned and have significant
degree of homology in the bHLH and PAS domains to each other and to drosophila gene
Dysfusion (Dys), Zebrafish (Danio rerio) NPAS4 and the Caenorhabditis Elegans (C.Elegans)
gene C15C8.2 (Cky-1) (Jiang and Crews 2003; Ooe, Saito et al. 2004; Ooe, Saito et al. 2007;
Ooe, Saito et al. 2009; Baxendale, Holdsworth et al. 2012). While NPAS4 is clustered in the
bHLH-PAS transcription factor family phylogenetic analysis of mouse PAS domain containing
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proteins suggests it has little similarity to any other bHLH-PAS factors (Ooe, Saito et al. 2004;
McIntosh, Hogenesch et al. 2010). This is also observed in C.elegans and drosophila (Jiang and
Crews 2003; Ooe, Saito et al. 2007). Within the bHLH and PAS A domain NPAS4 is most similar
to AhR/SIM2/NPAS1 (43%, 33% and 33% amino acid identity respectively) and within in the
PAS B domain is most similar to HIF1 /PER/BMAL1 (29%/27%/24% amino acid identity).
While the C-terminal region of the mammalian NPAS4 members is highly conserved, there is
little or no sequence homology to either C.elegans (Cky-1), Drosphila (Dys), or any of the
mammalian bHLH-PAS C-termini.
Drosophila Dysfusion
Drosophila SIM and trachealess (Trh) control transcription and development in the CNS
midline cells and tracheal cells, respectively, and both appear to do so through a common DNA
regulatory element known as the Central Midline Element (CME; ACGTG)(Crews 1998). The
NPAS4 related gene Dys was discovered by screening for bHLH containing proteins in
drosophila (Ledent and Vervoort 2001; Peyrefitte, Kahn et al. 2001). Expression of Dys was
subsequently shown in embryonic tissues such as foregut precursors, CNS cells which are
either part of the medial brain or frontal ganglion, tracheal fusion cells, the epidermal leading
edge, the hind gut and the anal pad from stage 11-12 of development (Peyrefitte, Kahn et al.
2001; Jiang and Crews 2003). Dys is expressed prior to tracheal fusion events and RNAi
knockdown or loss of function (LoF) Dys mutants results in aberrant tracheal fusion (Jiang and
Crews 2003; Jiang and Crews 2006). Development of the tracheal system is critical to the
complete formation of the oxygen delivering tubular network (Ghabrial, Luschnig et al. 2003)
and as such, inhibition of tracheal fusion by loss of Dys results in a lethal phenotype (Jiang and
Crews, 2003). The tracheal fusion cells where Dys is expressed extend actin-rich filopodia,
which are guided by cues in a similar fashion to that of axon growth cone guidance (Tanaka-
Matakatsu, Uemura et al. 1996; Englund, Steneberg et al. 2002; Ribeiro, Ebner et al. 2002).
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Tracheal branches undergo systematic migration from adjacent hemi-segments until they are
in close enough contact for the fusion cells to adhere to each other. Over expression of Dys
throughout the trachea results in inhibition of migration and ectopic branch fusion, implicating
Dys in the migration and fusion of actin-rich filopodia of the Drosophila tracheal system (Jiang
and Crews 2006). Like other members of the Drosophila bHLH-PAS family, Dys appears to
heterodimerise with Tgo to regulate target genes, in this case shotgun, CG13196, and
members only, all of which contribute to cell adhesion or other aspects of tracheal fusion or
migration (Wilk, Weizman et al. 1996; Jiang and Crews 2003; Jiang and Crews 2006). However,
due to the low degree of sequence similarity between NPAS4 and Dys, especially in the C-
terminus, and the absence of any reported expression of NPAS4 in the lung, NPAS4 may have
diverged in function in mammals.
NPAS4 Expression and Regulation
NPAS4 expression is highly restricted to the brain in rodents and humans, with a several
reports of weak expression within the testes (Flood, Moyer et al. 2004; Moser, Knoth et al.
2004; Ooe, Saito et al. 2004; Ramamoorthi, Fropf et al. 2011). Within the CNS, NPAS4
expression appears to be further restricted to neurons within the hippocampus, cortex,
olfactory bulb, and amygdala with very low or no expression seen in other brain areas or the
spinal cord (Moser, Knoth et al. 2004; Lin, Bloodgood et al. 2008; Ramamoorthi, Fropf et al.
2011). Unlike NPAS3 or Sim2, NPAS4 does not appear to be expressed in the developing
mouse embryo. Appreciable levels begin to appear perinatally and increase as SIM2 gradually
decreases up to 4 weeks post natal (Brunskill, Witte et al. 1999; Ooe, Saito et al. 2004).
A common theme among bHLH-PAS transcription factors is signal or stress responsiveness, for
example with HIF activated in hypoxia, AhR responsive to xenobiotics and SIM1 responsive to
appetite signals. The NPAS4 gene is also a signal or stress responsive, as expression has been
shown to be upregulated by seizure (Flood, Moyer et al. 2004; Baxendale, Holdsworth et al.
