Mammalian Circadian Clock: The Rolesof Transcriptional Repression and Delay
Yoichi Minami, Koji L. Ode, and Hiroki R. Ueda

Abstract The circadian clock is an endogenous oscillator with a 24-h period.
Although delayed feedback repression was proposed to lie at the core of the clock
more than 20 years ago, the mechanism for making delay in feedback repression inclock function has only been demonstrated recently. In the mammalian circadianclock, delayed feedback repression is mediated through E/E0 -box, D-box, andRRE transcriptional cis-elements, which activate or repress each other throughdownstream transcriptional activators/repressors. Among these three types ofcis-elements, transcriptional negative feedback mediated by E/E0 -box plays a criticalrole for circadian rhythms. A recent study showed that a combination of D-box and
RRE elements results in the delayed expression of Cry1, a potent transcriptional
inhibitor of the E/E0 -box. The overall interconnection of these cis-elements can besummarized as a combination of two oscillatory motifs: one is a simple delayed
feedback repression where only an RRE represses an E/E0 -box, and the other is a
repressilator where each element inhibits another in turn (i.e., E/E0 box represses an
RRE, an RRE represses a D-box, and a D-box represses an E/E0 box). Experimental
verification of the roles of each motif as well as post-transcriptional regulation of
the circadian oscillator will be the next challenges.

Y. MinamiLaboratory for Systems Biology, Center for Developmental Biology, RIKEN, Chuo-ku,
Kobe, Hyogo, Japan 650-0047
K.L. Ode
Laboratory for Synthetic Biology, Quantitative Biology Center, RIKEN, Chuo-ku,
Kobe, Hyogo, Japan 650-0047
H.R. Ueda (*)
Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, Chuo-ku,
Kobe, Hyogo, Japan 650-0047
Laboratory for Synthetic Biology, Quantitative Biology Center, RIKEN, Chuo-ku,
Kobe, Hyogo, Japan 650-0047
e-mail: hiroki.ueda@nifty.com
A. Kramer and M. Merrow (eds.), Circadian Clocks, Handbook of Experimental
Pharmacology 217, DOI 10.1007/978-3-642-25950-0_15,# Springer-Verlag Berlin Heidelberg 2013

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Keywords Phase vector model • Time delay • Clock controlled cis elements

1 Circadian Clock in Mammals
In mammals the master clock is located in the suprachiasmatic nucleus (SCN).
Transcript analyses have indicated that circadian clocks are not restricted to SCN,
but are found in several tissues including the liver (Yamazaki et al. 2000) andcultured cells such as rat fibroblasts Rat-1 (Balsalobre et al. 1998), mouse
fibroblasts NIH3T3 (Tsuchiya et al. 2003), or human osteosarcoma U-2OS cells(Isojima et al. 2009; Vollmers et al. 2008). Therefore, circadian rhythms are driven
by cell-autonomous oscillators. Studies across species have elucidated theconserved feature of molecular mechanisms underlying circadian rhythms: at thecore of the clock lies a transcriptional/translational negative feedback loop. For
example, in mice the transcription factors CLOCK and BMAL1 dimerize andactivate transcription of the Per and Cry genes. PER and CRY proteins accumulate
in the cytosol become phosphorylated and return to the nucleus where they inhibit
the activity of CLOCK and BMAL1. The turnover of PER and CRY proteins leads
to a new cycle of activation by CLOCK and BMAL1 via E/E0 -box (Dunlap 1999;
Griffin et al. 1999; Kume et al. 1999; Reppert and Weaver 2002; Young and Kay2001). In this process, PER and CRY form a negative feedback loop that inhibits
their own transcription. However, reciprocal activation of positive (CLOCK and
BMAL1) and negative (PER and CRY) regulators in a negative feedback loop is not
sufficient: there must be a delay or immediate self-inhibition of CRY and PER
would result in the stable lower expression of these factors rather than oscillation.
What molecular mechanism imposes this time delay? This chapter summarizes thetranscription network of the mammalian circadian clock and provides insights into
how the network together with post-translational regulation of clock proteins works
as a delayed negative feedback loop.

