characterization of the role of ubiquitin protein ligase

Characterization of the Role of Ubiquitin Protein Ligase E3 Component N-recognin 4 (UBR4) in the Murine Circadian

Clock

by

« Harrod Ho Pak Ling »

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Cell and Systems Biology University of Toronto

© Copyright by Harrod Ho Pak Ling 2014

ii

Characterization of the Role of Ubiquitin Protein Ligase E3

Component N-recognin 4 (UBR4) in the Murine Circadian Clock

Harrod Ho Pak Ling

Master of Science

Department of Cell and Systems Biology

University of Toronto

2014

Abstract

Ubiquitination is an important post-translational modification in the molecular clock that

regulates degradation of clock proteins through ubiquitin ligases. However, no ubiquitin ligase

has been implicated in the photic entrainment pathways in mammals thus far. In this thesis, I

characterized the physiological and molecular phenotype of a new ubiquitin ligase named UBR4

in the murine circadian clock. UBR4 is expressed throughout the suprachiasmatic nucleus in a

time-of-day-dependent fashion and is light-inducible in the early subjective night. Homozygous

ubr4 knockout are embryonic lethal, therefore heterozygous ubr4 mice (ubr4+/-

) were used to

study its physiological role in the circadian clock. I found that mice with reduced expression of

UBR4 show differential phenotype in circadian paradigms that involve chronic light disturbances.

Furthermore, preliminary data suggest that casein kinase 2 beta subunit is a potential substrate of

UBR4, affecting molecular clock component PER in vivo and in vitro.

iii

Acknowledgments

I would like to take this opportunity to thank all the people who have helped me along the way

during this thesis. First, I would like to thank my supervisor, Dr. Mary Cheng for everything that

she has done for me during the past two years. Her around the clock guidance and support,

expertise in the field of circadian and molecular biology have been extremely helpful and made

me learn and grow a lot as a scientist. I consider myself privileged to have this opportunity to

complete my studies with her.

I am also thankful to my supervisory committee members Dr. Joel Levine and Dr. Ian Orchard

for their valuable input and advice to my research that helped to shape it along the way.

I would like to thank my lab mates Lucia, Neel, Pascale and Steve and all the undergraduate

students especially Saina for their support in the past two years. I have been very fortunate to

have the opportunity to work with and learn from all of them. I would also like to thank my

wonderful roommate Jade for the support and guidance that she has given me in the past two

years.

Last but not least, I would like to thank my family for their encouragement and support despite

the long distance separating us during the past couple of years. The weekly calls I received from

them meant a lot to me and provided the much needed support for me to complete my research.

Thank you all for everything.

iv

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ............................................................................................................................... vii

1 Introduction ................................................................................................................................ 1

1.1 Circadian Rhythms .............................................................................................................. 1

1.2 The Suprachiasmatic Nucleus as the master clock ............................................................. 1

1.2.1 The SCN and its organization ................................................................................. 2

1.2.2 Function of neuropeptides in the SCN .................................................................... 3

1.3 Photic entrainment .............................................................................................................. 4

1.4 Core molecular clock feedback loop ................................................................................... 5

1.5 Post-translational control .................................................................................................... 6

1.5.1 Phosphorylation regulation by CK1 in the circadian clock .................................... 6

1.5.2 Phosphorylation regulation by CK2 in the circadian clock .................................... 7

1.5.3 Other kinases in the circadian clock ....................................................................... 9

1.5.4 Ubiquitination by FBXL family in the circadian clock ........................................ 10

1.5.5 Ubiquitination by β-TRCP family in the circadian clock ..................................... 10

1.5.6 Other ubiquitin ligases implicated in the circadian clock ..................................... 11

1.6 Ubiquitin Proteasome System ........................................................................................... 13

1.7 The N-end Rule pathway and N-recognins ....................................................................... 13

1.8 UBR4 and its known function ........................................................................................... 14

1.9 Rationale ........................................................................................................................... 15

1.10 Thesis Objective ................................................................................................................ 16

2 Methods and Materials ............................................................................................................. 17

2.1 Animals ............................................................................................................................. 17

v

2.2 Behavioral analysis and circadian paradigms ................................................................... 17

2.2.1 Constant darkness paradigm and phase shifting light pulse ................................. 17

2.2.2 Phase shift experiments ......................................................................................... 18

2.2.3 Chronic jetlag paradigm ........................................................................................ 18

2.2.4 Constant light paradigm ........................................................................................ 19

2.3 Cell culture and transfection ............................................................................................. 19

2.4 Tissue harvesting .............................................................................................................. 20

2.5 Western blotting ................................................................................................................ 20

2.6 Immunofluorescence ......................................................................................................... 21

2.7 Immunohistochemistry ..................................................................................................... 21

2.8 Immunocytochemistry ...................................................................................................... 22

2.9 Microscopy imaging ......................................................................................................... 22

2.10 Image Processing and Quantification ............................................................................... 23

2.11 Statistical Analysis ............................................................................................................ 24

3 Results ...................................................................................................................................... 25

3.1 Temporal and spatial expression of UBR4 in the SCN .................................................... 25

3.2 Co-localization of UBR4 with neuropeptides in the SCN ................................................ 29

3.3 The response of ubr4+/-

mice to constant darkness paradigm and phase shifting light

pulses ................................................................................................................................. 31

3.4 ubr4+/-

mice response to phase shift paradigm ................................................................. 34

3.5 ubr4+/-

mice response to chronic jetlag paradigm ............................................................. 36

3.6 ubr4+/-

mice response to constant light paradigm ............................................................. 38

3.7 Molecular clock protein PER1 and PER2 expression in ubr4+/-

mice SCN ..................... 40

3.8 SCN neuropeptide expression in ubr4+/-

mice .................................................................. 42

3.9 CK2β as a potential target of UBR4 in vivo and in vitro .................................................. 44

3.10 UBR4 affects PER1/2 expression in vitro ........................................................................ 46

vi

3.11 ubr4+/-

per2-/-

mice in constant darkness condition ........................................................... 48

4 Discussion ................................................................................................................................ 50

4.1 Reduced expression of UBR4 effect on circadian paradigms .......................................... 50

4.2 Constant light disruption in ubr4+/-

mice and implications .............................................. 51

4.3 Reduced expression of UBR4 disrupts the molecular clock ............................................. 52

4.4 UBR4 potential interaction with AVP and role in SCN synchrony .................................. 55

4.5 Importance of UBR4 augmented in per2-/-

background ................................................... 55

4.6 Conclusion ........................................................................................................................ 56

Bibliography ................................................................................................................................. 57

Copyright Acknowledgements ...................................................................................................... 70

vii

List of Figures

Figure 1. Simplified model of mammalian molecular clock with ubiquitin ligases ..................... 12

Figure 2. UBR4 time-of-day dependent expression profile in the SCN ....................................... 27

Figure 3. Light inducibility of UBR4 in the SCN in different time of day ................................... 28

Figure 4. UBR4 co-localization with neuropeptides in the SCN .................................................. 30

Figure 5. UBR4 expression in the SCN of ubr4+/-

mice ............................................................... 32

Figure 6. The response of ubr4+/-

mice to constant darkness and phase shifting light pulses ...... 33

Figure 7. The response of ub4+/-

mice to phase shift paradigm .................................................... 35


mice to chronic jetlag paradigm .............................................. 37


mice to constant light. ............................................................. 39

Figure 10. PER1 and PER2 expression in the SCN of ubr4+/-

mice ............................................. 41

Figure 11. AVP and VIP expression in the SCN of ubr4+/-

mice ................................................. 43

Figure 12. Effect of UBR4 deficiency on CK2β expression in vivo and in vitro ......................... 45

Figure 13. The effect of UBR4 on PER1 and PER2 expression in vitro ...................................... 47

Figure 14. Behavioral and molecular phenotype of ubr4+/-

per2-/-

mice ....................................... 49

Figure 15 The potential role of UBR4 in a simplified model of the mammalian molecular clock

with known ubiquitin ligases ........................................................................................................ 54

1

1 Introduction

1.1 Circadian Rhythms

Organisms on earth evolve by adapting to changes in the environment to survive. One of the

most conserved environmental stimuli present on earth is sunlight; hence, most organisms on

earth, ranging from cyanobacteria to plants, all the way up to complex animals such as humans,

have evolved to adapt to the environmental light-dark cycle and exhibit rhythms of about 24

hours that are called circadian rhythms (Dunlap, 1999). Circadian rhythms ensure that different

functions or behaviors are displayed at the appropriate time of day. Under constant conditions in

the absence of external cues, circadian rhythms continue to oscillate and are powered by various

endogenous clocks in the organism, which can be traced down to the single cell level, where

most cells possess the necessary molecular machinery to operate a functional clock (Reppert and

Weaver, 2002). However, endogenous clocks in peripheral tissues are under the control of a

master circadian pacemaker, the suprachiasmatic nucleus (SCN) in mammals (Hastings et al.,

2003).

1.2 The Suprachiasmatic Nucleus as the master clock

The suprachiasmatic nucleus (SCN) has been proven to be the master circadian pacemaker in

mammals originally from lesion studies, as animals with electrolytically lesioned SCN are

arrhythmic at the physiological and behavioral level (Lehman et al., 1987; Moore and Eichler,

1972; Stephan and Zucker, 1972). The idea of the SCN as the master clock has been further

confirmed from a study that transplanted SCN with shorter period onto an SCN-lesioned animal

with a previously normal period and restored the locomotor rhythm of the recipient animal based

on the donor’s short circadian period (Ralph et al., 1990). The SCN is a bilateral structure located

in the hypothalamus directly above the optic chiasm. Photic signals received from the retina are

transmitted to the SCN via the retinohypothalamic tract (RHT), which synapses directly onto

SCN neurons (Moore and Lenn, 1972). Other non-photic signals can also be transmitted to the

SCN from other brain regions such as intergeniculate leaflet (IGL) through the

2

geniculohypothalamic tract (GHT) (Harrington, 1997). These signals are integrated in the SCN

and synchronize the SCN timing to the environments. SCN neurons can oscillate individually

and have different phases and periods, but are tightly coupled and produce a single rhythmic

output from the master clock that aligns peripheral clocks throughout the body (Dibner et al.,

2010; Liu et al., 1997).

1.2.1 The SCN and its organization

The SCN is a heterogeneous structure composed of approximately 20,000 neurons that can be

divided into different regions. The murine SCN can be classified into two anatomically and

functionally distinct regions designated as the ventrolateral ‘core’ SCN and the dorsomedial

‘shell’ SCN. The core and shell SCN are characterized by their location in the SCN, but the

neurons in the two regions also express distinct sets of neuropeptides, primarily vasoactive

intestinal peptide (VIP) and arginine-vasopressin (AVP), respectively (Abrahamson and Moore,

2001).

The core SCN is situated near the base, or ventral region, of the SCN, directly above the optic

chiasm, and receives direct innervation from the retina (Abrahamson and Moore, 2001). The core

SCN is the retinorecipient area of the SCN, which integrates external input and shows only low

amplitude rhythms of clock gene expression (Hamada et al., 2001; Yan and Okamura, 2002). A

light pulse at night can induce rapid expression of immediate early gene and subsequent clock

gene expression in the region (Aronin et al., 1990; Rusak et al., 1990; Yan and Silver, 2002; Yan

et al., 1999). Furthermore, the core SCN is populated mainly by VIP neurons, which project to

the shell allowing them to communicate information to the rest of the SCN (Abrahamson and

Moore, 2001).

On the other hand, the shell SCN is located dorsal to, and surrounds, the core. The shell is not

directly innervated by projections from the retina, but is innervated by the core neurons and

contains projections to different brain regions (Leak and Moore, 2001). The shell SCN is

populated by AVP neurons and considered to be the rhythmic portion of the SCN, where clock

gene expression in this region has robust oscillation even in constant conditions (Hamada et al.,

2001; Yan and Okamura, 2002). When a light signal is presented at night, the shell relies on the

3

core SCN to convey photic information, as it lags behind the SCN core in clock gene induction

after light pulse (Nagano et al., 2003; Yan and Silver, 2004). Together, the shell and core SCN

need to act in concert in order to integrate external signal, reset the clock and produce a

synchronized rhythmic output that projects to other brain regions or organs.