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2012) ischemia (Shamloo, Soriano et al. 2006; Leong, Klaric et al. 2013), several
psychostimulants and opioid drugs (Piechota, Korostynski et al. 2010; Guo, Xue et al. 2012;
Martin, Jayanthi et al. 2012). In addition, physiological stimuli such as contextual fear
conditioning (CFC) (Ploski, Monsey et al. 2011; Ramamoorthi, Fropf et al. 2011), light
stimulation (Lin, Bloodgood et al. 2008; Maya-Vetencourt, Tiraboschi et al. 2012) or neuronal
depolarisation (Lin, Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011) induce expression
of NPAS4.
At a mechanistic level these stimuli are thought to act primarily through stimulation of voltage
gated calcium channels which initiates, calcium dependent gene expression. NPAS4 induction
can be blocked by calcium chelation, calcium channel blockade, NMDA receptor blockade or
AMPA receptor blockade but does not appear to be upregulated by neurotrophin signals (Lin,
Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011). Neuronal depolarisation results in an
extremely rapid (within 5 mins for CFC), robust and transient increase in NPAS4 mRNA and
protein through activation of VDCC channels in a protein synthesis independent manner
(Ramamoorthi, Fropf et al. 2011). NPAS4 induction following depolarisation peaks at 0.5-2 hrs
post depolarisation and is rapidly down regulated to near basal levels by 4-6hrs (Lin,
Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011).
Unlike other genes transiently stimulated by neuronal activity, NPAS4 protein is not activated
by neurotrophic factors, growth factors, or forskolin (a protein kinase A activator) making it
uniquely situated to respond only to learning and memory cues (Lin, Bloodgood et al. 2008;
Ramamoorthi, Fropf et al. 2011; Ebert and Greenberg 2013). While the molecular mechanisms
underlying both the restricted brain and activity-dependent expression of NPAS4 remain
unexplored, it is clear that the latter requires calcium dependent signalling events. Activation
of NPAS4 expression, which has been suggested to be mediated by calmodulin dependent
kinase IV (CaMK IV) and cAMP-response element binding protein (CREB), can be blocked by
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inhibition of nuclear calcium signalling (Greer and Greenberg 2008; Zhang, Zou et al. 2009). In
addition, it has also been suggested that PI3K and ERK/MAPK may also play a role in
upregulation of NPAS4 following NMDA receptor activation (Coba, Valor et al. 2008; Ooe,
Kobayashi et al. 2009). Activity dependent up regulation of NPAS4 is initially protein synthesis
independent and may involve other activity induced transcription factors such as CREB and
MEF2A/D (Flavell, Kim et al. 2008; Ramamoorthi, Fropf et al. 2011; Ebert and Greenberg
2013). Following the initial up regulation of NPAS4 following depolarisation, it’s proposed that
NPAS4 may strengthen its own expression through a feed forward pathway. Indeed, NPAS4
has been shown to bind and activate its own promoter, however the ramifications of this are
unknown (Ooe, Saito et al. 2004; Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010). While
there is cursory information about stimulus dependent activation, developmental regulation
of NPAS4 expression has not been investigated. Furthermore, it is not clear how NPAS4
protein and mRNA are down regulated following initial induction.
In other contexts an increase of intracellular calcium through endoplasmic reticulum stressors
can also induce NPAS4 expression (Ooe, Motonaga et al. 2009). Recently it has been shown
that pancreatic beta cells are capable of depolarisation induced NPAS4 expression (as
demonstrated by qRT-PCR and immunofluorescence experiments), indicating that NPAS4 may
act more broadly outside the nervous system as a calcium induced transcription factor in a
limited number of tissues (Sabatini, Krentz et al. 2013). However, the pervasiveness or
consequences of NPAS4 upregulation outside the nervous system or in response to ER stress
or calcium induction have yet to be fully elucidated.
Very little is known about the mechanisms which regulate NPAS4 expression, however
recently it has been shown that the NPAS4 promoter is methylated at several CpG sites and
mRNA expression may be repressed by this methylation (Rudenko, Dawlaty et al. 2013). Ten-
eleven translocation 1 (Tet1) is a dioxygenase enzyme which catalyses the addition of a
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hydroxy group onto methylated CpGs to mediate demethylation (Wu and Zhang 2011). It has
been proposed that demethylation may be important for regulation of activity inducible genes
and indeed knockout of Tet1 in mice leads to hypermethylated and suppressed NPAS4
expression in the hippocampus and cortex (Kaas, Zhong et al. 2013; Rudenko, Dawlaty et al.
2013). Interestingly, all bHLH-PAS transcription factor binding sites contain a CpG dinucleotide
between the Class I and Class II half sites which could potentially be methylated, however the
effect of non-methyl versus methyl-CpG bHLH-PAS response elements on binding of
heterodimers has not been explored.
NPAS4–dependent Transcriptional Regulation
NPAS4 is able to heterodimerise with the partner proteins ARNT and ARNT2 to bind to many
bHLH-PAS regulatory sequences by in vitro DNA binding assays and luciferase reporter
experiments (Ooe, Saito et al. 2004). Due to overlapping expression between NPAS4 and
ARNT2 within the CNS, and the suggestion that NPAS4 may interact more strongly with ARNT2
than ARNT, NPAS4 is anticipated to function in vivo with ARNT2 (Hirose, Morita et al. 1996;
Aitola and Pelto-Huikko 2003; Ooe, Saito et al. 2004; Ooe, Saito et al. 2009). bHLH-PAS
regulatory elements which NPAS4/ARNT2 have been shown to bind contain the core CME /
HRE (ACGTG) or the XRE (TNGCGTG) (Moser, Knoth et al. 2004; Ooe, Saito et al. 2004),
however the preferred DNA binding sequence of NPAS4/ARNT2 heterodimers are unknown.