2 Identification of the Circadian Transcriptional Network
2.1

Transcriptional Network Based on Three
Clock-Controlled Elements

2.1.1

The E/E0 -Box, the D-Box, and the RRE

The overall topology of mammalian circadian transcription network can be understood by the combination of three clock-controlled elements (CCEs), short consensus DNA sequences typically located near the promoter region of clock genes.
These CCEs are called the E/E0 -box (CACGT(T/G)) (Gekakis et al. 1998;
Hogenesch et al. 1997; Ueda et al. 2005; Yoo et al. 2005), the D-box (DBP responseMammalian Circadian Clock: The Roles of Transcriptional Repression and Delay361

element) (TTATG(C/T)AA) (Falvey et al. 1996; Ueda et al. 2005), and the RRE[RevErbA response element, also called as ROR response element (RORE)] [(A/T)
A(A/T)NT(A/G)GGTCA] (Harding and Lazar 1993; Preitner et al. 2002; Ueda
et al. 2002, 2005).
By performing transcriptome analysis, expression of 24-h periodic genes wasreported in cultured cells (Grundschober et al. 2001), the SCN (Panda et al. 2002;
Ueda et al. 2002), and other tissues such as heart (Storch et al. 2002), liver (Panda
et al. 2002; Storch et al. 2002; Ueda et al. 2002), aorta (Rudic et al. 2005), adipose
tissues (Zvonic et al. 2006), calvarial bone (Zvonic et al. 2007), and hair follicle(Akashi et al. 2010). Although there are differences in the rhythmicity of circadianexpressed genes in each tissue, the following mammalian clock genes most commonly have circadian oscillation: Period1 (Per1), Per2, Per3, Dec1 (Bhlhb2), Dec2
(Bhlhb3), Cryptochome1 (Cry1), Clock, Npas2, Bmal1 (Arntl), Dbp, E4bp4 (Nfil3),
RevErbAa (Nr1d1), RevErbAb (Nr1d2), and Rora. The temporal expression of eachgene is controlled by a different combination of CCEs. Evolutionary conserved
E/E0 -boxes are located in the noncoding regions of nine genes (Per1, Per2, Cry1,
Dbp, Rorγ, RevErbAa, RevErbAb, Dec1, and Dec2), D-boxes are contained in eightgenes (Per1, Per2, Per3, Cry1, RevErbAa, RevErbAb, Rorα, and Rorβ), and RREs
in six genes (Bmal1, Clock, Npas2, Cry1, E4bp4, and Rorc). The expressed geneproduct positively or negatively regulates transcription activity by acting on CCEs:
CCEs and these clock genes form a closed network structure (Fig. 1) as described
below.

2.1.2

Transcription Regulation via the E/E0 -Box and Clock GenesThe E/E0 -box is positively regulated by Bmal1, Clock, and Npas2 and negatively
regulated by Per1–3, Cry1–2, and Dec1–2. CRY and PER are hypothesized toautoregulate their own expression by repressing the heterodimeric complex of thebasic helix–loop–helix (bHLH) PER-ARNT-SIM (PAS) domain transcriptional
activators CLOCK and BMAL1, which bind to E/E0 -box elements in the Cry1and Per1–2 promoters. Although both positive regulators (Bmal1, Npas2, Clock)and negative regulators (Per1–3 and Cry1–2) have circadian rhythmic expression
patterns, peak time of positive regulators are antiphase to that of negative regulators(delayed negative feedback).

2.1.3

Transcription Regulation via the D-Box and Clock GenesThe D-box is positively regulated by PAR-bZIP (proline- and acidic amino acidrich basic leucine zipper) transcription factors (Dbp, Tef, and Hlf) and negatively
by E4bp4. Like the E/E0 -box, an antiphase relationship of gene expression
between negative and positive regulators can be observed. In the D-box case,

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aBioluminescencedLuc

Luciferin+ATP
CCE

CCE

CCE

SV40
SV40

CCE: Clock-controlled element
SV40: SV40 basic promoter

Luc: Luciferase
PEST: degradation sequence

Per1 D box

Per1 E box
Bioluminescence(103 counts/min)

b

PEST

Luc

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Bmal1 RRE
76

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76

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E box

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Day

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REVERBAb
E4BP4
RORa
RORb
RORc