1.2.2 Function of neuropeptides in the SCN

VIP has been suggested to be involved in photic gating, as VIP levels in the SCN can be

decreased by light pulse and application of exogenous VIP can phase shift the clock in vitro and

in vivo, mimicking a light pulse (Piggins et al., 1995; Reed et al., 2001; Shinohara and Inouye,

1995). The more significant role of VIP in the circadian clock has been demonstrated in

transgenic mice lacking either VIP or its receptor VIPR2, where vip-/-

or vipr2-/-

mice have

multiple rhythms or become arrhythmic in constant darkness (Aton et al., 2005; Colwell et al.,

2003; Harmar et al., 2002). At the cellular level, VIP is important for molecular timekeeping

within individual SCN neurons, and is also critical for synchrony among SCN neurons, as vipr2-/-

mice has dampened clock gene oscillations in SCN neurons and neurons are not synchronized

within the SCN (Maywood et al., 2006).

AVP in the SCN was previously considered solely to be a rhythmic output signal from the SCN,

due to the fact that it is released rhythmically and AVP neurons in the SCN project to other brain

regions (Abrahamson and Moore, 2001; Jin et al., 1999; Schwartz and Reppert, 1985). Recently,

gene-targeted mice have revealed additional roles of AVP in the SCN. Under constant darkness,

gene-targeted mice lacking AVP receptor V1a (V1a-/-

) show greater cycle-to-cycle variability in

activity onset with dampened circadian period amplitude, with some of these mice eventually

becoming arrhythmic (Li et al., 2009). Interestingly, mice lacking both V1a and V1b receptors

are resistant to jetlag, such that they can immediately entrain to an 8-hour shift in the LD cycle.

At the cellular level, V1a-/-

V1b-/-

mice have a less synchronized SCN clock (Yamaguchi et al.,

2013). Another study utilizing SCN co-culture techniques demonstrates that both VIP and AVP

signalling are important for SCN neuronal synchrony, but VIP has a more dominant effect over

AVP (Maywood et al., 2011). However, the role of AVP in synchrony may be more important

over long term in constant conditions, as AVP, but not VIP, remains rhythmic in constant

darkness and constant light condition (Isobe and Nishino, 1998; Okamura et al., 1995; Tominaga

4

et al., 1992). In summary, both VIP and AVP are important neuropeptides in the SCN that work

in conjunction with one another to synchronize SCN neurons and produce a rhythmic output.

1.3 Photic entrainment

The master pacemaker itself runs at a near but not exact 24 hour pace, and therefore requires

daily input from the surroundings to align the internal clock through a process called

entrainment. The most dominant signal to influence the clock is light, where changes in the daily

light-dark cycle can be transmitted to the SCN and reset the clock (Golombek and Rosenstein,

2010). As mice are nocturnal animals, light at night has a profound impact on their circadian

clock. Researchers have constructed the phase response curve as a tool to explain the phase-

dependent responsiveness of the circadian clock in constant darkness. The subjective day is

considered the ‘dead zone’, since light in the daytime does not phase shift the circadian clock.

Light in the early subjective night causes the circadian phase of the subsequent cycle to delay

(i.e. activity onset is later the next day), whereas light in the late subjective night causes a

circadian phase advance (Johnson, 1999). This provides the basis for how nocturnal mice can

entrain to a light-dark cycle, where mice can adjust its endogenous clock when exposed to light

during light-dark transition at either dusk or dawn.

At the molecular level, photic signals are received through photosensitive retinal ganglion cells

in the retina (Peirson and Foster, 2006), which project to the SCN and cause pituitary adenylate

cyclase activating peptide (PACAP) and glutamate to be released from the synapses (Hannibal,

2002). This neurotransmitter stimulation activates downstream signaling events primarily

through the cAMP response element binding protein (CREB) pathway (Meijer and Schwartz,

2003), which in turn activates transcription of immediate early genes (i.e. c-fos, egr1) (Aronin et

al., 1990; Rusak et al., 1990; Slade et al., 2001), and subsequently clock genes (Per1 and Per2)

(Yan and Silver, 2002; Yan et al., 1999), in order to reset the clock following a light pulse.

5

1.4 Core molecular clock feedback loop

Most cells in the body have the necessary components of the molecular clock called core clock

genes that can oscillate in a 24 hour manner. The molecular clock is controlled by an interlocking

transcriptional/translational feedback loop that drives the cyclic oscillation. In mammals, the

feedback loop is composed of a primary feedback loop and a secondary feedback loop

(Takahashi et al., 2008). The positive limb of the primary feedback loop is composed of CLOCK

and BMAL1, two basic helix-loop-helix transcription factors that heterodimerize and activate

transcription of genes constituting the negative limb via binding to their E-box enhancers

(Bunger et al., 2000; Gekakis et al., 1998; Hogenesch et al., 1998; King et al., 1997). The

negative limb of the primary feedback loop consists of three Period genes (Per1, Per2, and Per3)

and two Cryptochrome genes (Cry1 and Cry2). The transcribed and translated PER and CRY

proteins accumulate in the cytoplasm, heterodimerize, and translocate back to the nucleus where

they interact with the CLOCK-BMAL1 complex to inhibit their transcription of per and cry

genes, thereby closing the primary feedback loop (Kume et al., 1999; Okamura et al., 1999;

Shearman et al., 2000). The system is further fine-tuned by the secondary feedback loop, which

consists of retinoic acid receptor-related orphan receptor REV-ERBα and RORα. The

transcription of Rev-erbα and Rorα is similarly activated by CLOCK-BMAL1 dimers in an E-

box-dependent fashion. Their protein products can then translocate back to the nucleus and

regulate transcription of Bmal1 by binding to retinoic acid-related orphan receptor response

element (RRE) on the Bmal1 gene, completing the secondary feedback loop (Akashi and Takumi,

2005; Preitner et al., 2002; Sato et al., 2004). The level of Bmal1 transcription is determined by

the balance between activation from RORα and inhibition from REV-ERBα (Sato et al., 2004).

The activation and repression of the primary and secondary feedback loops happen daily, causing

these core clock genes and proteins to oscillate in a sinusoidal manner over 24 hours and thus to

serve as the clock machinery in a cell. Each of the core clock proteins itself are also transcription

factors that can activate their own set of downstream targets, activating signalling events

depending on the cell type, tailored for specific purposes of the tissue or organ at the appropriate

time of day (Akhtar et al., 2002; Panda et al., 2002).

6

1.5 Post-translational control

In addition to control at the transcriptional and translational level for core clock proteins, post-

translational modifications can also play an important role in fine-tuning molecular clock

oscillations. Two of the better studied post-translational modifications in the mammalian

circadian clock are phosphorylation and ubiquitination, where disruption in specific kinases and

ubiquitin ligases in mutant mice can cause significant changes in circadian period (Gallego and

Virshup, 2007).

1.5.1 Phosphorylation regulation by CK1 in the circadian clock

Phosphorylation can control many different aspects of a clock protein’s function, including

activity, subcellular localization and stability (Gallego and Virshup, 2007; Vanselow and

Kramer, 2007). In fact, the first naturally occurring circadian clock mutant identified in

mammals was the tau mutant hamster, which has a shortened period of 20 hours in hamsters with

the homozygous tau mutation (Ralph and Menaker, 1988) and was later found to carry a

mutation in casein kinase 1 epsilon (CK1ε), an isoform of the serine/threonine kinase CK1

(Lowrey et al., 2000). CK1ε tau mutation is a gain-of-function mutation that causes PER to be

hyperphosphorylated and promotes their degradation, thus shortening the clock (Gallego et al.,

2006; Meng et al., 2008). Another naturally occurring mutation in CK1 was found in a human

population with familial advanced sleep phase syndrome (FASPS), where FASPS patients have

an advanced phase in sleep-wake cycle that is about 4 hours earlier than the normal population

(Jones et al., 1999). Linkage analysis showed that FASPS can be caused by either a mutation in

Casein kinase 1 delta (CK1δ) (a homolog of CK1), leading to a reduction in kinase activity, or

by another mutation in the CK1ε/δ binding site of PER2 (Toh et al., 2001; Xu et al., 2005).

Despite the early discovery of CK1ε in the mammalian circadian field, CK1δ has recently

received more attention. Unlike CK1ε null mutant mice, which have a normal period, a non-

functional mutation of CK1δ in mice leads to period lengthening of the liver clock in vivo

(Etchegaray et al., 2009). Pharmacological inhibition of two CK1 isoforms in vitro and SCN

explant studies provide additional evidence that CK1δ plays a more dominant role than CK1ε in

PER phosphorylation under physiological conditions (Etchegaray et al., 2010; Walton et al.,

7

2009). Furthermore, CK1ε can also control the nuclear entry of PER1, affecting when PER:CRY

complexes can initiate the inhibition of CLOCK-BMAL1-activated transcription in the nucleus

(Akashi et al., 2002; Takano et al., 2004; Vielhaber et al., 2000). Lastly, phosphorylation of PER

proteins by CK1ε can determine its stability by priming it for degradation by the 26S proteasome

(Akashi et al., 2002; Camacho et al., 2001; Eide et al., 2005; Keesler et al., 2000). Together,

isoforms of CK1 can contribute to circadian clock timing by regulating various functions of PER

in vivo and in vitro.

1.5.2 Phosphorylation regulation by CK2 in the circadian clock

Casein Kinase II (CK2) is another serine/threonine kinase that has been implicated in circadian

clock control. CK2 is ubiquitously expressed in different cells in mammals and localized in

different cellular compartments, where it controls a wide variety of cellular functions, notably

cell cycle regulation and cell survival (Filhol and Cochet, 2009). Structurally, CK2 is composed

of two catalytic subunits termed CK2α, along with two regulatory beta subunits (CK2β), which

together form a tetramer (α2β2) that can phosphorylate more than 300 known substrates (Meggio

and Pinna, 2003).

CK2 has been identified in two behavioral screens for fly mutants with periods that differ

significantly from 24 hours. The CK2α subunit mutant fly was termed Timekeeper (Tik). Tik

homozygous flies do not live to adulthood, but Tik heterozygotes exhibit a period that is 1.5

hours longer than controls (Lin et al., 2002). This long period originates from a loss of enzymatic

function of CK2 in the fly circadian pacemakers, thus stabilizing its substrate PER in drosophila

clock neurons and delaying PER:TIM complex (TIM is the functional homolog of mammalian

CRY in drosophila) entry into the nucleus. The other mutant screen identified a CK2β subunit

mutant fly named Andante, which also exhibits a longer period (Akten et al., 2003). The Andante

mutation is located in the CK2β gene, where it perturbs CK2 subunit dimerization, thus affecting

CK2 kinase activity as a whole. With deficits in the CK2β subunit, Andante flies exhibit elevated

PER and TIM abundance, as well as abnormal cellular distribution and delayed nuclear entry of

PER:TIM complexes, mimicking the molecular and behavioral phenotype of CK2α Tik mutants.

Interestingly, both studies identified the respective CK2 subunit to be highly expressed only in a

small subsets of neurons in the ventral lateral network that corresponds to the pacemaker neurons

8

in drosophila, along with only ~2-8 other cells in the dorsal brain region, demonstrating the

specificity of CK2 localization in circadian pacemaker neurons in drosophila (Akten et al., 2003;

Lin et al., 2002).

CK2 has also been implicated in mammalian molecular clock machinery through three different

in vitro studies. First, a RNAi screen designed to identify novel kinases that modulate PER2

ultimately identified CK2 as a kinase in the mammalian circadian clock that affects PER2

stability (Maier et al., 2009). Knocking down the expression of CK2 using RNAi or inhibiting

CK2 function by pharmacological approaches lengthened the circadian period in Bmal1

promoter-driven luciferase activity in U-2OS cells, while overexpressing CK2 subunits caused

the circadian period to shorten. CK2 binds to PER2, phosphorylates its N-terminal residues and

stabilizes PER2 in the cytoplasm and nucleus. When the authors mutated a CK2-specific PER2

phospho-site, CK2 kinase activity on PER2 was disrupted, stabilizing PER2 and lengthening the

circadian period in a manner similar to RNAi or pharmacological approaches. A second study

using ex vivo SCN slices from Per2:luciferase knock-in mice, also demonstrated PER2 as a CK2

substrate. They demonstrated similar long circadian period with low amplitude phenotype when

a CK2 inhibitor was employed (Tsuchiya et al., 2009). However, the authors proposed a

contradictory mechanism whereby CK2 is normally responsible for degrading PER2, acting

synergistically with CK1ε to prime PER2 degradation. Further, using a mutant form of PER2

with CK2 phospho-site Ser-53 (s53) mutated, PER2-s53 was found to be resistant to CK1ε and

CK2 mediated degradation, thus lengthening the clock. Although these two studies propose

opposing theories regarding PER2 stability, CK2 can potentially affect PER2 in a complex

manner. The effects of CK2 on PER2 may depend on both spatial (e.g. subcellular localization)

and temporal (e.g. time of day) factors, along with functional specificity of different CK2

phospho-sites that may work in conjunction with CK1ε (Maier et al., 2009; Reischl and Kramer,

2011; Tsuchiya et al., 2009). The detailed mechanisms underlying CK2-dependent regulation of

PER2 remain to be fully explored. Lastly, CK2α can phosphorylate BMAL1 in vitro to control

its nuclear entry. Gene silencing of CK2α or mutating the CK2 phospho-site on BMAL1 can

impair rhythmicity of Per2:luciferase in mouse fibroblasts (Tamaru et al., 2009). Despite proof

of CK2 involvement in mammalian circadian clock in vitro, the physiological function of CK2 in

adult circadian clock in vivo is still unknown, since CK2α and CK2β are crucial for early embryo

development (Buchou et al., 2003; Lou et al., 2008).

