Resource

Knockout-Rescue Embryonic Stem Cell-Derived
Mouse Reveals Circadian-Period Control by Quality
and Quantity of CRY1
Graphical Abstract

Authors
Koji L. Ode, Hideki Ukai,
Etsuo A. Susaki, ...,
Masato T. Kanemaki, Hiroshi Kiyonari,
Hiroki R. Ueda

Correspondence
uedah-tky@umin.ac.jp

In Brief
Ode et al. establish an efficient method to
conduct gene-rescue experiments inmutant mice. Applying the method toknock in a series of CRY1 mutants into
Cry1 / :Cry2 / mice, they discover thatmultisite phosphorylation around theflexible loop domains of CRY1 determine
the period length of mammalian circadianclock in vivo.

Highlightsd

A KO-rescue ES mouse method was developed to produce 20
different KO-rescue strainsdMultisite phosphorylation of CRY1 can serve as a cumulative
timer

d

CRY1-PER2 interaction confers a robust circadianrhythmicity in micedFlexible loops of CRY1 determine circadian period in mice
without a turnover change

Ode et al., 2017, Molecular Cell 65, 1–15
January 5, 2017 ª 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.molcel.2016.11.022

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

Molecular Cell

Resource
Knockout-Rescue Embryonic Stem Cell-Derived
Mouse Reveals Circadian-Period Control
by Quality and Quantity of CRY1
Koji L. Ode,1,2,8 Hideki Ukai,2,8 Etsuo A. Susaki,1,2,3,8 Ryohei Narumi,2,9 Katsuhiko Matsumoto,2 Junko Hara,2
Naoshi Koide,2 Takaya Abe,4 Masato T. Kanemaki,3,5,6 Hiroshi Kiyonari,4,7 and Hiroki R. Ueda1,2,10,*
1Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan
2Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
3PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan
4Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047,
Japan
5Division of Molecular Cell Engineering, National Institute of Genetics, Research Organization of Information and Systems, Yata 1111,
Mishima, Shizuoka 411-8540, Japan
6Department of Genetics, School of Life Science, SOKENDAI, Mishima, Shizuoka 411-8540, Japan
7Animal Resource Development Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan
8Co-first author
9Present address: Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki,
Osaka 567-0085, Japan
10Lead Contact*Correspondence: uedah-tky@umin.ac.jp
http://dx.doi.org/10.1016/j.molcel.2016.11.022

SUMMARY

To conduct comprehensive characterization of molecular properties in organisms, we establishedan efficient method to produce knockout (KO)rescue mice within a single generation. We applied
this method to produce 20 strains of almost
completely embryonic stem cell (ESC)-derived mice(‘‘ES mice’’) rescued with wild-type and mutant
Cry1 gene under a Cry1 / :Cry2 / background. A
series of both phosphorylation-mimetic and nonphosphorylation-mimetic CRY1 mutants revealed
that multisite phosphorylation of CRY1 can serve as
a cumulative timer in the mammalian circadian clock.
KO-rescue ES mice also revealed that CRY1-PER2
interaction confers a robust circadian rhythmicity
in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation
feedback loops, the residues surrounding the flexible
P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rateof CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.

INTRODUCTION
A gene-rescue experiment under mutant background is powerfuland has been used in mammalian genetics (Antoch et al., 1997),

but the research procedures often require several generationsof animal crosses to obtain genetically modified mice. To overcome these problems, it is ideal if one can perform next-generation mammalian genetics, which can be defined as a production
and phenotype analysis of genetically modified mice within asingle generation. We previously reported that an injection of
three-inhibitor (3i)-treated embryonic stem cells (ESCs) into earlystage embryos at eight-cell stage can produce chimera micewith efficient contribution of ESC-derived cells (Kiyonari et al.,2010). Using this technique, it will be plausible to analyze thephenotype of gene-rescued mouse within a single generationby using mutant ESCs as a host cell line for the rescued gene.
Among various applications for this technological platform, themammalian circadian clock is an ideal model system because of
its underlying complex and dynamic molecular networks. The
E/E’-box-mediated transcriptional program has a critical rolein the core autoregulatory loop of the mammalian circadian clock(Mohawk et al., 2012). In this loop, basic helix-loop-helix (bHLH)PAS (Per-ARNT-Sim) transcription activators such as BMAL1
and CLOCK form heterodimers that bind to E/E’-box cis-elements in the promoter regions of their target genes including
the Per and Cry genes; CRYs in turn form repressor complexes
including PERs and other binding partners (Brown et al., 2005;
Duong et al., 2011; Kim et al., 2015) that physically associatewith the BMAL1/CLOCK complex to inhibit E/E’-box-mediatedtranscription. This delayed feedback repression mediated by
CRYs, especially CRY1, plays a pivotal role in the cell autonomous circadian oscillation in mammals (Khan et al., 2012;
Ukai-Tadenuma et al., 2011).
This model of transcriptional/translational feedback repression leads to theoretical predictions that the increased turnoverrate of transcription repressor mRNAs or proteins results in
Molecular Cell 65, 1–15, January 5, 2017 ª 2016 Elsevier Inc. 1