DEC1

DEC2

DBP

E-box

D-box

RRE

CKI
CKI
PER2

PER1

PER3

CRY1
CRY2
NPAS2
FBXL3
CLOCK
BMAL1

Fig. 1 Schematic representation of the transcriptional network of the mammalian circadian clock.
(a) In vitro cycling assay. Cultured mammalian cells (Rat-1) were transfected with dLuc under thecontrol of a clock-controlled element (CCE) and SV40 basic promoter (Ueda et al. 2005).
(b) Representative circadian rhythms of bioluminescence from a wild-type Per1 E-box CCE
fused to the SV40 basic promoter driving a dLuc reporter (left panel) and compared to bioluminescence rhythms driven by a Per1 D-box (center panel) and a RRE (right panel). Original figures
are reproduced from Ueda et al. (2005). (c) Genes and CCEs are depicted as ellipsoids andrectangles, respectively. Transcriptional/translational activation is shown by arrows (!) andrepression is depicted by arrows with flat ends (┤)

Mammalian Circadian Clock: The Roles of Transcriptional Repression and Delay363

the expression phase of the positive regulator Dbp is similar to that of Per1,
whereas the expression phase of the negative regulator E4bp4 is similar to that of
Bmal1 (Mitsui et al. 2001).

2.1.4

Transcription Regulation via the RRE and Clock Genes

RRE is positively regulated by Rora, Rorb, and Rorc and negatively regulated by
RevErbAa and RevErbAb. In the SCN, Rora and Rorb have circadian rhythms but
not Rorc (Ueda et al. 2002). Liu et al. reported that RevErbAa and RevErbAb arefunctionally redundant and necessary for oscillation of the RRE-regulated gene
Bmal1. By contrast, Rors contribute to Bmal1 amplitude, but are not required forgenerating oscillation (Liu et al. 2008).

2.1.5

Timing of Each CCE

The circadian timing at which each element becomes active for transcription canbe monitored with an in vitro cell culture system in which a destabilized firefly
luciferase (dLuc) reporter is driven by different clock-controlled promoters. After
cells are synchronized (i.e., with dexamethasone, forskolin, or serum), oscillations
in gene expression are recorded by bioluminescence (Nagoshi et al. 2004; Ueda
et al. 2002, 2005; Welsh et al. 2004). Using this in vitro cycling assay, the“phase” of each CCE can be determined (Ueda et al. 2002, 2005) (Fig. 1).
Note that the term “phase” used in this chapter represents relative peak timingof each circadian gene expression within single circadian cycle. Each CCE
is responsible for the gene expression at distinct circadian times: the peak timeof E/E0 -box-driven expression is followed by D-box-driven expression after aninterval of ~5 h. Then, RRE-driven expression follows D-box expression after~8 h. E/E0 -box-driven expression begins to appear again ~11 h after RRE-driven
expression. In the case of the SCN, the subjective time drawn by each CCE canbe illustrated as “morning-time” for the E/E0 -box, “evening-time” for the D-box,
and “nighttime” for the RRE (Ueda et al. 2005).

2.2

Importance of Gene Regulation via the E/E0 -Box

2.2.1

Circadian Clock Perturbation via CCEs

The three CCEs have different impacts on cellular circadian rhythms: perturbation of
E/E0 -box regulation abolishes circadian rhythms; perturbation of RRE regulation has
an intermediate but significant effect; and D-box disruption has almost no effect.
A study using Rat-1 cell showed this by overexpressing regulatory genes withrepressive activity to different CCEs (Ueda et al. 2005) (Fig. 2). The Per2 promoter
is regulated via an E/E0 -box and a D-box, and the Bmal1 promoter is regulated via

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Cry1Over Expression

E4bp4
Over Expression

RevErbAa
Over Expression

E/E'box

D-box

RRE

Bioluminescence (counts/min)

Per2-dLuc
45,00080,00080,000

5,000

5,000

5,000

Bmal1-dLuc
20,00020,00015,000

5,000

1,000

1,000

Fig. 2 Importance of the E/E0 -box. Effect of repression on each CCE. The E/E0 -boxes, D-box, and
RRE were repressed by overproduction of CRY1, E4BP4, and REVERBAa, respectively. The
consequences of those repressions were monitored by bioluminescence from Per2 and Bmal1
promoter driving a destabilized luciferase (Per2-dLuc and Bmail1-dLuc). Original figures are
reproduced from Ueda et al. (2005). The different shades of gray in the plot indicate different
amounts of transfected vector