9

1.5.3 Other kinases in the circadian clock

Glycogen synthase kinase 3 beta (GSK3β) has been implicated in the mammalian circadian clock

and interacts with many core clock proteins, including CLOCK, BMAL1, CRY2, REV-ERBα

and PER2, regulating the circadian clock in a number of ways. GSK3β can phosphorylate

CLOCK and BMAL1 and control the rate of their degradation (Sahar et al., 2010; Spengler et al.,

2009). GSK3β can also phosphorylate CRY2 and regulate its stability; however, this

phosphorylation requires synergistic action of another kinase, dual-specificity tyrosine-

phosphorylated and regulated kinase 1A (DYRK1A) to prime CRY2 for subsequent GSK3β

phosphorylation (Harada et al., 2005; Kurabayashi et al., 2010). GSK3β can phosphorylate and

stabilize REV-ERB, thus inhibiting Bmal1 transcription indirectly (Yin et al., 2006). Lastly,

GSK3β can phosphorylate PER2 and regulate its nuclear entry (Iitaka et al., 2005).

Physiologically, inhibition of GSK3β in vitro by small molecular inhibitor or lithium can cause

the period to either shorten or lengthen, respectively. Contradictory results obtained from

different types of inhibitors likely arise from differences in the specificity of the inhibitor used

and the complexity of regulation by GSK3β on both positive and negative elements of the

circadian clock (Hirota et al., 2008; Iitaka et al., 2005; Yin et al., 2006). Physiologically, GSK3β

heterozygous mice exhibit longer circadian locomotor activity rhythms (Lavoie et al., 2013).

Taken together, GSK3β can interact with different clock proteins as shown in numerous in vitro

studies. However, its precise physiological role in circadian clock in vivo remains to be clearly

defined.

Another kinase involved in the circadian clock is adenosine monophosphate (AMP)–activated

protein kinase (AMPK), a key regulator of metabolic function (Mihaylova and Shaw, 2011).

AMPK can regulate the phosphorylation of CRY1 and its subsequent degradation by FBXL3

(Lamia et al., 2009). AMPK can also indirectly regulate PER2 by phosphorylating and activating

CK1ε, leading to an increase in CK1ε activity and its degradation effect on PER2 (Um et al.,

2007). The implication of AMPK involvement in circadian clock opens up the possibility of

crosstalk between metabolism and circadian clock mechanisms.

10

1.5.4 Ubiquitination by FBXL family in the circadian clock

Ubiquitination in the mammalian circadian clock has only been discovered during the past

decade, and only a handful of ubiquitination ligases have been identified so far. FBXL3 was

identified using a forward genetic mutagenesis screen in two separate studies, in which the

overtime and afterhour mutants exhibit an abnormally long period of 26-27 hours (Godinho et al.,

2007; Siepka et al., 2007). These mice carry a loss-of-function mutation in FBXL3, the ubiquitin

ligase responsible for degrading CRY. Studies have demonstrated that a decrease in FBXL3

activity stabilizes CRY, which attenuates oscillations of other clock genes and proteins. Without

the timely degradation of CRY proteins, inhibition of CLOCK-BMAL1-mediated transcription is

extended, thus prolonging the circadian cycle and period (Busino et al., 2007; Godinho et al.,

2007; Siepka et al., 2007). Another F-box protein that is a FBXL3 paralog, called FBXL21, also

regulates CRY turnover. In contrast to nuclear localized FBXL3, FBXL21 is expressed both in

the cytoplasm and nucleus, playing a dual role of promoting CRY degradation in the cytoplasm

during the day, and antagonizing the stronger degradation effect of FBXL3 in the nucleus at

night. This causes Fbxl21 mutant mice to have a shorter period than wild-type mice (Yoo et al.,

2013), and the fbxl21 mutation can partially rescue the long period phenotype of FBXL3 mutant

mice (Hirano et al., 2013; Yoo et al., 2013). These studies demonstrate the complexity of CRY

protein degradation in the circadian clock, where a single protein can be degraded by different

ligases depending on the time of day and subcellular location.

1.5.5 Ubiquitination by β-TRCP family in the circadian clock

β-TRCP1 and β-TRCP2 are components of Skp1-Cul1- F-box protein (SCF) ubiquitin ligase,

which ubiquitinates and degrades PER1 and PER2 in a CK1ε dependent manner in vitro (Eide et

al., 2005; Ohsaki et al., 2008; Reischl et al., 2007; Shirogane et al., 2005). Inhibiting β-TRCP1/2

by expressing a dominant negative form or by RNAi knockdown in fibroblasts can lead to

stabilization of PER proteins and a longer period with a dampened rhythm (Ohsaki et al., 2008;

Reischl et al., 2007; Shirogane et al., 2005). However, β-TRCP1 deficient mice show

comparable period to wild-type controls with normal response to phase shifting light pulses

(Ohsaki et al., 2008), suggesting that β-TRCP1 is dispensable for clock timing in the SCN,

potentially as a result of functional redundancy with β-TRCP2.

11

1.5.6 Other ubiquitin ligases implicated in the circadian clock

In addition, ubiquitin ligase Arf-bp1 and Pam have been shown to ubiquitinate and degrade the

secondary core clock feedback loop component, REV-ERBα. By knocking down Arf-bp1 and

Pam, REV-ERBα is stabilized and, in turn, suppresses expression of BMAL1 and its downstream

activation of other clock genes (Yin et al., 2010). Although most identified ubiquitin ligases act

on the repressors of the circadian clock, an ubiquitin ligase, UBE3A, has been shown to

ubiquitinate BMAL1 in vitro and trigger its degradation (Gossan et al., 2014). Knocking down

UBE3A in vitro can lead to period lengthening, reduction in amplitude and eventual loss of

rhythms altogether. In summary, regulation of protein degradation within both the positive and

negative limbs of the molecular core clock is crucial for circadian clock timing.

12

Figure 1. Simplified model of mammalian molecular clock with ubiquitin ligases

CLOCK and BMAL1 are transcription factors at the center of the primary feedback loop that

activates transcription of the elements of the negative limb, per and cry. PER and CRY proteins

then heterodimerize and translocate to the nucleus to inhibit CLOCK-BMAL1-activated

transcription, completing the primary feedback loop. The secondary loop consists of rev-erbα

and rorα, whose transcription is activated by CLOCK-BMAL1. RORα and REV-ERBα feedback

to the primary loop by activating or repressing the transcription of bmal1, respectively. The

protein kinases CK1 and CK2 are crucial for phosphorylation of PER proteins, leading to their

subsequent degradation by β-TRCP1/2. Other ubiquitin ligases involved in the circadian clock

are also highlighted. Together, FBXL3 and FBXL21 control degradation of CRY proteins.

Degradation of REV-ERBα is controlled by Arf-bp1 and Pam. Ubiquitin ligase UBE3A has also

recently been implicated in the degradation of BMAL1.

13

1.6 Ubiquitin Proteasome System

Protein degradation is an important process in controlling the appropriate levels of protein in a

cell, and thus plays a fundamental role in controlling clock oscillations at the single cell level

(Gallego and Virshup, 2007). One of the major degradation pathways in eukaryotes is the

ubiquitin proteasome pathway. This pathway recognizes specific proteins for degradation

through ubiquitination, a process that involves 3 distinct enzymes: the ubiquitin-activating

enzyme (E1), which covalently attaches a ubiquitin protein onto itself; the ubiquitin-conjugating

enzyme (E2), to which the ubiquitin moiety is transferred from E1; and the ubiquitin ligase (E3),

which recognizes a specific substrate protein and, with the aid of E2, transfers ubiquitin onto its

final target. The ubiquitinated protein bears a poly-ubiquitin tag that is recognized by the 26S

proteasome and is ultimately degraded (Glickman and Ciechanover, 2002). The specificity of

this pathway is determined by the E3 ubiquitin ligase: there are more than 600 known E3 ligases

that each recognizes a specific subset of substrates and follows distinct rules of recognition (Li et

al., 2008; Nagy and Dikic, 2010).

1.7 The N-end Rule pathway and N-recognins

The evolutionarily conserved N-end rule pathway is a specific mode of recognition in the

ubiquitin proteasome system that recognizes a specific residue on the N-terminus of the target

protein (Varshavsky, 1997). The N-end rule states that the stability of the substrate protein is

dictated by the nature of a specific residue on their N-terminus (Bachmair et al., 1986). Ubiquitin

ligases that mediate the N-end rule pathway recognize their specific substrate by binding to an N-

terminal degradation signal, termed the N-degron. The N-degron is composed of a destabilizing

N-terminal residue, an internal Lys residue for the attachment of poly-ubiquitin chain and an N-

terminal extension (Varshavsky, 1996). These N-degrons are recognized by ligases that are

collectively termed N-recognins. In mammals, there are four N-recognins identified so far

(Tasaki et al., 2005). These N-recognins belong to a family of UBR proteins (UBR1-7) and are

characterized by the distinct UBR box, a domain that encodes for a ~70 residue long zinc finger

like domain that serves as the substrate recognition domain in the N-end rule pathway (Tasaki et

al., 2009). Although all UBR family proteins contain a UBR box, only UBR1, 2, 4 and 5 can

participate in the N-rule pathway that recognizes N-degrons (Tasaki et al., 2009). N-recognins

14

can also contain other substrate binding sites in addition to the UBR box domain, called N-

domain, which together can bind to both type 1 basic and type 2 bulky hydrophobic destabilizing

N-terminal residues (Tasaki et al., 2009). Besides containing the UBR box, N-recognins

generally contain signature sequences, such as RING and HECT domains, which are unique to

E3 ubiquitin ligases or substrate recognition subunits of an E3 complex. The exception is UBR4,

which contains no known E3 recognition sequences apart from the UBR box (Tasaki et al.,

2012).

1.8 UBR4 and its known function

Ubiquitin protein ligase E3 component N-recognin 4 (UBR4) is an exceptionally large protein

(~570kDa) whose physiological function in adult mammals is largely unknown. Furthermore,

apart from my thesis work, UBR4 has not yet been implicated in the circadian timing mechanism.

In the past decade, various studies have implicated its importance at the cellular level and during

animal development. UBR4 is extremely important for embryogenesis, as knockout of UBR4

leads to embryonic lethality caused by defects in brain, vascular and cardiac development

(Nakaya et al., 2013; Shim et al., 2008; Tasaki et al., 2013). As an N-recognin, UBR4 can

participate in the N-end rule pathway by recognizing both type 1 and type 2 degrons and target

specific substrates with N-degrons for degradation (Tasaki et al., 2005). However, UBR4 has

recently been shown to be involved in non-selective lysosomal degradation: in the yolk sac,

UBR4 associates with autophagic cargoes that mediate bulk/non-specific proteolysis during

development (Tasaki et al., 2013).

At the cellular level, UBR4 has been implicated in integrin-mediated signaling that affects

membrane morphogenesis and cell apoptosis (Nakatani et al., 2005). UBR4 also plays a role in

cell metabolism, where it degrades ATP-citrate lyase (ACLY), a protein that couples energy

metabolism with fatty acid synthesis and supports cell growth (Lin et al., 2013). In neurons,

UBR4 has been described as a microtubule-associated protein responsible for neuronal

differentiation and migration (Shim et al., 2008). In addition, UBR4 can complex with the

calcium sensor protein, calmodulin, to influence neuronal survival, and can regulate

neurogenesis by affecting mitotic spindle orientation in neuronal progenitor cells (Belzil et al.,

2014; Belzil et al., 2013). Another study found that UBR4 ubiquitinates and degrades a renal

15

calcium channel named transient receptor potential cation channel subfamily V member 5

(TRPV5), playing a role in calcium homeostasis in renal epithelial cells (Radhakrishnan et al.,

2013).