Comparison of activities of NPAS4 on bHLH-PAS responsive reporters and in vitro DNA binding
suggests that NPAS4 binds most strongly to TCGTG and GCGTG containing sites (Ooe, Saito et
al. 2004). Analysis of NPAS4 Chromatin immunoprecipitation -sequencing (ChIP-Seq)
experiments in depolarised mouse cortical neurons, together with in vitro site selection
experiments to identify the preferred NPAS4/ARNT DNA binding motif, also supports this
notion (Kim, Hemberg et al. 2010)( D.C. Bersten, V. Bhakti, and M.L. Whitelaw unpublished
results).
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Initial functional experiments suggested that NPAS4 may control expression of the cytoskeletal
binding protein Drebrin and the apoptosis inducing gene Bax (Ooe, Saito et al. 2004; Hester,
McKee et al. 2007). However, examination of NPAS4 dependent target genes during neuronal
depolarisation suggest that these maybe either context dependent or artefacts of
overexpression in cell lines (Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010; Pruunsild,
Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). ChIP-seq and microarray analysis of NPAS4
bound and differentially regulated target genes following KCl-depolarisation has been
performed and reveals extensive occupancy of NPAS4 at promoters and enhancers to regulate
many activity dependent genes involved in inhibitory synapse development (Lin, Bloodgood et
al. 2008; Kim, Hemberg et al. 2010; Ramamoorthi, Fropf et al. 2011; Bloodgood, Sharma et al.
2013). NPAS4 occupancy at enhancer sites correlates well with CBP occupancy and supports
existing data suggesting CBP/p300 acts as an NPAS4 transactivator (Ooe, Saito et al. 2004; Kim,
Hemberg et al. 2010). A number of confirmed NPAS4 target genes have been examined
including BDNF, c-Fos, Egr1 and Arc (Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010;
Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). It is proposed that NPAS4
predominantly acts through enhancer recruitment of RNA Pol II to activate transcription of its
target genes (Kim, Hemberg et al. 2010; Ramamoorthi, Fropf et al. 2011). NPAS4 binds directly
to BDNF promoter I, promoter III and promoter IV to act as one of the main drivers of activity
induced expression of BDNF (Lin, Bloodgood et al. 2008; Pruunsild, Sepp et al. 2011;
Bloodgood, Sharma et al. 2013). Deletion of promoter IV NPAS4 response element
significantly weakens activity induced expression of BDNF although it does not alter the initial
responsiveness of BDNF to depolarisation (Pruunsild, Sepp et al. 2011). It has therefore been
proposed that NPAS4 acts to enhance or strengthen activity induced gene expression
(Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). In addition, it has been
suggested by several groups that NPAS4 may act directly on its own promoter to strengthen its
activation (Ooe, Saito et al. 2004; Lin, Bloodgood et al. 2008). Importantly, ARNT2 has also
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been shown to bind to BDNF promoters and regulate BDNF expression, although the extent of
its contribution to activity dependent or NPAS4 dependent gene expression remains
unexplored (Pruunsild, Sepp et al. 2011).
NPAS4 Function Activity-Dependent Transcription in the CNS
Neuronal activity plays key roles in neuron survival, proliferation, migration, axon/dendrite
guidance and growth, synapse formation, maintenance and elimination, and neurotransmitter
specification (Spitzer 2006; Greer and Greenberg 2008; Spitzer 2012). These processes guide
neuronal activity profiles in the prospective central nervous system and underlie the cellular
and molecular basis of memory and learning. In addition, genetic aberrations in neuronal
activity are thought to be critical to many neuropsychiatric disease states and may be the
underlying cause of autism and schizophrenia (Ramocki and Zoghbi 2008; Ebert and Greenberg
2013). Importantly, over excitation due to disruption of neuron homeostasis may also
underlie the pathogenesis of many neurodegenerative disorders such as dementia,
Alzheimer’s disease, stroke and seizure(Saxena and Caroni 2011).
Sensory experience drives synaptic plasticity and learning in animals. One seminal experiment
outlining this phenomena showed that ocular occlusion during critical periods around birth,
when neuron development and synapse formation occur, leads to cortical circuit
rearrangements to favour the non-occluded eye (Wiesel 1982). It is now well established that
sensory experience and neuronal activity drive synaptic plasticity during critical periods and
throughout adulthood. Electrical activity in neurons can take many forms, of which the best
established being activation of voltage-gated calcium channels leading to action potentials
(Greer and Greenberg 2008). During early stages of neuron development GABA and glycine
are able to generate depolarising chloride currents due to efflux of intracellular chloride ions
(Spitzer 2006). Later in neuron development this phenomena is reversed and leads to chloride
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influx and inhibition of neuronal activity. In addition, glutamate generates depolarisation via
activation of voltage gated calcium and sodium channels. Calcium influx through voltage
gated calcium channel triggers rapid activation of gene expression, which in turn acts to
modulate synapse formation and plasticity. Calcium activated gene expression in neurons is
mediated by a number of activity-dependent transcription factors including CREB, MEF2A/D,
USF1/2, NF B, MeCP2, SRF and NPAS4, many of which are regulators of synapse formation
and function (Greer and Greenberg 2008).