an RRE. When E/E0 -box activity is perturbed by overexpression of the Cry1 gene,both Per2-promoter-driven reporter gene (Per2-dLuc) and Bmal1-promoter-driven
reporter gene (Bmal1-dLuc) lose circadian rhythms. When an RRE is perturbed
through RevErbAa overexpression, Bmal1-dLuc loses circadian rhythms and theamplitude of Per2-dLuc rhythmic expression is decreased. The impact of RRE
perturbation through RevErbAa overexpression appeared to be more significant inmice liver. Kornmann et al. showed that liver-specific overexpression of RevErbAa
abolishes the rhythmicity of PER2::Luc expression in liver explants (Kornmann
et al. 2007). Contrary to the case of E/E0 -box and RRE, D-box perturbation throughE4bp4 overexpression causes both Per2-dLuc and Bmal1-dLuc transcriptional
activity to have normal circadian rhythms (Ueda et al. 2005). These varying effects
are difficult to explain by mere quantitative differences in the strength of the three
repressors, which suggests that there is some qualitative difference between E/E0 box, D-box, and RRE regulation in circadian rhythmicity.

2.2.2

Circadian Feedback Repression: Heart of the CircadianTranscriptional Network

PER and CRY play key roles in the circadian clock transcriptional network byclosing the negative feedback loop of E/E0 -box regulation. CRY has stronger

Mammalian Circadian Clock: The Roles of Transcriptional Repression and Delay365

repressor activity than PER (Kume et al. 1999). Sato et al. reported that interferenceof CRY1’s repressor activity on E/E0 -box-mediated transcription can abolish circadian transcriptional oscillations. They screened both human CLOCK and BMAL1
alleles that were insensitive to CRY1 repression but maintained normal transcriptional activity. Selected clones have normal transcriptional activities similar to wild
type in the absence of CRY1, but have greater reporter activity in the presence of
CRY1. By analyzing either Per2-dLuc or Bmal1-dLuc, they observed that
cotransfection of either CLOCK or BMAL1 mutant alleles resulted in substantial
impairment of circadian rhythmicity after one or two cycles of oscillation;
cotransfection of both CRY-insensitive mutant CLOCK and BMAL1 together
resulted in the loss of circadian promoter activity. This suggests that transcriptional
repression of CLOCK/BMAL1 by CRY1 is required for circadian regulation via
both an E/E0 -box and an RRE (Sato et al. 2006) (Fig. 3).

3 Minimal Circuit of the Mammalian Circadian Clock3.1

Two Delayed Negative Feedback Loops

How is the negative feedback to an E/E0 -box delayed? Although there is an E0 -box
and an E-box in Cry1’s regulatory region (Fustin et al. 2009; Ueda et al. 2005), thepeak of Cry1 expression is evening-time, which is substantially delayed relative toother genes with an E/E0 -box (Fustin et al. 2009; Ueda et al. 2005). Cry1 has twofunctional RREs in one of its introns (Ueda et al. 2005) and also D-box in its
promoter region (Ukai-Tadenuma et al. 2011). Ukai-Tadenuma et al. experimentally confirmed that the combination of daytime elements (D-box) and nighttime
elements (RREs) within its intronic enhancer gives rise to Cry1’s delayed eveningtime expression. Interestingly, the observed delayed expression was well explainedby a simple phase-vector model that enabled artificially designed delayed
expressions (Ukai-Tadenuma et al. 2011) (Fig. 4).
Based on this simple phase-vector model (Fig. 4), they generated an array of
Cry1 constructs that have different phases and used these in a genetic complementation assay to restore circadian oscillation in arrhythmic Cry1/:Cry2/ cells
established from Cry1/:Cry2/ double-knockout mice (van der Horst et al.
1999). These experiments reveal that substantial delay of Cry1 expression isrequired to restore single-cell-level rhythmicity and that prolonged delay of Cry1
expression can slow circadian oscillations (Fig. 5). These results suggest that phasedelay in Cry1 transcription is required for mammalian clock function and theseresults provide formal proof that the design principle of the mammalian circadianclock transcriptional network is negative feedback with delay (Ukai-Tadenuma
et al. 2011).