Furthermore, UBR4 seems to be a common target hijacked or utilized by viruses during infection

in cells. UBR4 can interact with cancer related proteins, including retinoblastoma tumor

suppressor protein pRB and human papillomavirus type 16 E7 and E6 oncoprotein, playing a role

in anchorage-independent growth and cancer cell growth (DeMasi et al., 2005; Huh et al., 2005;

Thomas et al., 2013; White et al., 2012). UBR4 also plays a role in dengue virus infection, where

it is leads to STAT2 degradation and assists the virus in evading immune system detection

(Morrison et al., 2013).

Homologs of UBR4 exist in other model organisms including Drosophila (named PUSHOVER

or POE) and Arabidopsis (named BIG). PUSHOVER in drosophila is involved in

neurotransmitter release process, neuronal excitability and synaptic vesicle fusion in

neuromuscular junction and perineural glial growth (Richards et al., 1996; Xu et al., 1998; Yager

et al., 2001). BIG in Arabidopsis was identified by two independent gene mutations, in which the

big mutant exhibit altered response to hormones and light with impaired auxin transport (Gil et

al., 2001). Interestingly, a recent linkage analysis identified ubr4 as candidate gene that causes

episodic ataxia in humans, indicating a potential function of UBR4 in humans (Conroy et al.,

2014). Taken together, UBR4 is involved in multiple functions during development and in cells

and has important physiological functions in different organisms.

1.9 Rationale

Although different studies have implicated the ubiquitin-proteasome pathway in the regulation of

clock timing mechanism, how this specific degradation pathway might play a role in photic

entrainment of the circadian clock remains elusive. To this end, in a collaboration study between

the laboratory of Dr. Figeys and ours, proteome-wide screens for proteins in the murine SCN that

are differentially regulated after nocturnal light exposure have been performed (Tian et al.,

2011). The expression of an E3 ubiquitin ligase that belongs to the N-end rule pathway named

ubiquitin protein ligase E3 component N-recognin 4 (UBR4) was greatly upregulated upon light

16

stimulation at night time, suggesting a possible role for this ubiquitin ligase in the regulation of

photic entrainment. Hence, we decided to characterize the role of UBR4 in circadian clock

functioning and photic entrainment in this thesis.

1.10 Thesis Objective

The goal of this thesis is to characterize this newly identified ubiquitin ligase UBR4 in the

murine SCN. First, UBR4 expression across the circadian cycle and in response to light

stimulation will be determined in the SCN. At the physiological level, I will use subject ubr4

heterozygous mutant mice to different circadian paradigms to characterize its circadian

behavioral phenotype. At the molecular level, I will utilize in vivo and in vitro methods to find

out how UBR4 might participate in the regulation of molecular clock timing mechanisms and to

identify potential targets of UBR4 that may link it directly to the molecular clock.

17

2 Methods and Materials

2.1 Animals

Mice with ubr4 disruption were generated using a gene-trap approach by inserting the NEO

cassette into the ubr4 gene locus. No ubr4 knockout mice (ubr4-/-

) have been obtained as they are

embryonic lethal (Nakatani et al., 2005; Shim et al., 2008; Tasaki et al., 2013) and all ubr4

mutant animals used in experiments were ubr4 heterozygous (ubr4+/-

) mice that display no overt

phenotype relative to their wild-type littermate (ubr4+/+

) controls. per2-/-

(Zheng et al., 1999)

mouse strains were crossed with ubr4+/-

to obtain the per2-/-

ubr4+/-

mice, with per2-/-

ubr4+/+

mice

being used as controls in indicated experiments. Except for behavioral experiments, mice were

group-housed (up to 5 animals per cage) in polycarbonate cages with ad libitum access to food

and water throughout the experiment. All procedures followed the guidelines of the Canadian

Council on Animal Care and animal protocols were approved by the Local Animal Care

Committees at the University of Toronto Mississauga.

2.2 Behavioral analysis and circadian paradigms

Male mice aged 6-8 weeks at the beginning of the experiment were individually housed in cages

with free access to a running wheel placed inside a light-tight ventilated circadian activity

chamber with computer-controlled light schedules (Phenome Technologies Inc.) and wheel-

running activity was monitored by Clocklab (Actimetrics). All mice were initially entrained to a

12h-light:12h-dark (LD) cycle for at least a week before any subsequent behavioral experiments.

Light intensity at the cage-level was ~30 lux unless stated otherwise.

2.2.1 Constant darkness paradigm and phase shifting light pulse

After entrainment to a fixed LD cycle, mice were released to complete constant darkness (DD) to

measure free running period. Circadian time (CT) 12 was defined as onset of locomotor activity

in constant darkness. After 2 weeks in DD, mice received a brief light pulse (~80 lux) for 15

18

minutes at CT 15 in a separate cabinet and thereafter returned to the recording chamber (in DD).

After another 2 weeks in DD, the same cohort of mice received another brief light pulse (~80

lux) for 15 minutes at CT 22. Period was measured with chi-square periodogram with 10 days of

locomotor activity after 5 days of initial adjustment to phase changes with Clocklab software

(Actimetrics). Arrhythmicity in DD was defined as the absence of a single significant peak in the

chi-square periododram. Light pulse-induced phase shift was measured using the Clocklab

software by fitting regression lines through the 10 days of locomotor activity onset before and

after the light pulse (e.g. day 5 – 15 after the light pulse) and quantified as the displacement

between the two regression lines.

2.2.2 Phase shift experiments

After entrainment to the initial LD cycle (8am lights on -8pm lights off), the LD cycle was

abruptly advanced by 7 hours by shortening the duration of the last dark phase of the original LD

cycle to 5 hours (i.e. 12h-light:5h-dark)(8am lights on – 8pm lights off – 1am lights on). The LD

cycle was then resumed to 12h-light:12h-dark with the light and dark onsets occurring 7 hours

earlier than in the initial cycle (1am lights on – 1pm lights off). After stable entrainment to the

new LD cycle for at least 2 weeks, the LD cycle was then delayed by 7 hours by extending the

last dark phase of the current LD cycle for an additional 7 hours (i.e. 12h-light:19h-dark) (1am

lights on – 8pm lights off – 8am lights on) and then resuming to a 12h-light:12h-dark cycle (8am

lights on: 8pm lights off). Cumulative phase shifts in the advanced or delayed LD cycle were

determined by measuring the difference between daily onset of locomotor activity and predicted

onset based on the previous LD cycle (phase advance was plotted as positive phase shift and

phase delay was plotted as negative phase shift) for 10 days. Days to entrain were determined

when onset of locomotor activity is stable near the dark onset of the new LD cycle schedule.

2.2.3 Chronic jetlag paradigm

After entrainment to the initial LD cycle, mice were subjected to a Chr6/2 chronic jetlag protocol

detailed elsewhere (Casiraghi et al., 2012). In short, LD cycle was abruptly advanced by 6 hours

every 2 days by shortening the dark phase of the second cycle by 6 hours. Mice were maintained

19

in the jetlag protocol for 4 weeks and subsequently released into DD. Period and circadian

amplitude were measured with chi-square periodogram with 3 weeks of locomotor activity

discarding the first week of jetlag protocol with Clocklab software. DD period was measured as

stated earlier in constant darkness paradigm.

2.2.4 Constant light paradigm

After stable entrainment to a fixed LD cycle, mice were released to constant light (LL) (12h-

light:12h-light) with stepwise increase in light intensity starting from 5 lux. Light intensity was

then increased every 2 weeks in LL to 10lux, 20lux and 30lux at ZT 12 from the initial LD cycle.

Period and circadian amplitude were measured using the Clocklab software with 2 weeks of

locomotor activity for the each light intensity. Arrhythmicity in LL was determined when a

significant peak was not obtained from the chi-square periodogram.

2.3 Cell culture and transfection

Mouse neuroblastoma N2A cells were maintained in Dulbecco’s Modified Eagle Medium

(DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Wisent) and penicillin

streptomycin (Gibco) in 37oC with 5% CO2 incubator. Cells were plated onto 6 well plates

(western blotting) or on coverslips in 24 well plates (immunocytochemistry) the day before

transfection to reach 80-90% confluence on the day of transfection. Cells were transfected using

Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction with the following

constructs or siRNA: V5-Per1, V5-Per2, V5-Cry1, V5-Cry2 (generous gift from N. Cermakian),

siRNA specific to UBR4 (ON-TARGET plus SMARTpool Human ZUBR1, ThermoFisher

Scientific), and a non-specific control siRNA (ON-TARGET plus Non-targeting control pool,

ThermoFisher Scientific). Subsequent processing or drug treatments were done 24 hours post

transfection. Tetrabromocinnamic acid (TBCA) (Sigma) dissolved in DMSO was applied for 3

hours to the culture, 24 hours after transfection with V5-Per1 and siRNA.

20

2.4 Tissue harvesting

All mice were entrained to 12:12h LD cycle for at least two weeks, and transferred to DD for 2

cycles prior to tissue harvest for immunohistochemistry, immunofluorescence and western

blotting studies. Mice were killed by cervical dislocation, and decapitated under dim red light.

Eyes were covered with black electrical tape during brain dissection, and subsequently immersed

in cold oxygenation media and sliced into a 800m coronal section containing the SCN using a

vibratome. For immunohistochemistry and immunofluorescence studies, coronal brain sections

were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH7.4, for 6 hours at

room temperature (tissues for UBR4 immunostaining were fixed for 4 hours at 4oC), transferred

to 30% sucrose in PBS (4oC, overnight incubation), thin sectioned (30m) on a freezing

microtome, and stored in 30% sucrose PBS (4oC) until use. For Western blotting, SCN tissues

were manually dissected with a razor blade immediately after vibratome sectioning, frozen on

dry ice and stored in -80oC until use.

2.5 Western blotting

SCN tissues were homogenized using 26G X 5/8 needle in ice-cold RIPA lysis buffer (50mM

Tris-Cl pH8, 150mM NaCl, 2mM EDTA pH8, 1% NP-40, 0.5% sodium deoxycholate, 0.1%

SDS) supplemented with protease inhibitor cocktail (Sigma) and 1mM sodium orthovanadate

immediately before use. Homogenized lysates were incubated on ice for 20 mins, then

centrifuged at 17,000g 4oC for 20 mins and the supernatant was kept as protein samples on ice.

Protein samples were quantified by Bradford protein assay using Coomassie Plus Protein Assay

Reagent (Fisher Scientific). Protein samples were prepared with SDS loading buffer with 1mM

DTT and heated at 95oC for 5 mins. Samples (20-40ug) were resolved using 5% - 12% Tris-

Glycine sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted onto PVDF

membrane (Immobilon-P, Fisher Scientific) for 2 hours or overnight by wet transfer. Blotted

membrane was blocked in 5% milk in Tris-buffered saline pH7.4 with 0.05% Triton-X (TBST)

for 1 hour and incubated in primary antibody overnight in 4oC. The following antibodies were

used: rabbit anti-UBR4 (Sigma-Aldrich: HPA021046, 1:500), rabbit anti-actin (Sigma-Aldrich:

A2066, 1:20000), rabbit anti-CK2β (abcam: ab133576, 1:2000) and mouse anti-V5 (abcam:

ab27671, 1:2000). The following day, membrane was washed five times with TBST for 5 mins

21

each and incubated in HRP-conjugated goat anti-mouse or anti-rabbit IgG (H+L) secondary

antibody (Thermo Scientific: 31430, 31460, 1:200,000) for 2 hours in room temperature.

Membrane were then washed five times in TBST for 5 mins each and developed. Signals were

detected by chemiluminescence with SuperSignal West Femto maximum sensitivity substrate

(ThermoFisher Scientific) on films.

2.6 Immunofluorescence

SCN sections were permeabilized by washing five times in phosphate-buffered saline pH7.4 with

0.05% Triton-X (PBST) for 5 mins each. Sections were blocked in 10% horse serum in PBST for

1 hour and incubated in primary antibody overnight in 4oC. The following antibodies were used:

rabbit anti-UBR4 (Sigma-Aldrich: HPA021046, 1:100), guinea-pig anti-AVP (Peninsula

Laboratories: T-50548, 1:4000), guinea-pig anti-VIP (Peninsula Laboratories: T-5030, 1:3000).

