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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
Figure 8. The response of ubr4+/-
mice to chronic jetlag paradigm .............................................. 37
Figure 9. The response of ubr4+/-
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
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
Figure 6. The response of ubr4+/-
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
Representative actograms of ubr4+/+
(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
Figure 8. The response of ubr4+/-
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
Figure 9. The response of ubr4+/-
mice to constant light.
Representative actograms of ubr4+/+
(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
Representative actograms of ubr4+/+
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