NPAS4 and synapse formation
Initial experiments in cultured hippocampal neurons showed that NPAS4 was able to regulate
the number and strength of inhibitory inputs on excitatory pyramidal neurons both
perisomatically and dendritically (Lin, Bloodgood et al. 2008). In contrast, NPAS4 seemed not
to have any effect on excitatory synapse number or dendritic patterning of the neurons (Lin,
Bloodgood et al. 2008). More recently, using conditional knockout NPAS4 mice where NPAS
has been sparsely ablated in CA1 hippocampal neurons, recording of inhibitory post synaptic
potentials in layers containing either dendrites or soma has revealed that NPAS4 has opposing
actions on inhibitory synapse formation in the two compartments, increasing inhibition within
the soma and decreasing inhibition in dendrites (Bloodgood, Sharma et al. 2013). The function
of compartment specific regulation of inhibitory synapses is as yet unclear, however, somatic
inhibition may be more effective at limiting action potential spikes than dendritic inhibition,
while increased activity in dendrites may aid more effective dendritic synapse plasticity
(Sylwestrak and Scheiffele 2013). The downstream mechanism(s) by which NPAS4 imparts
compartment specific neuron inhibition is also unclear. It has been shown that specific
transcripts of BDNF are regulated by NPAS4 binding and activation at promoter I, III and IV
(Lin, Bloodgood et al. 2008; Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011;
Bloodgood, Sharma et al. 2013). While BDNF has been shown to be involved in inhibitory
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synapse formation (Marty, Wehrle et al. 2000; Lin, Bloodgood et al. 2008) and some
transcripts can be can be targeted to different cellular compartments (Pattabiraman, Tropea
et al. 2005; An, Gharami et al. 2008; Dean, Liu et al. 2012), the contribution of NPAS4 to
regulating subcellular levels of BDNF through transcript specific regulation is yet to be
addressed.
NPAS4 in memory, learning and behaviour.
The selective and transient induction of NPAS4 following sensory experience and neuron
depolarisation prompted several groups to investigate the function NPAS4 in memory and
learning in mice (Ploski, Monsey et al. 2011; Ramamoorthi, Fropf et al. 2011; Coutellier, Beraki
et al. 2012; Maya-Vetencourt, Tiraboschi et al. 2012). In independent mouse knockout models
NPAS4 has been shown to be involved in contextual memory formation in adult mice. Either
global or conditional knockout of NPAS4 in the CA3 region of the hippocampus attenuated
long-term memory formation in mice, which is dependent on NPAS4 transcriptional activity
(Ramamoorthi, Fropf et al. 2011; Coutellier, Beraki et al. 2012). However, short-term memory
was affected in only in the global NPAS4 knockout mice (Ramamoorthi, Fropf et al. 2011).
Additional behavioural deficits such as increased aggression and hyperactivity are observed in
the NPAS4 null mice and decreased contact with unfamiliar mice are observed in animals
heterozygous null for NPAS4 (Coutellier, Beraki et al. 2012). Both heterozygous and
homozygous null NPAS4 mice also have defects in sensorimotor gating which may reflect
impaired cognitive processing of sensory information (Coutellier, Beraki et al. 2012). Taken
together this outlines the importance of NPAS4 in sensory induced memory, learning and
cognitive abilities which results in behavioural and social defects in NPAS4 null mice.
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NPAS4 in Neuroprotection
NPAS4 null mice have been shown to have much reduced life span, with only 20-30% of mice
surviving to 16 months (Ooe, Motonaga et al. 2009). This reduction in life span was associated
with a marked increase in age related neurodegeneration in knockout animals in addition to
increased susceptibility of NPAS4 null mice to glutamate induced neurotoxicity (Ooe,
Motonaga et al. 2009). It is therefore hypothesised that induction of NPAS4 expression may
confer neuroprotection during hyperexcitation or ischemia. This is supported by experiments
where overexpression of NPAS4 (Hester, McKee et al. 2007) increased cell survival or
knockdown decreased cell survival in post-mitotic neurons induced to undergo apoptosis
(Zhang, Zou et al. 2009). While priming post-mitotic neurons with elevated NPAS4 by
overexpression or increased neuron depolarisation may provide neuroprotection, sustained
overexpression in cultured cell lines appears to induce apoptosis (Hester, McKee et al. 2007).
Taken together, this suggests that moderate transient upregulation of NPAS4 maybe
protective while sustained high level NPAS4 expression maybe detrimental to cell survival.