The following day, sections were washed five times with PBST for 5 mins each and incubated in

Alexa Fluor 488-conjugated rabbit (Invitrogen: A11034, 1:1000) and DyLight 594-conjugated

guinea-pig (Jackson ImmunoResearch: 706515148, 1:500) secondary antibodies for 2 hours in

room temperature. Sections were washed five times in PBST for 5 minutes each and mounted on

microscope slides with mounting media Dako fluorescent mounting medium (Dako).

2.7 Immunohistochemistry

SCN sections were permeabilized by washing five times in PBST for 5 mins each. Sections were

then incubated in 0.3% hydrogen peroxide in PBS for 20 mins and washed five times in PBST

for 5 minutes each. Sections were blocked in 10% goat serum in PBST for 1 hour and incubated

in primary antibody overnight in 4oC. The following antibodies were used: rabbit anti-Per1

(generous gift from S. Reppert), rabbit anti-Per2 (generous gift from D. Weaver), guinea pig

anti-AVP (Peninsula Laboratories: T-50548, 1:75000), guinea pig anti-VIP (Peninsula

Laboratories: T-5030, 1:25000). The following day, sections were washed five times with PBST

for 5 mins each and incubated in biotinylated anti-rabbit or anti-guinea pig IgG (H+L) secondary

antibodies (Vector Laboratories: BA-1000, BA-7000, 1:300) for 2 hours in room temperature.

Sections were washed five times in PBST for 5 minutes each and detection was achieved using

22

HRP-ABC method with Vectastain Elite ABC kit and peroxidase DAB substrate kit (Vector

Laboratories) according to manufacturer’s instructions.

2.8 Immunocytochemistry

Cells on coverslips were fixed in 4% paraformaldehyde for 20 mins at room temperature 24-48

hours post transfection or drug treatment. After two quick washes with PBS, cells were

permeabilized by washing three times with PBST for 5 mins each. Cells were then blocked with

10% horse serum in PBST for 1 hour and incubated overnight in 4oC in mouse anti-V5 antibody

(abcam: ab27671, 1:2000). The following day, cells were washed five times in PBST for 5 mins

each and incubated in Alexa Fluor 594-conjugated mouse secondary antibodies (Invitrogen:

A21203, 1:1000) for 2 hours in room temperature. Cells were washed five times in PBST for 5

mins each and incubated with DAPI fluorescent stain in PBS (1:10000) for 5 minutes. Cells were

washed 3 times with PBS for 5 mins each and mounted on slide with mounting media Dako

fluorescent mounting medium (Dako).

2.9 Microscopy imaging

Images were acquired using a Zeiss Axio Observer Z1 inverted microscope equipped with a

Laser Scanning Microscope (LSM) 700 module and Zeiss LSM510 laser scanning confocal

microscope (Carl Zeiss MicroImaging GmbH). Images were acquired with ZEN 2008 and ZEN

2010 software (Carl Zeiss MicroImaging GmbH). For immunofluorescence and

immunocytochemistry studies, low magnification images were obtained with a 20x objective

(Plan-Apochromat 20X/0.8 M27 and EC Plan-Neofluar 20x/0.5) and high magnification images

were obtained with a 63x oil immersion objective (Plan-Apochromat 63x/1.4). For 20X

magnification for immunofluorescence studies, a Z-stack was acquired with 2.3-µm optical

sections with 7 optical slices. Z-stacks of the same magnification were acquired with all confocal

parameters (laser intensity, gain, pinhole size, scanning speed, and image averaging) held

constant for each experiment. For immunocytochemistry, a single plane image was acquired with

2µm optical sections. For immunohistochemistry studies, images were obtained with 10X and

23

20X magnification with light microscopy in LSM700 and acquired with the same light intensity

setting for each antibody staining.

2.10 Image Processing and Quantification

Microscope images were processed and analyzed with Image J (Rasband, W.S., ImageJ, U. S.

National Institutes of Health, http://imagej.nih.gov/ij/). For immunofluorescence study of UBR4

expression, each Z-stack was separated into individual acquisition channels (488 nm or 594 nm

for UBR4 and AVP, respectively), and each channel was then re-stacked. For the 20x images, a

maximum intensity projection of the entire Z-stack was produced of the SCN section for

quantification. For each SCN at 20x, fluorescence intensity values for individual UBR4-

expressing cells were obtained by measuring the grayscale intensity in a fixed circular shaped

region of interest for each visible cell. Background of each SCN section was obtained by

measuring the grayscale intensity in a region of interest without any visible UBR4 cells. Mean

normalized fluorescence intensity for each SCN section was calculated by subtracting the

background intensity from mean of fluorescence intensity values for all cells measured. Numbers

of UBR4-expressing cells in the SCN were also manually counted at 20x using maximum

intensity projection of the Z-stack. Every cell with immunofluorescent signal exceeding

background staining was included in the count. Three sections were chosen per animal in each of

the rostral, middle and caudal portion of the SCN for each time point analyzed. The mean

normalized fluorescence intensity of individual cells and mean cell count of SCN were analyzed

separately for the rostral, middle or caudal sections and mean values were computed and reported

according to time point or treatment and sections. For immunohistochemistry quantification,

average grayscale intensity was obtained for the area of the outlined SCN regions specified in

each different staining using the polygon tool. Background of each SCN section was obtained by

measuring grayscale intensity on each side of each SCN in the region of interest without specific

staining. Mean normalized grayscale intensity for each SCN was calculated by subtracting the

inverse of background intensity from inverse of outlined region intensity. For

immunocytochemistry quantification, average fluorescence intensity was obtained by outlining

each cell that had fluorescent signal exceeding the background staining. Mean intensity shown

was calculated for each treatment by averaging intensity of all cells quantified. For western blot

http://imagej.nih.gov/ij/

24

quantification, films were scanned and average grayscale intensity was quantified for each

indicated specific band using the polygon tool in ImageJ to trace the outline of band. Mean

intensity for each protein was normalized to actin as the loading control for each specific sample.

2.11 Statistical Analysis

For behavioral analysis, independent samples t-test was used to compare differences for

circadian parameters (period, phase shift and amplitude) between genotypes. For

immunofluorescence studies, mean intensity and cell counts were analyzed by a one-way

ANOVA comparing between different CT followed by Fisher’s least significant difference

(LSD) post hoc test where appropriate. Changes in mean cell intensity and cell count of UBR4-

expressing cells following light stimulation were analyzed by a two-way ANOVA with CT and

light treatment as the independent variables. Significant interactions were explored with

independent samples t-test with Bonferonni correction. For immunohistochemistry studies, mean

intensity was analyzed by two-way ANOVA comparing differences for time, genotypes and their

interactions. Each individual time point was analyzed with independent samples t-test between

genotypes. For immunocytochemistry, mean intensity was analyzed by one-way ANOVA

comparing between treatments (drug or siRNA). Alpha was set at 0.05 for all statistical analyses.

25

3 Results

Results from section 3.1 and 3.2 and Figure 2, 3 and 4 were extracted from published manuscript:

Ling HH, Beaulé C, Chiang C-K, Tian R, Figeys D, Hai-Ying M. Cheng (2014) Time-of-Day-

and Light-Dependent Expression of Ubiquitin Protein Ligase E3 Component N-Recognin 4

(UBR4) in the Suprachiasmatic Nucleus Circadian Clock. PLoS ONE 9(8): e103103.

doi:10.1371/journal.pone.0103103

My supervisor Hai-Ying Mary Cheng and I contributed to experimental design, conducted the

experiments, carried out the data analysis, made the figures and contributed to the writing for the

published manuscript. Results used in section 3.1 and 3.2 and Figure 2, 3, 4 from the published

manuscript are modified in this thesis. The mass spectrometry screen and analysis in the

published manuscript was done by Drs. Cheng-Kang Chiang, Ruijun Tian and Daniel Figeys.

Mass spectrometry screen in section 3.9 was done by collaborator Drs Cheng-Kang Chiang and

Daniel Figeys from University of Ottawa.

3.1 Temporal and spatial expression of UBR4 in the SCN

To examine the spatiotemporal expression of UBR4, I used immunofluorescence staining to

visualize UBR4 expression in the SCN of wild-type C57/BL6 mice. I quantified the expression

of UBR4 levels in the rostral, middle and caudal sections of the SCN across a circadian cycle.

Following 2 days in constant darkness, mice were killed at 3 hour intervals across a 24 hour

cycle. As shown in Fig. 2A and B, mean intensity of UBR4 expression in individual SCN cells

fluctuates in a time-of-day dependent manner as one-way ANOVA revealed a significant effect

of time on UBR4 fluorescent intensity. However, the time of day fluctuation in individual cell

intensity was only seen in the middle and caudal sections of the SCN (p<0.05), but not rostral

sections (p>0.05). UBR4 expression decreases throughout the day where it reaches nadir at CT10

before rising again in the early subjective night (CT13). Total number of UBR4 expressing cells

26

in the SCN was also quantified in the sections. Number of UBR4 expressing cells in the SCN

does not fluctuate according to the time of day regardless of the section (Fig. 2C) (p>0.05).

To confirm the mass spectrometry study that prompted our study of UBR4 in the SCN (Tian et

al., 2011), I investigated the light inducibility of UBR4. Two separate cohorts of mice were

adapted in DD for two days; one group received a brief light pulse (LP) (15min, 100lux) at CT6,

15 or 22 on day 3 of DD, whereas the other group that did not receive a light pulse served as dark

controls (DD). SCN tissues were harvested 4 hours post light pulse and compared with the DD

controls killed at the same time. Mean UBR4 expression intensity in individual SCN cells did not

significantly differ between LP and DD mice at all three time points tested (Fig. 3A,B)(p>0.05).

However, the total number of UBR4-expressing cells reveals a significant effect of light

treatment (p<0.05), where a light pulse in the early subjective (CT15) increased the abundance of

UBR4 expressing cells (p<0.05), but light in the mid subjective day (CT6) or late subjective

night (CT 22) did not (Fig. 3C) (p>0.05). Together, the data demonstrate that UBR4 expression

in the murine SCN fluctuates throughout the day and can be regulated by early night light pulse.

27

Figure 2. UBR4 time-of-day dependent expression profile in the SCN

(A)Representative micrographs showing the temporal and spatial expression profile of UBR4 in

the SCN. Mice were dark adapted for 2 consecutive days prior to tissue harvest at the indicated

circadian times. (B) Quantification of mean UBR4 intensity in individual cells in different

sections as a function of circadian time. Values were normalized to background staining. y-axis

represents mean intensity of UBR4 staining in individual cells in grayscale intensity units. n = 4

animals per time point. (C) Quantification of the number of UBR4-expressing cells in different

SCN sections as a function of circadian time. y-axis represents number of counted UBR4-

positive cells. n = 4 animals per time point. (Scale bar = 50 µm).

28

Figure 3. Light inducibility of UBR4 in the SCN in different time of day

(A) Representative micrographs showing the expression of UBR4 in rostral-middle section of the

SCN following a light pulse. Light pulses (LP) (15 min, 100 lux) were administered at CT 6, 15

and 22 and tissues were harvested 4 h later. Dark (DD) controls were killed at the same time

without prior light treatment. (B) Quantification of mean UBR4 intensity in individual cells in

the rostral-middle section of the SCN after a light pulse given at the indicated time. Values were

normalized to background staining. y-axis represents mean intensity of UBR4 staining in

individual cells in grayscale intensity units. x-axis represents the CT when the light pulse was

administered. (C) Quantification of the number of UBR4-expressing cells in the rostral-middle

section of the SCN after a light pulse given at the indicated time. y-axis represents number of

counted UBR4-positive cells. n = 4 animals per time point. *p<0.05 LP vs. DD control. (Scale

bar = 50 µm).

29

3.2 Co-localization of UBR4 with neuropeptides in the SCN

The SCN is regionally subdivided based on distinct expression patterns of different

neuropeptides. The two major regions of the SCN are termed the shell and core, characterized by

the expression of arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP),

respectively (Abrahamson and Moore, 2001). Since UBR4 has a distinct pattern reminiscent of

AVP expression in the shell SCN, I investigated whether UBR4 is expressed in AVP neurons in

the SCN. To confirm this, I used double immunofluorescence labelling to visualize the co-

localization between UBR4 and AVP. As shown in Fig 4A and B, UBR4 was detected in

virtually all AVP-positive cells in the SCN, but not in VIP neurons. To verify that the co-

localization was not due to cross-reactivity of our antibodies, I looked at other regions that have

high AVP expression such as the paraventricular nucleus (PVN) and the supraoptic nucleus

(SON) and saw no detectable co-localized UBR4 and AVP expression, suggesting that UBR4 is

specifically expressed within AVP-positive cells only in the SCN (Fig 4A).