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

A

B

C

D

Figure 1. The Phenotype Analysis of KO-Rescue MEFs
(A) A systems framework to reveal the critical residues of CRY1 for controlling the circadian period in vivo.
(B) Identified (black) and predicted (blue) phosphorylation sites, and conserved residues involved in canonical electron-transfer pathway in 6-4 photolyase(green). Identified phosphorylated peptides are shown in Table S1.
(legend continued on next page)2 Molecular Cell 65, 1–15, January 5, 2017

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

circadian-period shortening (Forger, 2011). Several lines of evidence support this prediction that circadian period of mammalian clocks can be controlled by protein stability of CRY1/2. For
example, FBXL3, a component of Skp1-Culin-F-box-protein(SCF) ubiquitin ligase complex that guides CRY1/2 to proteasome-mediated proteolysis, shortens circadian period (Busino
et al., 2007; Godinho et al., 2007; Siepka et al., 2007). By
contrast, KL001, which binds to the flavin adenine dinucleotide(FAD)-binding pocket of CRY1/2 and stabilizes the protein,
lengthens the circadian period (Hirota et al., 2012; Nangle
et al., 2013).
However, there are some results that contradict with thetheoretical prediction. For example, destabilization of CRY1 by
AMPK-dependent phosphorylation should results in the shortening of circadian period, but stimulation of AMPK results inperiod lengthening (Lamia et al., 2009). A recent chemicalbiology study even succeeded to synthesize a compound boundto the CRY1/2 FAD-binding pocket, that shortens the circadianperiod, but surprisingly, stabilizes CRY1 (Oshima et al., 2015).
These results imply that the stability-independent period determination of circadian clocks, demonstrated in other organisms(Larrondo et al., 2015; Nakajima et al., 2005), might be also
true for mammalian circadian clocks.
In this study, we established an efficient method to produce
knockout (KO)-rescue mice within a single generation (KOrescue ES mouse method) by the 3i-8-cell method. We thenapplied this method to the production of 20 strains of different
Cry1 wild-type/mutants knockin mice under a Cry1 / :Cry2 /double mutant background. The mutagenesis of CRY1 revealed
that residues surround the flexible P loop and C-lid domains of
CRY1 determine the period length of circadian clock, and mostof them have only a marginal effect on the degradation rate of
CRY1, suggesting the presence of period determination mechanism independent of CRY1’s degradation rate. This highthroughput knockin mouse strategy would accelerate circadianand other fields of biology using various mouse strains harboring
modified genes or reporters and thus may help to shift the conventional way of mammalian genetics, which depends largely on
the crossing of animals.
RESULTS
The Phenotype Analysis of KO-Rescue Mouse
Embryonic Fibroblasts
We intended to analyze complex and dynamic molecular networks in organisms by focusing on CRY1 protein in mammalian
circadian clocks as a model system (Figure 1A). The first step isthe identification of critical residues of CRY1 for circadian-period
determination. Phospho-peptides derived from CRY1-overex-pressed 293T cells were analyzed with mass spectrometer(Figure S1A). We identified 27 phosphorylation sites (Figure 1B;
Table S1) including previously reported residues targeted by
mitogen-activated protein kinase (MAPK S247) (Sanada et al.,2004), AMPK (S71 and S280) (Lamia et al., 2009), and DNA-PK(S588) (Gao et al., 2013). We did not identify phosphorylation at
S404 but included this residue as a possible phosphorylationsite based on database prediction as a protein kinase C (PKC)-targeted site (Lamia et al., 2009). Three groups of potential functional
residues in CRY1 (tryptophan triplet W320, W374, and W397; histidine/tyrosine triplet H355, H359, and Y413; and asparagine
N393) were also selected based on a canonical electron-transfer
pathway proposed in its evolutionally most-related protein, 6-4
photolyase (Figure S1B) (Ozturk et al., 2007; Sancar, 2004).
Each phosphorylated residue was then mutated to phosphorylation-mimetic aspartic acid (D) or non-phosphorylationmimetic alanine (A). The conserved electron-transfer motifs
were mutated to alanine. The mutant CRY1 was then expressedin Cry1 / :Cry2 / mouse embryonic fibroblast (MEF) under
the control of Cry1 promoter combined with intronic enhancer
Rev-Erb/ROR-binding element (RREs) (Ukai-Tadenuma et al.,2011). The expressed CRY1 rescued circadian rhythmicity in
the MEF with various period and amplitude depending on themutations (Figure 1C). A numbers of mutants failed to rescue
the detectable rhythmicity; we noticed that there are three classes of these arrhythmic mutants, namely, dominant-short, hyperrepression, and hyporepression types (Figure 1D). Each classof mutant affected the circadian rhythmicity in a qualitatively
different manner when it was overexpressed in wild-type NIH
3T3 cells (Figure S1C). When wild-type CRY1 was overexpressed in NIH 3T3 cells, the expression greatly suppressed
the amplitude of E-box driven reporter without affecting theperiod length. Arrhythmic hyporepression mutants failed to suppress the amplitude suggesting the reduced transcriptionrepressor activity. In contrast, mutants classified as arrhythmic
hyperrepression further suppressed the amplitude comparedwith wild-type CRY1, suggesting the enhanced transcriptionrepression activity. Unlike these classes, arrhythmic dominantshort mutants accelerated the speed of circadian oscillation
when they were overexpressed in NIH 3T3 cells. We interpreted
that intrinsic period of these arrhythmic dominant-short mutants
are extremely short.
The overall in vitro phenotype results are summarized in
Figure 1D and Table S2. Although mutation on the conserved
electron-transfer motifs caused different effects, we expectthat these motifs in mammalian cryptochrome may not have anelectron transfer activity, at least in the regulation of circadianrhythmicity, because phenylalanine substitution of tryptophan(W374F:W397F) (23.0 ± 0.3 hr) or cysteine substitution of(C) Examples of circadian oscillation in Cry1 / :Cry2 / MEF monitored by transiently transfected reporter plasmid expressing destabilized luciferase driven by
Per2 gene’s promoter (P(Per2)-dLuc). For the Cry1 / :Cry2 / MEF rescue experiments in this and the other figures, the luciferase signal was normalized suchthat the maximum value of each time course data is one, and shown as average (solid line) ± SD (shade). Note that the same data for wild-type rescue was shownin each panel for comparison.
(D) Period and amplitude of rescued circadian rhythmicity in Cry1 / :Cry2 / MEF was calculated for each mutant CRY1, and shown as average ± SD. The
arrhythmic mutants were classified based the experiments using NIH 3T3 cells as shown in Figure S1C. The value of period and relative amplitude, and number ofexperiments are shown in Table S2. Wild-type/mutant CRY1 indicated in red were used for KO-rescue ES mouse experiment in Figures 4 and 5.
See also Tables S1 and S2 and Figure S1.