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Project Rationale
NPAS4 expression is mostly restricted to the brain and exhibits transient increases to dampen
neuronal activity by upregulating inhibitory synapse number and strength on excitatory
neurons. This homeostatic regulation of inhibitory synapses implicates NPAS4 in various
neuropsychiatric diseases including autism as well as neurodegenerative diseases in which
excitotoxicity plays a role in the aetiology of the disease. While these are of key interest in the
field, the mechanisms of regulation of NPAS4 gene expression, the potential contribution to
human disease and effective modelling of behaviour in mice are yet to be explored. These
aspects were therefore the focus of the research in this thesis, with the aims of;
1. Exploring the molecular mechanisms which control and restrict NPAS4 expression to
the brain
2. Investigating the contribution of human single nucleotide variants to the function of
the NPAS4/ARNT2 transcriptional dimer
3. Generating technological platforms to effectively model NPAS4 function in vitro and in
mice
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Chapter 2: Results
2.1 Elucidation of molecular mechanisms, which restrict NPAS4 expression to the brain.
NPAS4 is an activity dependent bHLH-PAS transcription factor that controls the expression of
genes involved in inhibitory synapse formation and maintenance. NPAS4 expression is
predominantly restricted to cortical and hippocampal regions of the brain and is highly
induced following neuronal depolarisation, paradigms of learning, seizure or ischemia. The
molecular mechanisms underlying restricted expression pattern were addressed in paper II,
entitled “ Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs”.
For the study in paper II we generated antibodies against NPAS4 protein and confirmed both
activity-induced expression in primary cultured neurons and restriction of mRNA expression to
predominantly cortical/hippocampal brain regions. Paper II identified conserved binding sites
for the transcriptional repressor RE-1 silencing transcription factor (REST) within the promoter
and Intron I of the NPAS4 gene. We found REST to be strongly bound to these sites to repress
NPAS4 expression in non-neuronal and undifferentiated cells. Deletion of these REST binding
sites derepressed NPAS4 expression in embryonic stem cells, and reporter gene experiments
in HEK293T cells showed that both intron I and promoter RE-1 sites are important for
repression. We also showed that REST binding correlates with CTCF occupancy, known to
induce DNA looping and mediate insulator function, this suggesting that CTCF may aid
silencing of NPAS4.
In addition, we also showed that the highly conserved 3’UTR of NPAS4 might also play a role in
regulating NPAS4 expression. We established that NPAS4 can be repressed by miR-224 and
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miR-203 and went on to show that miR-224 is enriched with the hypothalamic/midbrain
regions; being expressed from an intron of the GABA A receptor epsilon gene along with miR-
452. We propose that miR-224 may fine tune NPAS4 expression to restrict expression to
cortical regions of the brain. The work describes the first mechanistic explanation of the brain-
restricted expression of NPAS4.
2.2 Human variants in the NPAS4/ARNT2 transcriptional complex can disrupt function
Neuropsychiatric disease has been suggested to derive, in part, from defects which disrupt
neuronal excitation/inhibition balance (Rubenstein and Merzenich 2003; Kehrer, Maziashvili et
al. 2008; Rubenstein 2010; Yizhar, Fenno et al. 2011). Loss of function mutations in synapse
effectors also suggests synapse dysfunction may play a key role in disrupting this homeostatic
balance in these diseases (Ramocki and Zoghbi 2008; Sudhof 2008; Ebert and Greenberg
2013). In addition, glutamatergic hyperexcitation, which may also arise from disruption of the
excitatory/inhibitory balance or other neuronal stressors, may promote the progression of
neurodegenerative disorders (Bezprozvanny and Mattson 2008; Saxena and Caroni 2011).
Given the links between NPAS4 function and neurological disease like phenotypes in mice, we
sought to investigate whether non-synonymous single nucleotide variants found within human
NPAS4, or its heterodimeric partner protein ARNT2, may disrupt function of the dimer. If so,
we reasoned that a rationale for exome sequencing of NPAS4 in neurological disease affected
patient cohorts might emerge.
In paper III, “Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS)
Transcription Factor Complex NPAS4/ARNT2 Disrupt Function” we found that several variants
in NPAS4 or ARNT2 were able to reduce or ablate a bHLH-PAS responsive luciferase reporter
gene. We also showed that one variant, NPAS4.F147S, failed to upregulate endogenous BDNF
expression due to disruption of dimerisation with ARNT2. We also showed that an ARNT2
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variant, ARNT2.R46W, had reduced activity due to disrupted nuclear localisation. Structural
modelling of the bHLH-PAS regions of the NPAS4/ARNT2 heterodimer, based on the related
CLOCK/BMAL structure (Huang, Chelliah et al. 2012), indicated that the conserved F147
residue lies at the PAS A dimer interface and may directly participate in heterodimer
formation. This is also supported by the finding that mutations which weaken AhR-ARNT
dimerisation lie within this region (Hao, Whitelaw et al. 2011). Paper III shows the potential for
de novo discovery of disease relevant variants by selecting polymorphisms from databases for
screening in function based assays.
2.3 Generation and validation of Inducible and reversible systems for lentiviral and ES cell (Col1a1-RMCE) mediated manipulation of NPAS4 levels.
Inducible and reversible systems for modulating gene expression are critical for investigating
biological pathways. These systems are of particular importance when studying genes critical
for essential cell processes. For example, removal or knockdown of genes critical for cell
survival or proliferation results in cell death, making generation of animal models and cell lines
redundant. In addition, inducible and reversible manipulation of gene expression is of
particular interest in neuroscience, immunology, developmental and cancer biology disciplines
where induction and reversal of genes proposed to underpin a phenotype would be
advantageous for studying disease progression or biological processes.