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Per2-dLuc% activity of wild type

300
Clock/Bmal1
Clock/Bmal1-MT
Clock-MT /Bmal1
Clock-MT /Bmal1-MT

250
200
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100
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0

0

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day
Bmal1-dLuc% activity of wild type

500

Clock/Bmal1
Clock/Bmal1-MT
Clock-MT /Bmal1
Clock-MT /Bmal1-MT

400
300
200
100
0

0

1

2

3
day

4

5

6

Fig. 3 The impairment of CRY-mediated repression. Coexpression of CLOCK/BMAL1 mutant
heterodimers that are insensitive to CRY repression ablates circadian E-box and RRE activities in
NIH3T3 cells. Plasmids expressing Flag-tagged CLOCK and BMAL1 alleles were transiently
cotransfected with the Per2-dLuc (upper panel) or Bmal1-dLuc reporter plasmid into NIH3T3
cells (lower panel). Per2 or Bmal1 promoter activities in NIH3T3 cells transfected with single or
double CRY1-insensitive CLOCK, and BMAL1 mutants (MT) were monitored over 5 (upper
panel) or 6 days (lower panel). All reporter activities were normalized such that the median wildtype luciferase activity over the time course was 100 %. Original figures are reproduced from
Sato et al. (2006)

Based on these results, they hypothesized that the transcriptional network can be
simplified into a model consisting of two transcriptional activations and fourtranscriptional repressions on three regulatory DNA elements (Fig. 6). Notably,
this diagram can be envisaged as a composite of two distinct oscillatory network
motifs (1) a repressilator, which is composed of three repressions, and (2) a delayed
negative feedback loop, which is composed of two activations and one repression.
Both oscillatory network motifs include delayed feedback repression and cangenerate autonomous oscillations independently (Elowitz and Leibler 2000;
Stricker et al. 2008).

Mammalian Circadian Clock: The Roles of Transcriptional Repression and Delay

367

Pc
P2

Φ2
Φc’
Φ1

P1

Phase

Amplitude

Amplitude

Fig. 4 Phase-vector model. A new phase results from the combinatorial synthesis of two
transcriptional regulators or two clock-controlled DNA elements, which can be illustrated to afirst-order approximation by a phase-vector model. This combinatorial regulatory mechanism forgenerating new circadian phases of transcription represents a general design principle underpinning complex system behavior. Assume wave function fx(t) ¼ Ax cos(θ(t) + ϕx). The amplitudeof wave A is represented by the length of a phase vector P, and the phase of wave ϕ isrepresented by the angle of P. The component waves f1 and f2 are displayed by phase vectors P1
and P2. Pc is the summed phase vector of P1 and P2. Original graph is reproduced from UkaiTadenuma et al. (2011)

3.2

Genetic Evidence for the Importance of CCEs

The minimal circuit model implies that all of three CCEs have substantial importance for the circadian oscillator. The importance of an E/E0 -box-mediated regulation is manifested by the phenotypes of several clock gene-knockout mice. The
circadian clock governs physiological phenomena like day–night variation ofactivity, so changes in behavioral rhythms reflect differences in the endogenous
clock of mutant mice. Accordingly, disruption of Bmal1, a positive regulator of
E/E0 -box-mediated regulation, directly results in the loss behavioral rhythms inmice (Bunger et al. 2000; Shi et al. 2010). Disruption of Clock gene did not result inloss of behavioral rhythms (DeBruyne et al. 2007) probably because Clock and anothergene Npas2 have redundant roles: Clock and Npas2 double-knockout mice havearrhythmic behavioral patterns (DeBruyne et al. 2007), while Npas2-disrupted mice
have normal behavioral rhythms (Dudley et al. 2003). Loss of negative regulator of E/
E0 -box-mediated transcription also results in arrhythmic phenotypes. Both Per1 andPer2 disrupted mice have loss of circadian rhythmicity of behavioral activity (Bae et al.
2001; Zheng et al. 2001), and Cry1/:Cry2/ mice have arrhythmic behavioral
patterns (van der Horst et al. 1999; Vitaterna et al. 1999).
The minimal structure shown in Fig. 6 implies that D-box and RRE also play
an essential role to maintain the delay time for negative feedback. For example,
the double knockout of RevErbAa and RevErbAb mice has arrhythmic

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abPromoter + Intron

24/0

15

22

ox

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RRE

Intronxbo
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Promoter

10
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Amplitude

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0
No CCE
15

Phase Delay

Bioluminescene (106 counts/min)

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10

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' bo
E
E/

/E
E

5
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'b

20

Intron

10
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0

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1

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3 4
Day

5

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Period Length

Fig. 5 The biological relevance of delayed Cry1 expression in circadian clock function. (a) Per2dLuc bioluminescence levels in transfected Cry1/:Cry2/ cells. The Per2-dLuc reporter and aCry1 expression construct were cotransfected into Cry1/:Cry2/ cells. (b) Cry1 expression
phases under different promoters (Ueda et al. 2005; Ukai-Tadenuma et al. 2011) that contain eithera D-box (Cry1 promoter), RRE (Cry1 intron), or both (promoter + intron). (c) Substantial delay infeedback repression is required for mammalian clock function. The decreased delay dampens theamplitude of circadian oscillations (top panel), and the prolonged delay in feedback repression
slows the frequency of circadian oscillations (bottom panel) compared to wild type (middle panel).
Original figures are reproduced from Ukai-Tadenuma et al. (2011). Different trace shades represent results from triplicated experiments