30

Figure 4. UBR4 co-localization with neuropeptides in the SCN

(A) Expression of UBR4 (left-most column) and AVP (middle column) in the SCN,

paraventricular nucleus (PVN) and supraoptic nucleus (SON) were assessed by

immunofluorescence staining. The right-most column shows the merged image indicating co-

localized expression. UBR4 is expressed in AVP-positive cells of the SCN but was not detected

in AVP-positive cells of the PVN or SON. (B) Expression of UBR4 (left-most column) and VIP

(middle column) in the SCN. UBR4 is not expressed in VIP-positive cells in the SCN. For (A)

and (B), the boxed regions of the SCN are shown in higher magnification in the lower panels.

(Scale bar = 50 µm).

31

3.3 The response of ubr4+/-

mice to constant darkness paradigm and phase shifting light pulses

To study the effects of deficiency of UBR4 on the circadian clock, I used the ubr4+/-

mice as

homozygous ubr4-/-

mice are embryonic lethal. I confirmed that UBR4 expression in the SCN

was lower in ubr4+/-

mice using both Western blotting (Fig 5A) and immunofluorescence (Fig

5B, C). To determine whether UBR4 is important for circadian clock control and photic

entrainment, I examined how ubr4+/-

mice behave in constant darkness and in response to brief

light pulses at different times of the subjective night. All mice were rhythmic under constant

darkness and ubr4+/-

mice display an initial circadian period (first 2 weeks in DD) that was

comparable to ubr4+/+

control mice (ubr4+/+

:23.59 ± 0.06 hours, ubr4+/-

:23.58 ± 0.06 hours) (Fig

6A-E). Light exposure in the early and late subjective night can cause the circadian phase to

delay and advance, respectively (Johnson, 1999). A 15 minute light pulse was given at either

CT15 or CT22 under stable free-running conditions. Amount of phase shift was calculated as the

displacement between two fitted regression lines on the activity onset over multiple days before

and after the light pulse. Compared to wild-type controls, ubr4+/-

mice exhibit comparable phase

shifts in response to both early (CT15) (ubr4+/+

:-2.12 ± 0.23 hours, ubr4+/-

:-2.35 ± 0.20 hours)

(p>0.05) and late (CT22) (ubr4+/+

:1.75 ± 0.20 hours, ubr4+/-

:1.69 ± 0.34 hours) (p>0.05)

subjective night light pulses (Figure 6E), indicating that deleting a single copy of ubr4 is not

sufficient to alter light-induced phase shifting of the circadian clock. However, a significant

difference was observed in circadian period in the later portions (post CT22LP) of the

experiment between ubr4+/-

mice and ubr4+/+

controls (ubr4+/+

:23.49 ± 0.11 hours, ubr4+/-

:23.13

± 0.08 hours) (p<0.05) and also pre and post CT22LP in ubr4+/-

mice (ubr4+/-

post CT15LP/pre

CT22LP:23.47 ± 0.08 hours, ubr4+/-

post CT22LP:23.13 ± 0.08 hours) (p<0.05) (Figure 6F). This

suggests that either long-term constant darkness or multiple light disturbances in constant

darkness can significantly shorten the period in ubr4+/-

mice. To investigate this, we housed a

separate cohort of ubr4+/-

and ubr4+/+

mice in constant darkness for an extended period of time

(total of 6 weeks) without subjecting them to any light pulse and observed no difference in

circadian period (ubr4+/+

:23.57+0.07hours, ubr4+/-

: 23.64+0.13hours) (p>0.05) (data not shown).

Together, our results indicate that deleting one copy of the ubr4 gene is not sufficient to alter the

pace of the circadian clock under short-term free-running conditions, or to alter its phase-shifting

32

ability to nocturnal light exposure. Nevertheless, multiple light disturbances in constant darkness

have a greater period-shortening effect on ubr4+/-

mice than on controls.

Figure 5. UBR4 expression in the SCN of ubr4+/-

mice

(A) Western blot analysis of UBR4 protein levels in the SCN of ubr4+/+

and ubr4+/-

mice. The

intensity of UBR4 band in ubr4+/-

mice was greatly diminished. Actin was used as loading

control. Immunofluorescence staining of UBR4 in the SCN of ubr4+/+

(B) and ubr4+/-

(C).

Fluorescence intensity of UBR4 in the SCN was lower in ubr4+/-

mice.

33


mice to constant darkness and phase shifting light pulses

Representative actograms of ubr4+/+

(A, C) and ubr4+/-

(B, D) mice are shown. Mice were

entrained to a 12:12h LD schedule for 2 weeks before releasing to DD. Mice received a 15 min

(80 lux) light pulse at CT 15 (first red dot) and CT 22 (second red dot) when their circadian

period are stable. (B) Phase shift after CT15 and CT22 LP are shown. Phase delay is shown in

negative values and phase advance in positive values. (C) Circadian period before CT15 LP,

after CT15 LP before CT22 LP and after CT22 LP was calculated by chi-square periodogram. n=

9 animals per genotype. All values represent mean ± SEM. (*p<0.05)

34

3.4 ubr4+/-

mice response to phase shift paradigm

Since no apparent differences in phase shifting ability of short-term light pulse was observed in

the subjective night, I used another circadian behavioral paradigm with longer and more

disruptive light changes to observe whether UBR4 is important for photic entrainment. I

challenged ubr4+/+

and ubr4+/-

mice to an abrupt shift of 7 hours in the LD cycle. Both ubr4+/+

and ubr4+/-

mice were stably entrained to a LD cycle before an abrupt 7 hour advance of the LD

cycle was applied. After the mice had been stably entrained to the new advanced light cycle for

at least 2 weeks, the LD cycle was delayed 7 hours back to the original LD cycle (Fig 7A, B).

The rate of entrainment to the new LD cycle was assessed by the total number of days required

to stably entrain to the new LD cycle and by the cumulative phase shifts on each day following

the change in the LD cycle. I observed that ubr4+/-

mice required, on average, about 6.38 ± 0.58

days to advance to the new LD cycle, compared to 4.38 ± 0.30 days in ubr4+/+

mice (p<0.05).

Furthermore, the difference in re-entrainment rate was attributed to smaller shifts per day after

the LD cycle advancement as shown in Fig 7C. However, ubr4+/-

mice exhibit no difference in

entraining to a 7 hour delay in the LD cycle, taking a comparable 5.57 ± 0.45 days versus 5.28 ±

0.27 days in ubr4+/+

mice to entrain (p>0.05) and showing a similar entrainment rate (Fig 7D).

Together, these results demonstrate that UBR4 is important for entraining to advances, but not

delays, of the LD cycle.

35

Figure 7. The response of ub4+/-

mice to phase shift paradigm


(A) and ubr4+/-

(B) mice are shown. Light phases are

shaded in grey. Mice were initially entrained to 12:12h LD schedule before a 7 hour advance in

the LD cycle. After stable entrainment to the new LD cycle, mice were subjected to a 7 hour

delay of the LD cycle. Cumulative phase shift after advance (C) and delay (D) of the LD cycle

was calculated by comparing activity onset of each day after phase shift with predicted activity

onset based on the previous LD cycle. n = 8 animals per genotype. All values represent mean ±

SEM. (*p<0.05)

36

3.5 ubr4+/-

mice response to chronic jetlag paradigm

Since ubr4+/-

mice are phenotypically different from ubr4+/+

mice in their ability to adapt quickly

to an advance of their LD cycle (a model of acute jetlag), I wanted to observe how ubr4+/-

mice

would behave in a chronic jetlag setting that applies a more chronic stress to the circadian clock.

I subjected ubr4+/-

mice to a chronic jetlag protocol for 4 weeks, where the light-dark cycle was

advanced by 6 hours every 2 circadian cycles by shortening the dark phase of the second cycle

by 6 hours. Previous studies have shown that about half of the mice that underwent this 6/2

chronic jetlag protocol would have a desynchronized clock that displays two split rhythms: a

short period (~21hours) that follows the jetlag schedule and a longer period that is approximately

24 hours. The rest would either have 3 different periods (21, 23 and 24 hours), only the short

period or only the long period (Casiraghi et al., 2012). For ubr4+/-

mice that experienced this

jetlag protocol (Fig 8A-D), I observed that ubr4+/-

mice showed comparable period to wild-type

mice with either the ~21 hour period that followed the advanced light-dark schedule (ubr4+/+

:

21.08 ± 0.03hours (4/9 mice), ubr4+/-

: 21.08 ± 0.05hours(4/9 mice)) or the ~24 hour period that

does not entrain to the jetlag schedule (ubr4+/+

: 23.95±0.15 hours (5/9 mice), ubr4+/-

: 23.60 ±

0.14hrs (5/9 mice)) (Fig 8E). However, the amplitude of the period during jetlag obtained from

chi-square periodogram analysis is lower in ubr4+/-

mice in both the 21 and 24 hour groups, and

reaching statistical significance in the 24 hour period group (Fig 8F) (p<0.05). When the chronic

jetlagged mice were released to constant darkness at the end of the experiment, ubr4+/+

and

ubr4+/-

mice again showed a comparable period (ubr4+/+

: 23.58±0.10 hours, ubr4+/-

: 23.56 ± 0.06

hours). This indicates that the circadian clock in ubr4+/-

mice is disrupted only when exposed to

chronic jetlag setting, but can quickly return to normal when released back to constant darkness.

37


mice to chronic jetlag paradigm

Representative actograms of (A) ubr4+/+

mice with 21 hour period (B) ubr4+/-

mice with 21 hour

period (C) ubr4+/+

mice with 24 hour period (D) ubr4+/-

mice with 24 hour period, during the

chronic jetlag protocol. Light phase was shaded in grey. Mice were entrained to a 12:12h LD

schedule before release to the chronic jetlag paradigm. During the jetlag paradigm, the LD cycle

was advanced by 6 hours every 2 days by shortening the dark phase by 6 hours for a total of 4

weeks. (E) Summary of circadian periods obtained from chi-square periodogram analysis during

the jetlag paradigm for both genotypes, separated by whether mice exhibit a 21 or 24 hour

period. (F) Summary of circadian period amplitude obtained from chi-square periodogram

analysis during the jetlag paradigm. n = 9 animals combined for both hour group per genotype.

All values represent mean ± SEM. (*p<0.05)

38

3.6 ubr4+/-

mice response to constant light paradigm

To further explore the behavioral phenotype of ubr4+/-

mice in chronic light disturbances, I

examined ubr4+/-

mice in constant light (LL) conditions to observe how chronic and constant

light would disturb the circadian clock in mice with only one copy of the ubr4 gene. In LL,

nocturnal mice would begin to free-run with a lengthened period of more than 24 hours with

decreased period amplitude that will eventually become arrhythmic after extended period of time

in constant light (Aschoff, 1960; Golombek and Rosenstein, 2010). I subjected both ubr4+/+

and

ubr4+/-

mice to a prolonged period in LL condition starting at 5 lux light intensity, with a gradual

increase in light intensity every 2 weeks for a total of 9 weeks to a maximum of 30lux (Fig 9A-

F). ubr4+/+

mice continued to lengthen their period throughout LL with increasing light intensity

(Figure 9A,D), whereas ubr4+/-

mice exhibited a wide range of phenotypes during the constant

light paradigm, ranging from shorter than 24 hour period (Figure 9C), constantly varying period

(Figure 9F) to complete arrhythmicity at a quicker rate (Figure 9B,E). In addition, when I

analyzed the circadian amplitude from chi-square periodogram, a significant difference was

detected between genotypes starting at all light intensities (10 lux, 20 lux and 30 lux p<0.05)

except 5 lux (p>0.05). ubr4+/-

mice had a much lower circadian amplitude under LL, similar to

the chronic jetlag paradigm (Fig 9G). Collectively, chronic light disturbances have profound and

unpredictable consequences on the circadian clock of ubr4+/-

mice, causing them to behave in a

vastly different and more unstable way than control mice.

39


mice to constant light.


(A, D) and ubr4+/-

(B, C, E, F) mice are shown. Mice were

entrained to a 12:12h LD schedule (30lux) prior to release to constant light (LL). The light

intensity in LL started at 5 lux and increased every 2 weeks to 10 lux, 20 lux and 30 lux for a

total of 9 weeks in LL. (G) Summary of circadian period amplitude obtained from chi-square

periodogram analysis during constant light paradigm at different light intensities. n = 8 animals

per genotype. All values represent mean ± SEM. (*p<0.05)

40

3.7 Molecular clock protein PER1 and PER2 expression in ubr4

+/- mice SCN

PERIOD1 and PERIOD2 are core clock proteins that act as repressors of the molecular clock.