Several tools for inducible and reversible control of gene expression have been developed in
the past and have been used with limited success. The best studied example being the
tetracycline inducible system combined with either shRNA-mediated knockdown or cDNA
mediated ectopic or over expression. The use of the tetracycline inducible system has been
hampered for several technical reasons. Initial systems employed tetracycline repressor TetR
variants, which repressed transcription from a constitutive promoter containing tet operator
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34
elements, but these systems were generally very leaky in cell lines and animals. While the use
of Tet activator variants and modified tet operator elements reduced background expression
in the absence of doxycycline, most of the variants still have poor sensitivity to Dox, which is a
limitation for in vivo experiments, eg in tissues where DOX transport is poor (i.e. brain). Most
currently used systems still contain significant background expression and generally the tet
responsive promoter and tet activator are delivered on separate plasmids, leading to mosaic
inducible expression in cell lines.
Reliable and reproducible generation of inducible cell lines and mice has been inadequate due
to technical limitations such as cell lines being refractory to transfection and, particularly in
mice, random integration effects. Furthermore, the use of shRNA systems for use in
generating inducible and reversible knockdown in cells and mice relies heavily on efficient
selection of potent shRNA sequences, which are able knockdown gene expression in single
copy.
In manuscript IV, “Inducible and reversible lentiviral and recombination mediated cassette
exchange (RMCE) systems for controlling gene expression”, we attempt to overcome many of
the limitations of previous inducible and reversible systems for ectopic expression and
knockdown of gene expression. We design and test single inducible and reversible lentiviral
constructs for reliable generation of cell lines, which utilise Tet activator variants with
dramatically improved sensitivity to doxcycline and no detectable background expression.
These constructs allow for uniform inducible and reversible expression in cell lines. In
addition, we overcome limitations of reliable targeting into embryonic stem cells for the
generation of mice by adapting a Recombination Mediated Cassette Exchange (RMCE) strategy
which results in ~100% efficient targeting of single inducible and reversible constructs into the
Col1a1 locus. We also design and test constructs which allow for tissue specific Dox inducible
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expression either by Cre mediated gain of Tet activator expression or using tissue specific
promoters.
Using published high throughput data which tested the efficiency of ~60,000 shRNA
sequences, we built an algorithm to predict shRNA sequences with high knockdown potency.
We demonstrate that this is a potentially useful tool for predicting shRNA targets by selecting
and testing sequences for knockdown of ARNT and ARNT2, using the single integration RMCE
embryonic stem cells. We find that the majority of predicted shRNA sequences against either
ARNT or ARNT2 are able to efficiently knockdown endogenous protein expression in
embryonic stem cells.
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Chapter 3: Discussion
NPAS4 has recently been revealed as a critical gene in the regulation and maintenance of
neuronal activity in excitatory neurons. NPAS4 protein and mRNA are transiently upregulated
by neuronal activity to homeostatically regulate inhibitory synapse inputs, preferentially in the
soma of neurons, to dampen neuronal activity. The signal regulated nature of NPAS4 has many
similarities with other bHLH-PAS domain containing proteins such as the AhR, which is signal
regulated by binding to polyaromatic hydrocarbons, resulting in up regulation of genes
involved in cellular detoxification. In response to hypoxic signals, the HIF proteins are
stabilised and become transcriptionally active to regulate genes, which respond to low oxygen
tension by altering metabolism and promoting increased angiogenesis. Responding to
physiological or stress signals to induce genetic programs for protection, adaption or
maintaining homeostasis seems to be unifying theme for bHLH-PAS transcription factors.
Tight temporal and spatial control of NPAS4 expression is likely to be critical for the correct
specification of inhibitory synapses and the homeostatic activity balance of neurons within the
CNS. Sustained overexpression in cell lines and by pathological inducers such as seizure and
ischemia may indeed be detrimental to neuronal cellular function. Therefore the precise
mechanisms which control NPAS4 expression are likely to be highly regulated and may have
importance in disease states. In paper II we describe a mechanism that restricts NPAS4
expression to the brain by finding the repressor REST silences expression in non-neuronal and
undifferentiated tissues (Figure 2.). REST binds strongly to multiple sites within the NPAS4
gene to mediate silencing of NPAS4 expression outside the CNS, implying that repression of
NPAS4 expression may be functionally important during development. Silencing the NPAS4
gene outside the CNS may also be aided by CTCF, which flanks the NPAS4 gene and overlaps
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37
with REST binding sites, perhaps implying that looping may enhance silencing. This possibility
remains to be explored. Interestingly, CTCF binding at sites flanking the NPAS4 gene has been
reported for brain tissue and CTCF may act to insulate activity inducible expression of NPAS4
from adjacent genes, which are not depolarisation regulated.