Mammalian Circadian Clock: The Roles of Transcriptional Repression and Delay

E4BP4

369

The Minimal Circuit
for the Mammalian Circadian Transcriptional Network

DBP
TEF
HLF

E/E'box

BMAL2

D-box

BMAL1

CLOCK

N P AS2

RRE
E/E'boxC R Y2D-box

C R Y1

REVERBAa
DEC1

REVERBAb
DEC2

The Repressilator
E/E'boxD-box

The Delayed
Negative-Feedback Loop
E/E'box

D-box

ROR
PER1
ROR
CKI

PER2

CKI

ROR
PER3

RRE

RRE

RRE

Fig. 6 The minimal circuit for the mammalian circadian transcriptional network. (a) The transcription network of the mammalian circadian clock (Ueda et al. 2005; Ukai-Tadenuma et al.
2011). (b) The minimal circuit (top panel) can be illustrated as a composite of two distinct
oscillatory network motifs: a repressilator (bottom left panel) and a delayed negative feedback
loop (bottom right panel). Transcriptional activation (arrows); transcriptional repression (arrows
with flat ends); regulatory DNA elements (rectangles; E/E0 -box, morning; D-box, daytime; RRE,
nighttime). Original graph is reproduced from Ukai-Tadenuma et al. (2011)

behavioral phenotypes and arrhythmic clock gene expression (Bugge et al. 2012;
Cho et al. 2012).
The importance of D-box transcriptional regulators is still unclear because noreport shows that dysfunctional mice for D-box regulators have completely arrhythmic behavioral patterns. Lopez-Molina et al. reported that Dbp knockout mice havenormal behavioral rhythms compared to wild type (Lopez-Molina et al. 1997). Hlf
or Tef disrupted mice also have almost normal behavioral rhythms (Gachon et al.
2004). Even triple knockout of PAR-bZIP transcriptional factor mice have almost
normal behavior rhythms (Gachon et al. 2004). Although E4bp4 knockout mice wasconstructed (Gascoyne et al. 2009), behavioral rhythms of the mice were notreported.

3.3

Generation of Various Phases by the Combination of CCEs

From DNA microarray data, more than 10 % of expressed genes have circadianrhythms with a wide range of peak timings (Delaunay and Laudet 2002); thedistribution of peak timing is not limited to three circadian times corresponding
to the expression timing of each CCE. How do these “intermediate” expression
timings arise? One possibility is that the combination of three CCEs generates
various circadian phases.
Ukai-Tadenuma, Kasukawa et al. adopted a synthetic approach to physicallysimulate the correlation between CCEs combinations and the peak timing of

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abArtificial Activator

dGAL4VP16

22
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SV40
SV40

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E/

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SV40
SV40

Repressor

PEST

Gal4

dGAL4VP16

dGAL4

UAS

UAS

UAS

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ox

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4.0
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ox
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D -boxD

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16Artificial Promoter

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bo

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x

CCE

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Output

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Luc

d

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Noon

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Repressor

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4

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Morning6

Daytime

RRE
Dx

bo

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16

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14

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8.9

Dawn

10
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Activator
Late Night

Night time

Evening

Fig. 7 Combinatorial regulation of circadian phases by a synthetic system. (a) The artificialtranscription system. Activator and repressor are driven under clock-controlled elements (CCEs).
Details are described in main text. (b, c) Promoter activities of an activator, repressor, and output
in different artificial transcriptional circuits. The schemes summarize the representative promoter
activities of each artificial circuit monitored by bioluminescence from NIH3T3 cells, wherean activator, repressor, and output phases are indicated with their peak time (gray numbers).
Morning activator under E0 -box control and nighttime repressor under RRE control and(b) daytime activator under D-box control and morning repressor under E0 -box control (c).
(d) The relationship of the expression timings of the transcription factors and output. Various
expression timing is generated from three basic phases (morning, daytime, and nighttime). Blacklines indicate activation (arrows) and gray lines repression (arrows with flat ends). Original figures
and graphs are reproduced from Ukai-Tadenuma et al. (2008)