PER1 and PER2 are both expressed rhythmically in the SCN and peak in the early/mid

subjective night (Field et al., 2000). To see whether reduced levels of UBR4 alter the core

molecular clock, I examined the expression of PER1 and PER2 in the SCN throughout a 24 hour

circadian cycle after two days in constant darkness. Using immunohistochemistry (IHC), I

observed that PER1 expression in the whole SCN was comparable between ubr4+/+

and ubr4+/-

mice, where a significant time difference was detected as expected (p<0.05), but no difference

was detected for genotype or interaction (Fig 10A, B) (p>0.05). PER1 expression in the

respective shell and core SCN also showed a significant time effect (p<0.05), but again no

difference was detected for genotype or interaction (p>0.05). However, PER2 expression levels

in the whole SCN were altered in ubr4+/-

mice, as a significant difference was detected for time,

genotype and interaction (p<0.05). ubr4+/-

mice showed an earlier and significant increase in

PER2 expression at CT 10, with a broad peak and a higher expression at CT18 (Fig 10 D, E).

Upon closer examination, the difference in PER2 expression levels was attributed primarily to

the shell. Significant time effect was detected in both shell and core for PER2 expression

(p<0.05), where significant differences in genotype and interaction were only detected in the

shell SCN (p<0.05) but not the core (p>0.05). The expression levels of PER2 were significantly

different at CT10, CT18 and CT22 for the whole SCN, and CT10, CT18, CT20 and CT22 for the

shell SCN (p<0.05). As a result, our data suggests that UBR4 plays a role in the regulation of

core circadian clock, where mice that express lower levels of UBR4 exhibit a modest difference

in PER2 oscillation pattern.

41

Figure 10. PER1 and PER2 expression in the SCN of ubr4+/-

mice

Representative IHC staining of PER1 (A) and PER2 (D) in the SCN across the circadian cycle.

Quantification of relative PER1 (B) and PER2 (E) staining intensity in the whole SCN were

normalized to background staining and plotted as a function of circadian time for ubr4+/+

and

ubr4+/-

mice. (C) Outline of SCN regions (shell and core) and whole SCN used for measuring

intensity of staining. n = 3-4 animals per genotype per time point. All values represent mean ±

SEM of normalized grayscale intensity. (*p<0.05)

42

3.8 SCN neuropeptide expression in ubr4+/-

mice

AVP and VIP are important neuropeptides in the SCN (Abrahamson and Moore, 2001). Both

peptides play different roles in coupling of individual SCN neurons and are important for

producing a single rhythmic output (reviewed in section 1.2.2). As UBR4 co-localizes with AVP

in the SCN neurons (Fig 4A), I was interested in whether AVP rhythm would be altered when

the expression of UBR4 is reduced. Therefore, I used IHC to quantify the overall expression

intensity of AVP in SCN neurons across the circadian day after two days in constant darkness.

AVP neurons are localized to the shell of the SCN, and have projections along the third ventricle

(Fig 11A). Expression of AVP in the whole SCN changes throughout the day as a significant

time effect was detected (p<0.05), but no difference was detected in genotype or interaction

(p>0.05) (Fig 11C). Similar results have been obtained for AVP expression in the shell SCN,

where a significant time effect (p<0.05) was detected but not genotype or interaction (p>0.05).

VIP neurons are located within the core SCN and projects to the SCN shell (Fig 11B). VIP levels

are generally suppressed in the light portion of normal light-dark cycle, but remain stable in

constant conditions (Okamura et al., 1995). As expected, VIP expression levels in the whole and

core SCN after 2 days in constant darkness did not exhibit a circadian rhythm and were

comparable between ubr4+/+

and ubr4+/-

mice, as no difference was detected between time,

genotype or interaction (Figure 11D) (p>0.05). To conclude, neuropeptide expression in the

SCN does not appear to differ substantially between ubr4+/+

and ubr4+/-

mice in constant

darkness.

43

Figure 11. AVP and VIP expression in the SCN of ubr4+/-

mice

Representative IHC staining of AVP (A) and VIP (B) in the SCN across the circadian cycle.

Quantification of relative AVP (C) and VIP (D) staining intensity in the whole SCN were

normalized to background staining and plotted as a function of circadian time for ubr4+/+

and

ubr4+/-

mice. n = 3-4 animals per genotype per time point. All values represent mean ± SEM of

normalized grayscale intensity. (*p<0.05)

44

3.9 CK2β as a potential target of UBR4 in vivo and in vitro

To find out how UBR4 can help control circadian rhythmicity in the SCN, we conducted a mass

spectrometry screen to probe for targets of UBR4 using SCN tissues collected from ubr4+/+

and

ubr4+/-

mice after two days in constant darkness. This screen led us to investigate the regulatory

subunit of casein kinase 2, CK2β. CK2 is a kinase that has been implicated in the mammalian

circadian clock, and was previously shown to alter circadian period in vitro by affecting PER2

stability (Maier et al., 2009; Tsuchiya et al., 2009). CK2β was found to be higher in ubr4+/-

mice

in the mass spectrometry screen, indicating it may be a potential target of UBR4-directed

degradation. To confirm the mass spectrometry results, I conducted Western blot analysis with

an antibody specific to CK2β. SCN from ubr4+/-

mice housed in DD for 2 days were harvested

at CT14 and CK2β levels were found to be higher, but did not reach statistical significance (Fig

12A) (p>0.05). Interestingly, in SCN samples collected from ubr4+/-

mice that underwent long

term DD (Fig 12B) and long term LL (Fig 12C), CK2β expression was elevated in ubr4+/-

SCN

under both conditions, with the difference between genotypes reaching statistical significance in

the LL group (p<0.05) (Fig 12B). To further complement our in vivo data, I used siRNA specific

to ubr4 to knock down the expression of UBR4 in mouse neuroblastoma N2A cells as performed

previously (Ling et al., 2014). N2A cells that were transfected with ubr4-siRNA showed higher

levels of CK2β (Fig 12D), consistent with our in vivo results. Overall, our data suggest that

CK2β is a potential substrate of UBR4, such that when UBR4 is reduced in expression, levels of

CK2β are elevated.

45

Figure 12. Effect of UBR4 deficiency on CK2β expression in vivo and in vitro

Western blot analysis of CK2β levels in the SCN of ubr4+/+

and ubr4+/-

mice (A) after 2 days in

DD, sacrificed at CT14, (B) after long term DD, sacrificed at CT19, and (C) after long term LL,

sacrificed at CT19. (D) Expression of endogenous CK2β in mouse neuroblastoma N2A cells

after treated with no, negative control, or ubr4 specific siRNA. Actin was used as a loading

control. Quantification of relative CK2β level (adjusted to actin level) was shown for (B) and

(C). Values represent mean ± SEM of the CK2β band intensity after normalized to actin.

(*p<0.05)

46

3.10 UBR4 affects PER1/2 expression in vitro

As CK2 has been demonstrated to regulate the molecular clock machinery by affecting PER2

stability in mammals, I sought to find out how knocking down ubr4 with siRNA in vitro would

affect overexpressed PER expression. In co-transfection experiments in N2A cells, ubr4-siRNA

markedly reduced the abundance of V5-tagged PER1 and PER2 proteins relative to a non-

specific siRNA control, as determined by Western blot analysis (Fig 13A, B). Furthermore, the

decrease in PER1 levels was visualized using immunocytochemistry, where fluorescence

intensity level in cells transfected with V5-PER1 construct was decreased when cells were

treated with UBR4-siRNA compared to non-specific siRNA control (Figure 13C, D). To confirm

that the decreased abundance of PER1 proteins was mediated through CK2β, I utilized a

selective kinase inhibitor Tetrabromocinnamic acid (TBCA) that specifically targets CK2

(Pagano et al., 2007). After TBCA treatment for 3 hours, the decrease in V5-PER1 levels in

ubr4-siRNA treated samples was reversed when compared to DMSO treated controls; V5-PER1

expression was now comparable between non-specific siRNA and ubr4-siRNA samples (Figure

13C, D) (p>0.05).

47

Figure 13. The effect of UBR4 on PER1 and PER2 expression in vitro

Western blot analysis of N2A co-transfected with (A) V5-Per1 and (B) V5-PER2 with no,

control or ubr4 specific siRNA. Western blot was performed using an antibody specific to V5.

Actin was used as loading control. (B) Immunocytochemistry of N2A transfected with V5-PER1

(red) with control or UBR4 siRNA. Cells were treated for 3 hours in CK2 inhibitor TBCA, 24

hours post-transfection. DMSO treatment for 3 hours was used as a control (C) Summary of

individual V5 labelled cell fluorescence intensity with transfected V5-Per1 after TBCA treatment

or DMSO control. At least 100 cells were quantified and summarized in each condition. Values

represent mean ± SEM of cell fluorescence intensity (*p<0.05).

48

3.11 ubr4+/-

per2-/-

mice in constant darkness condition

Since UBR4 affects PER1 and PER2 expression in vitro and PER2 in vivo, I crossed ubr4+/-

with

per2 -/-

mice to observe how a single copy of ubr4 coupled with per2 knockout would impact the

circadian clock in DD. Under constant darkness, all per2-/-

mice exhibited a short period of

approximately 22 hours (Fig 14A) (Zheng et al., 1999). When coupled with a reduction in ubr4,

4 out of 5 ubr4+/-

per2-/-

mice lost circadian rhythmicity within a month in DD (Fig 14B,C),

whereas all ubr4+/+

per2-/-

controls still maintained rhythmicity during the entire course of DD

(5/5 ubr4+/+

per2-/-

control) (Fig 14A). To further probe the molecular mechanism underlying the

differences seen at the behavioral level, I harvested the SCN from this batch of mice after 45

days in DD to study molecular changes in the SCN. Using IHC, I found that all ubr4+/-

per2-/-

mice that are arrhythmic had a diffuse and low PER1 expression in the SCN compared to

ubr4+/+

per2-/-

control sacrificed at CT 4 or 16 (Fig 14D). This suggests that reducing UBR4

expression in a per2-/-

background has a profound impact on the circadian clock under DD.

49

Figure 14. Behavioral and molecular phenotype of ubr4+/-

per2-/-

mice


per2-/-

(A) and ubr4+/-

per2-/-

(B, C) mice. Light phases are

shaded in grey. Mice were entrained to a 12:12h LD schedule for 10 days before releasing to DD

for 45 days. All per2-/-

mice show a phase angle in 12:12h LD schedule. n = 5 animals per

genotype. (D) Representative IHC staining of PER1 in the SCN of mice after long term DD.

SCN were harvested at either CT 4 or 16 (determined based on behavior) for rhythmic

ubr4+/+

per2-/-

mice. SCNs from ubr4+/-

per2-/-

mice were harvested at the same time as

ubr4+/+

per2-/-

animals, because CT determination was not possible due to their arrhythmicity.

IHC staining in all ubr4+/-

per2-/-

mice show low and diffuse expression of PER1.

50

4 Discussion

The role of ubiquitin ligases and the timely degradation of clock proteins are crucial for circadian

clock timing in mammals. So far, no ubiquitin ligase has been implicated in photic entrainment

in mammals. In this thesis, I uncovered the role of a newly identified ubiquitin ligase UBR4 in

the murine SCN. Through mass spectrometry screen, UBR4 was identified as a significantly

upregulated protein after early subjective night light pulse. I confirmed the increased expression

of UBR4 in the murine SCN after early night light pulse with immunofluorescence. The increase

was due to recruitment of extra UBR4-expressing cells but not an increase in expression in

individual cells (Fig 3). I showed that UBR4 has a specific spatial and temporal expression

profile in the SCN that fluctuates in a time-of-day dependent manner in the middle and caudal

portions of the SCN (Fig 2). I have further addressed the role of UBR4 in the circadian clock

behaviorally and molecularly, as discussed below.

4.1 Reduced expression of UBR4 effect on circadian paradigms

Behaviorally, mice harboring a single copy of the ubr4 gene exhibit interesting phenotypes using

different circadian paradigms that involve multiple or long-term light disturbances. ubr4+/-

mice

did not show a difference in circadian period in DD initially, but exhibited a significantly shorter

period after two light pulses, with the phase-advancing light pulse being the more potent of the

two at reducing the period (Fig 6). ubr4+/-

mice showed a reduced rate of re-entrainment to an

advance, but not delay, of the LD cycle (Fig 7). The jetlag paradigm also involves constant phase

advancing every 2 days (Fig 8). All three paradigms involve light exposure during the late

subjective night, the phase advancing portion of the phase response curve (Johnson 1999).