We also show that miR-224 may play role in restricting NPAS4 expression to cortical brain
regions by targeting the NPAS4 3’UTR (Paper II). Given that NPAS4 can influence the number
of inhibitory inputs, miR-224 may allow for an altered activity profile in certain neuronal
subtypes and it would therefore be of interest to investigate the in vivo function of loss of miR-
224 in mice. Interestingly, we found that inhibition of both methyltransferases and histone
deacetylases in embryonic stem cells leads to an upregulation of NPAS4 expression, which is
supported by recent evidence that the NPAS4 promoter is methylated and regulated by the
demethylase promoting enzyme Tet1 (Rudenko, Dawlaty et al. 2013). While the mechanism of
how Tet1 is regulated in response to neuronal activation in unknown, it has been shown that
the methyl-CpG-Binding protein 2 (MeCP2) can be phosphorylated in response to
depolarisation, leading to dissociation of MeCP2 from methyl-CpGs and corepressors. This may
allow Tet1 access to catalyse demethylation and depression of transcription (Chen, Chang et
al. 2003; Ebert, Gabel et al. 2013).
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Figure 2. Schematic diagram of the regulation of NPAS4 expression by REST and miR-224. RE-
1 Silencing Transcription factor (REST) represses NPAS4 expression in embryonic stem cells
and non-neuronal cells by binding to multiple RE-1 sites in the promoter and intron I of the
NPAS4 gene. In unstimulated primary neurons, REST expression is down regulated
derepressing NPAS4 expression. microRNA 224 (miR-224) is expressed in midbrain and
hypothalamic brain regions and is able to repress NPAS4 expression, which may restrict NPAS4
expression to cortical and hippocampal neurons. Upon depolarisation of neurons, NPAS4
expression rapidly increases and binds its own promoter, which may further increase its own
expression. Reproduced from paper II Bersten et al, Biochim Biophys Acta. 2014.
Significant questions still remain in the regulation of NPAS4 expression. For example, while
strong binding of NPAS4 to its own promoter is present following depolarisation and NPAS4
has been shown to activate its own promoter in luciferase reporter gene assays, it is still
unclear what involvement NPAS4 has in stimulating its own expression ((Ooe, Saito et al.
2004)Paper II). For example, initial upregulation of NPAS4 mRNA following depolarisation is
unhindered by cyclohexamide treatment, implying that other proteins may be responsible for
the initial wave of NPAS4 expression (Ramamoorthi, Fropf et al. 2011). In addition, Cre
mediated knockout of NPAS4 in neurons does not affect depolarisation mediated NPAS4
promoter driven luciferase induction (Ramamoorthi, Fropf et al. 2011), but induction kinetics
have not been investigated in NPAS4 knockout GFP reporter mice . As such there is a critical
need to identify proteins, which initiate the transient induction of NPAS4 following neuronal
depolarisation. As with the BDNF promoter, NPAS4 may not be responsible for the initial
induction of its own expression, but rather strengthening the response after gene induction
(Pruunsild, Sepp et al. 2011). In addition, NPAS4 protein is rapidly down regulated after being
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39
initially induced by depolarisation, peaking in expression at ~2 hrs and almost absent after
6hrs. The mechanism by which this down regulation occurs remains unexplored.
Elucidation of NPAS4 function has revealed a critical homeostatic role in regulating neuronal
activity by controlling the balance between excitatory and inhibitory synaptic inputs. Mice
lacking NPAS4 have many phenotypic characteristics similar to those seen in both
neuropsychiatric diseases such as autism and schizophrenia and in neurodegenerative disease.
Hypotheses for the aetiology of both of these disease states include dysfunction in
excitatory/inhibitory balance within the brain, leading to cognitive dysfunction. At least in the
case of autism like behaviours, this has been elegantly shown in mice using optogenetic
techniques to disrupt excitatory/inhibitory balance in cortical neurons (Yizhar, Fenno et al.
2011). For aforementioned reasons NPAS4 and the regulation of NPAS4 expression may have a
role either directly or indirectly in these diseases.
In Paper III we investigate the potential contribution of human non-synonymous single
nucleotide variants on the function of the NPAS4/ARNT2 transcriptional complex. We find that
several naturally occurring variants have the ability to disrupt NPAS4/ARNT2 transcriptional
function and find biochemical mechanisms for the defective complex include either
attenuating heterodimer formation or nuclear localisation. The location of variants showing
loss of heterodimer formation was consistent with previous loss of dimerisation mutations
discovered by bacterial two hybrid screening (Hao, Whitelaw et al. 2011). We also recently
published a collaborative work describing loss of function for single nucleotide variants in
SIM1, which were associated with severe obesity (Bonnefond, Raimondo et al. 2013;
Ramachandrappa, Raimondo et al. 2013). The location of loss of function variants described in
these manuscripts is consistent with our data in Paper II in that they are commonly clustered
N-terminally in domains critical for heterodimerisation, DNA binding and nuclear localisation.
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40
Many of the variants described in paper III were discovered from high throughput genome
sequencing efforts, the implication of which is that many have not been independently
validated. In addition, while many of the variants studied in paper III are common variants
within the human population, those which display altered activity appear to be rare or de novo
variants. Anonymous donation of samples in these sequencing efforts makes linking genotype
to phenotype difficult. As such we cannot draw conclusions about the relevance of these
variants to disease, suffice to say that common variations do not appear to significantly alter
NPAS4 function in reporter experiments.
While links with disease phenotype was beyond the scope of Paper III, it suggests that the
identified loss of function variants may have implications for neuropsychiatric or
neurodegenerative disease. Furthermore, it also outlines the potential use of de novo
functional screening of human variants in genes with already defined roles in disease.