expression. They used three components: an artificial activator (dGAL4-VP16), an
artificial repressor (dGAL4), and a dGAL4-VP16-driven reporter gene (dLuc) as an
output (Fig. 7a). If the expression of artificial activator and repressor are controlled
by different CCEs, then the output may vary according to a combination of thevarious peak timings of each CCE. By taking the peak expression timing of clock
gene expression in mouse liver, phase of each CCE-driven gene expression can be
related with subjective circadian time: E/E0 -box-driven expression peak timing as“morning,” RRE-driven expression peak timing as “night,” and D-box-driven peaktiming as “daytime.” They created “daytime” expression by the combination of E/
E0 -box (morning)-driven activator and RRE (night)-driven repressor (Fig. 7b). This

Mammalian Circadian Clock: The Roles of Transcriptional Repression and Delay371

is similar to transcriptional regulation via D-box control; D-box is activated by E/
E0 -box-controlled Dbp and repressed by RRE-controlled E4bp4, and output phase is“daytime.” Next, they created “night” by the combination of a D-box-driven
activator and an E/E0 -box-driven repressor (Fig. 7c). This is similar to an RRE
with output phase “night”: RRE is regulated by D-box-driven activator (Rora) andE/E0 -box-driven repressor (RevErbAa), though RevErbAa is also controlled by Dbox. By combining these CCEs in different arrangements, Ukai-Tadenuma,
Kasukawa et al. also generated additional phases (Fig. 7d), which are not identicalto any of the original CCE timings (Ukai-Tadenuma et al. 2008).

4 Post-Translational Regulation, Another Layer of Delayor Another Oscillator?
4.1

Phosphorylation of PER

As we discussed above, accumulating evidence indicates that Cry1-mediateddelayed negative feedback plays a critical role in the circadian transcription network. If so, is the network structure of transcription activator/inhibitor relationshipsufficient for generating mammalian circadian properties? If we replace all transcription factors with artificial ones [such as GAL4-VP16 used in Ukai-Tadenuma
et al. (2008)] but keep the network structure, could we reproduce a robust circadiansystem? Natural circadian systems, however, seem to be more complex than thetranscription-translation network; post-translational regulation is also critical for
circadian function (Gallego and Virshup 2007). In particular, phosphorylation of
PERs by CKIδ/ε is one of the determinants of circadian period length (Lowrey et al.
2000; Toh et al., 2001; Xu et al., 2005). The first circadian mutant identified in
mammal was the tau-mutant hamster, which has a shorter behavioral period lengthcompared to a normal hamster (Ralph and Menaker 1988). Takahashi’s group
identified the tau mutation in the CKIe gene and found that PER phosphorylation
is lower in tau-mutant hamsters (Lowrey et al. 2000). The importance of PER
phosphorylation by CKIδ/ε for circadian rhythms is also true in humans. Toh et al.
discovered that familial advanced sleep-phase syndrome (FASPS) is caused by amutation in the CKIδ/ε binding site of PER2 (Toh et al. 2001). Likewise, Xu et al.
found that a mutation in CKIδ can also cause FASPS by modulating PER stability(Xu et al. 2005). Additionally, chemical biology approaches identified several
compounds that shorten or lengthen circadian period (Chen et al. 2012; Hirota
et al. 2008; Isojima et al. 2009). One remarkable example is a series of CKIδ/εinhibitors, which can lengthen molecular clock period from 24 h to 48 h at thecellular level (Isojima et al. 2009).
How PER phosphorylation controls circadian period is still mysterious, but
phosphorylation affects PER stability. PER protein is degraded by proteasomemediated proteolysis when phosphorylation of PER triggers recruitment of βTrCP,

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a subunit of the SCF ubiquitin ligase (Eide et al. 2005; Shirogane et al. 2005).
However, the FASPS mutation site is different from the region involved in βTrCP
recognition of PER (Eide et al. 2005). Furthermore, several results imply that
phosphorylation on FASPS-mutated site stabilizes PER protein (Shanware et al.
2011; Vanselow et al. 2006; Xu et al. 2007). Therefore, phosphorylation may
regulate the stability of PER in multiple ways. Recent studies of Drosophilamelanogaster PER and Neurospora crassa FRQ (a functional counterpart ofPER) show that multisite phosphorylation induces conformational changes inthese proteins (Chiu et al. 2011; Querfurth et al. 2011). A similar case might also
be true for mammalian PER: phosphorylation may control the stability of mammalian PER by changing its global structure, not just by creating a recognition site for
βTrCP at a specific location.
The stability control of PER also may contribute to delay for transcriptional
negative feedback. Unlike other clock genes, the expression peak of Per1 and Per2mRNA is ~4 h earlier than PER1/PER2 proteins (Pace-Schott and Hobson 2002).
This delay between mRNA and protein may be one of the determinants of period
length.