Collectively, these results suggest that UBR4’s role in photic entrainment may be phase-

restricted to the late subjective night.

Furthermore, ubr4+/-

mice show a wide range of phenotypes under long term LL (Fig 9).

Normally, the lengthening in period seen in LL is proportional to light intensity as predicted by

Aschoff’s rule in nocturnal animals, which has been demonstrated in mice under laboratory

51

settings (Aschoff, 1960; Pendergast et al., 2010; Steinlechner et al., 2002). The period

lengthening in constant light can be partially explained by the phase response curve, in that the

phase delaying portion is larger than phase advancing portion for mice, causing an overall

period lengthening effect when light falls on the entire phase response curve (Pendergast et al.,

2010). The period length in constant light, therefore, reflects the balance between overall phase

delaying and phase advancing ability of the animal (Pendergast et al., 2010; Steinlechner et al.,

2002). This provides a potential explanation for the phenotype observed in ubr4+/-

mice under

long term LL. The wide range of phenotypes exhibited by these animals is very unstable,

potentially due to altered phase advancing ability of ubr4+/-

mice. This further strengthens the

hypothesis that UBR4 regulates photic entrainment specifically in the phase advancing portion of

the phase response curve. However, it should be noted that ubr4+/-

mice do not phase shift

differently to a single short-term (15 mins) phase advancing light pulse in the late subjective

night, suggesting that this specificity and phenotypes observed in ubr4+/-

mice require light

disturbances of long duration.

4.2 Constant light disruption in ubr4+/-

mice and implications

Exposure to constant light is very disruptive to the circadian clock and can cause arrhythmicity

over long term. Behavioral arrhythmicity induced by constant light has been studied at the

cellular level in the SCN. Arrhythmicity is believed to arise from desynchrony among SCN

neurons, where individual SCN neurons can still maintain rhythmicity but the intercellular

coupling of SCN neurons has been disrupted, causing an overall disrupted output from the clock

(Ohta et al., 2005). Weak intercellular synchrony may be a potential explanation for the

behavioral phenotype of ubr4+/-

mice under constant light. Because ubr4+/-

mice show a wide

range of phenotypes that all have low amplitude (Fig 9G), I hypothesize that ubr4+/-

mice may

have a more desynchronized SCN under constant light. Our jetlag paradigm data also support

this hypothesis, as the circadian amplitude was lower in ubr4+/-

mice (Fig 8F). Previous studies

have shown that behavioral rhythmic power is negatively correlated with individual neuronal

phase variance and that behavioral rhythms are determined by the combined output of individual

SCN neurons (Chen et al., 2008; Ciarleglio et al., 2009; Low-Zeddies and Takahashi, 2001). The

collective data lead us to the proposition that the decrease in circadian amplitude and unstable

52

phenotype observed in ubr4+/-

mice under chronic light disturbances are likely due to a more

desynchronized circadian clock, in that the behavioral phenotype of individual mice is dictated

by the amount of variation among SCN neurons and the level of synchrony that the SCN is able

to achieve. Further studies are needed to elucidate the underlying molecular mechanisms that

govern UBR4’s effects on the circadian clock under constant light or chronic jetlag, and to define

the role of UBR4 in synchrony of SCN neurons.

4.3 Reduced expression of UBR4 disrupts the molecular clock

ubr4+/-

mice exhibit an altered core molecular clock in the SCN with differences in expression

level of PER2 but not PER1, with the PER2 difference being attributed to effects seen in the

SCN shell but not SCN core (Fig 10). The SCN is divided in two main regions, one of which, the

shell, is considered to be the rhythmic portion and exhibits strong oscillations of per expression

(Yan and Okamura, 2002; Yan et al., 1999). The localization of UBR4 to the shell region (Fig 2)

suggests a potential interaction between UBR4 and PER2 in that region. UBR4 declines

throughout the subjective day and reaches nadir at CT 10 (Fig 2), coinciding with the rising of

PER2 in the SCN starting at CT10 (Fig 10) (Field et al., 2000). Since PER2 levels are higher in

ubr4+/-

mice at CT10, it is tempting to speculate that UBR4 may regulate PER2 expression and

determines its turnover in vivo. It should be noted that UBR4 staining in the SCN is exclusively

cytoplasmic, whereas PER2 staining is predominantly nuclear, thus favoring the possibility that

UBR4 regulates PER2 expression indirectly.

In line with UBR4 affecting PER2 indirectly, I have identified CK2β as a potential substrate of

UBR4, as CK2β expression is elevated in ubr4+/-

mice and in ubr4 knockdown N2A cells (Fig

12). CK2β is the regulatory subunit of CK2, and as such is important for CK2 tetramer complex

formation, regulation of its kinase activity and overall stability of CK2 complex (Bibby and

Litchfield, 2005; Litchfield, 2003). CK2 is important in various cellular functions, including its

notable role in cell cycle regulation (Filhol and Cochet, 2009). Recently, CK2 has been

implicated in mammalian circadian clock mechanism, whereby knocking down or inhibiting

CK2 can alter circadian period and amplitude in vitro (Maier et al., 2009; Tamaru et al., 2009;

Tsuchiya et al., 2009). CK2 has been shown to interact with and phosphorylate PER2 in

mammalian cell culture: phosphorylation by CK2 controls PER2 subcellular localization and

53

stability (Maier et al., 2009; Tsuchiya et al., 2009). The phosphorylation mechanism is further

complicated by the fact that PER2 has multiple serine sites for phosphorylation that can be

phosphorylated by multiple kinases in mammals, including CK1δ and CK1ε (Albrecht, 2007;

Maier et al., 2009; Toh et al., 2001; Vanselow et al., 2006). The localization and stability of

PER2 is likely regulated by a combination of events that requires multiple and sequential

phosphorylation by different kinases at multiple sites, but the complete and precise mechanism is

still largely unknown (Merrow et al., 2006). Here, we limit our scope to the potential effects of

CK2 on phosphorylation of PER, leading to an enhanced rate of degradation. I speculate that

UBR4 normally controls the degradation of CK2β. With our data, when levels of UBR4 are

reduced by siRNA knockdown, CK2β expression is elevated. Augmented CK2 expression and

activity presumably increases phosphorylation of PER, leading to increased degradation and

lower PER abundance, as confirmed by our V5-PER1/2 experiments in vitro. However, it should

be noted that inconsistencies have been found in our in vivo and in vitro data. First, PER2

expression is increased in vivo but decreased in vitro when UBR4 levels are reduced. Second,

PER1 expression is not altered in vivo but is decreased in vitro with reduced UBR4 expression. A

potential explanation is that ubr4+/-

mice have compensatory changes that increase total levels of

PER2 but not PER1 to counteract the increased phosphorylation on PER2 and its degradation.

Alternatively, PER1 and PER2 may be regulated differently in vivo. It is also important to keep

in mind that the in vitro data presented used overexpressed versions of V5-tagged PER1 and

PER2, and not endogenously expressed PER1 and PER2. Furthermore, I did not distinguish

between phosphorylated or unphosphorylated forms of PER1/2. In this regard, I propose more in-

depth experiments regarding expression of phosphorylated and unphosphorylated forms of

PER1/2 in vivo and in vitro and their subcellular localization with reduced UBR4 expression to

better understand the mechanistic link between UBR4, CK2 and PER1/2.

54

Figure 15 The potential role of UBR4 in a simplified model of the mammalian molecular

clock with known ubiquitin ligases

UBR4 is incorporated into simplified model of the mammalian molecular clock. CK2 is

degraded by UBR4 under normal conditions, thereby decreasing phosphorylation of PER

proteins and enhancing their stability.

55

4.4 UBR4 potential interaction with AVP and role in SCN synchrony

Since UBR4 co-localizes with AVP in the SCN, I wanted to determine the expression pattern of

AVP in the SCN (Fig 11). AVP is localized to the shell SCN and its receptor V1a is highly

expressed throughout the shell and core SCN (Young et al., 1993). AVP transcription is directly

regulated by core clock components CLOCK and BMAL1,and its synaptic release follows a

circadian rhythm (Jin et al., 1999). Recently, AVP has been implicated in the synchronization of

SCN neurons (Maywood et al., 2011; Yamaguchi et al., 2013). With the behavioral phenotype

suggesting disrupted synchrony among SCN neurons and UBR4 localization to AVP neurons, I

propose a potential link between AVP and UBR4, where their downstream signaling events may

be connected. Recently, one study showed that UBR4 expression is altered upon AVP

stimulation in the rat kidney collecting duct through mass spectrometry screen (Lee et al., 2011),

confirming that AVP and UBR4 are interconnected, at least in other tissues. Although our results

showed no difference for AVP expression in the SCN (Fig 11), it should be noted that SCN

tissues were harvested from mice that had been housed in DD for two days, a condition that, to

our knowledge, does not elicit a behavioral phenotype in ubr4+/-

mice. Further experiments

regarding AVP expression in the SCN after chronic light disturbances remain to be done, in

order to provide a functional and mechanistic link between AVP and UBR4, as well as a

potential explanation for desynchrony seen at the behavioral level.

4.5 Importance of UBR4 augmented in per2-/-

background

With the exception of bmal1-/-

mice, mice harbouring mutation of a single core clock gene rarely

exhibit arrhythmicity under short-term DD (Bunger et al., 2000). Per2-/-

mice normally show a

shortened period of 22 hours in constant darkness, and some eventually become arrhythmic after

an extended period of time in DD (Zheng et al., 1999). In our preliminary data, when per2

knockout is coupled with reduced levels of ubr4, ubr4+/-

per2-/-

mice lost their rhythmicity in

constant darkness much more quickly than their ubr4+/+

per2-/-

littermates (Fig 14). This led us to

hypothesize that UBR4 and PER2 together are indispensable for circadian clock timing. Our in

vitro data showed that UBR4 is important in regulating expression of PER1 and PER2. As per1

and per2 have been shown to be partially redundant, but have specialized functions in core clock

56

timing mechanisms, the lack of per2 would likely magnify the importance of per1 on the

circadian clock. Per1 has been shown to be involved in precision and stability control of the

circadian clock as seen in per1 mutant mice, providing a potential molecular basis of the

arrhythmia displayed by ubr4+/-

per2-/-

mice (Albrecht et al., 2001; Zheng et al., 2001). Moreover,

the gradual loss of rhythmicity in ubr4+/-

per2-/-

mice further suggests a role of UBR4 on long-

term clock maintenance rather than short-term clock timing, echoing the phenotypes observed in

other long-term circadian paradigms. It would be interesting to know how ubr4+/-

per2-/-

mice

behave in response to phase shifting light pulses and constant light paradigms, as per2-/-

mice

have been shown to have an exaggerated response to phase advancing light pulse with no

response to phase delaying light pulse and a shortened period of 22-23 hours under constant light

(Albrecht et al., 2001; Steinlechner et al., 2002; Zheng et al., 2001). Therefore, more detailed

behavioral and molecular characterization of ubr4+/-

per2-/-

are needed to uncover this

interconnected relationship between PER1/2 and UBR4.

4.6 Conclusion

UBR4 belongs to the evolutionary conserved N-end rule pathway with homologs in drosophila

and Arabidopsis, each having diverse functions. Given the evolutionarily conserved nature of the

circadian clock across different organisms, our discovery that murine UBR4 participates in

photic entrainment and circadian clock mechanisms opens up the possibility that UBR4 might

also be important in circadian clock timing or photic entrainment in other organisms. In

conclusion, this thesis identifies a newly discovered ubiquitin ligase UBR4 in the murine SCN.

The importance of UBR4 is demonstrated through differential phenotypes observed behaviorally

and molecularly in ubr4+/-

mice. Finding out the precise role and mechanism of action of UBR4

in the SCN would introduce the N-end rule pathway and ubiquitin proteasome pathway as key

players in the photic entrainment process, and would lead to a better understanding of the

mammalian circadian clock.

57

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Copyright Acknowledgements

Results from section 3.1 and 3.2 and Figure 2, 3 and 4 were published as part of a manuscript:

Ling HH, Beaulé C, Chiang C-K, Tian R, Figeys D, Hai-Ying M. Cheng (2014) Time-of-Day-

and Light-Dependent Expression of Ubiquitin Protein Ligase E3 Component N-Recognin 4

(UBR4) in the Suprachiasmatic Nucleus Circadian Clock. PLoS ONE 9(8): e103103.

doi:10.1371/journal.pone.0103103

characterization of the role of ubiquitin protein ligase

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