Following our observations regarding the ability of naturally occurring variants to disrupt
NPAS4/ARNT2 function outlined in paper III and the tightly regulated mechanisms to control
NPAS4 expression outlined in paper II, we propose that screening for disease associated single
nucleotide and copy number variants within coding and regulatory regions of NPAS4 is
warranted, within neuropsychiatric or neurodegenerative disease populations.
As discussed above, bHLH-PAS transcription factors often act transiently to respond to signals
and perform protective or homeostasis related functions. Homeostatic regulation of key
signal response pathways is consistent with bHLH-PAS family members having important
developmental and disease related roles. Targeted disruptions of many of the bHLH-PAS genes
result in embryonic lethality and many have distinct roles in different tissues. It would
therefore be advantageous to study function following gene manipulation in an inducible and
reversible manner. Specifically, development of inducible, reversible and tissue specific gain or
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41
loss of HIF1 / HIF2 mouse models would significantly aid the elucidation and distinction for
roles of the HIFs in cancer initiation, progression and metastasis. In addition, An Inducible and
reversible loss of NPAS4 mouse model would be invaluable to the advancement of the study of
the basic mechanisms underlying synapse formation, memory and learning formation and the
investigation and modelling of neuropsychiatric disease.
We therefore embarked on designing and testing generic platforms in which bHLH-PAS
transcription factors could be manipulated in an inducible, reversible and tissue specific
fashion. Utilising optimised Tet-On variants with improved sensitivity to Dox and reduced
background we constructed lentiviral and recombination mediated cassette exchange
plasmids, which contained all the components necessary for Dox-inducible expression (see
paper IV and Figure 3.).
We demonstrated that lentiviral generation of cell lines affords homogeneous inducible and
reversible manipulation of gene expression with no detectable background. We also
generated Col1a1 targeted RMCE FLP-In embryonic stem cells and mice for efficient FLP
mediated targeting of FLP-INDUCER Dox regulated constructs into the Col1a1 locus. We
demonstrate tight, inducible and reversible expression from mES-FLP-INDUCER cells able to
knockdown gene expression or ectopically express cDNA similar to endogenous levels.
Modular design of FLP-INDUCER plasmids allow for promoter exchange to enable tissue
specific promoters to be used to gain tissue/cell specific expression. We also show that LoxP-
STOP-LoxP mES FLP-INDUCER cells may, in principle, be used to limit inducible expression to
certain tissues or cells (Figure 3.).
While there are other established (i.e. Cre/loxP) and emerging (CRISPR/TALEN) techniques to
allow temporal and/or spatial gene ablation, some of which can employ inducible knockout
strategies such as tamoxifen inducible nuclear translocation of oestrogen receptor (ER)-CRE
recombinase to remove flox’d genes. These approaches irreversibly knockout the gene and
require time consuming generation of flox’d genes and crossing into CRE driver lines.
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42
Furthermore, in many disease models it may be advantageous to reversibly modulate gene
expression to monitor the progression/reversion of disease. We note that the FLP-INDUCER
system may be well suited to these specific disease model applications while also being
compatible with established CRE-mediated tissue specific and/or temporal control of gene
expression.
Alternative RMCE targeting loci have also been successfully used in the past for transgenic
mouse experiments including inducible Tet-On expression systems (Tchorz, Suply et al. 2012;
Haenebalcke, Goossens et al. 2013). Several of these systems would be compatible with the
FLP-INDUCER system with minimal modification, presenting an opportunity to generate multi
allelic inducible mouse models (Tasic, Hippenmeyer et al. 2011; Tchorz, Suply et al. 2012).
While the Rosa26 locus has been used extensively for generating site-specific transgenesis,
expression from this locus appears to result in significant mosaicsim and lower inducible
transgene induction as compared to the Col1a1 locus (Haenebalcke, Goossens et al. 2013). In
addition to the Col1a1 and rosa26 sites of integration the Hipp11 and hprt loci have
successfully been used to drive ubiquitous expression in mouse models and may represent
useful sites for RMCE mediated FLP-INDUCER insertion (Bronson, Plaehn et al. 1996; Tasic,
Hippenmeyer et al. 2011).
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43
Figure 3. Lentiviral and Collagen 1a1 Recombination Mediated Cassette Exchange (RMCE)
systems for inducible and reversible knockdown or overexpression. A schematic showing the
single construct modular design of lentiviral and FLP-INDUCER vector systems used to
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44
generate stable cell lines or site-specific integration into germline competent mouse
embryonic stem cells.
In an attempt to more accurately predict high potency miR30 shRNAs able to knockdown gene
expression using the FLP-INDUCER system, we also designed and tested an shRNA prediction
tool. This tool incorporated experimental data assessing the potency of ~60,000 shRNAs from
high throughput experiments as well as mRNA secondary structure to score shRNA sequences
based on their putative targeting efficiency. We show by cloning and testing 10 shRNA targets
against ARNT or ARNT2 using the mES-FLP-INDUCER system that the prediction tool can
effectively identify high potency shRNA sequences.
We are now well situated to rapidly generate inducible and reversible knockdown mouse
models for studying the function of the bHLH-PAS transcription factors. As discussed above
this will be invaluable to studying the more complex adult roles for the bHLH-PAS transcription
factors in normal physiology and disease.
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45
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