4.2

Stability Control of CRY in Circadian Oscillations

Recently, researchers noticed that not only PER but also CRY stability is importantfor clock period. In 2007, two lines of ENU-mutant mice with long behavioral
rhythms were reported from different groups—Overtime (Siepka et al. 2007) and
Afterhours (Godinho et al. 2007). Both the Ovt and Afh mutations are located in thesame gene Fbxl3. Fbxl3 encodes an ubiquitin ligase E3 and controls CRY stability
by inducing CRY protein ubiquitination and degradation (Godinho et al. 2007;
Siepka et al. 2007). Delayed expression of CRY1 could be caused by the combinatorial effect of delayed transcription activation and active degradation. These datasuggest that temporal control of clock gene products (like PER and CRY) is also
important for generating circadian rhythms. Effects of the CKIεtau and Fbxl3Afhmutations are additive and independently contribute to circadian period (Maywood
et al. 2011).

4.3

Post-Translational Oscillation of the Mammalian
Circadian Clock

Phosphorylation-dependent degradation may be directly related to PER oscillation.
Two reports showed that PER2 protein translated from constitutively expressedmRNA undergoes circadian oscillation (Fujimoto et al. 2006; Nishii et al. 2006).
These studies imply that a layer of post-translational control blankets theMammalian Circadian Clock: The Roles of Transcriptional Repression and Delay373

transcription-translation circadian machinery. Consistent with this idea, several
studies have shown that circadian rhythmicity is robust against fluctuations inoscillating transcriptional activity. For example, the expression pattern of Bmal1
and Clock can be constant throughout the circadian cycle (von Gall et al. 2003).
Reducing the overall transcriptional activity only modestly affects the period lengthof circadian rhythms in cultured cells (Dibner et al. 2009). Even in for CRY,
rhythmic expression is dispensable for circadian oscillation to a certain extent;
weak circadian oscillations can be observed in Cry1/:Cry2/cells rescued byCry1 under constant expression (Ukai-Tadenuma et al. 2011) or a constant supply
of CRY proteins (Fan et al. 2007). Genetic studies in Drosophila show that flies
with constant expression of PER maintain circadian rhythmicity (Ewer et al. 1988;
Frisch et al. 1994; Vosshall and Young 1995; Yang and Sehgal 2001). Taken
together, these results suggest that circadian oscillations do not necessarily depend
solely on the transcriptional activity in the E/E0 -box feedback loop, because posttranslational control of clock proteins can compensate for loss of transcriptional
rhythms.
A post-translational circadian oscillator was also found in the cyanobacterium
circadian clock. Oscillations occur in the phosphorylation state of KaiC, a centralcomponent of cyanobacterial circadian clock, even after the termination of global
transcriptional activity (Tomita et al. 2005). This KaiC-phosphorylation rhythm canbe reconstituted in vitro by mixing KaiC and its regulatory factors KaiB and KaiC
together with ATP (Nakajima et al. 2005). In mammals, a recent study discovered
the presence of the circadian oscillations in the redox state of enucleated human red
blood cells (O’Neill and Reddy 2011). The circadian oscillation in redox status ofperoxiredoxin proteins is conserved from prokaryotes to eukaryotes (Edgar et al.
2012) and can regulate the neuronal activity of SCN (Wang et al. 2012). Although acore post-translational circadian oscillator in mammals remains to be identified,cooperation of transcription-translation oscillator and post-transcriptional oscillator
would provide a more robust circadian timekeeping system. The investigation ofcompatible interactions between delayed negative feedback loops mediated by the
CCEs and yet-unknown core post-translational oscillators will lead to a newunderstanding of mammalian circadian clocks.
Acknowledgements We thank Ms. Maki Ukai-Tadenuma and Drs. Arthur Millius and Rikuhiro
Yamada for figure preparation and valuable comments.

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