Molecular Cell 65, 1–15, January 5, 2017 3

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

28

5

1

225 - L E R K A W V A N F E R P R M N A N S L L A S P T G L S P Y L R F G
243 - L E R K A W V A N Y E R P R M N A N S L L A S P T G L S P Y L R F G
225 - L E R K A W V A N F E R P R M N A N S L L A S P T G L S P Y L R F G
244 - L E R K A W V A N Y E R P R M N A N S L L A S P T G L S P Y L R F G

26

25
2
25
4

7

9

24

24

3
24

A

LS
LS
LS
LS

R L F Y F K L T D L YK K V K K N S S P P L S L
R L F Y Y R L WD L YK K V K R N S T P P L S L
R L F Y F K L T D L YK K V K K N S S P P L S L
R L F Y Y R L WD L YK K V K R N S T P P L S L

B
S158D
S285A

0.9

S243A

S243A
S285A

0.9

WT

WT

WT

0.6

0.6

0.6

0.3

0.3

0.3

0.0

0.0
0

1

2

3

4

5

0.0
0

S158D:S285A
WT

0.9

1

2

3

4

0

5

S243A:S285A
WT

0.9
0.6

0.6

0.3

0.3

0.3

0.0

0.0
1

2

3

4

2

3

4

5

S243A:S261A
WT

0.0
0

5

1

0.9

0.6

0

S261A

0.9

1

2

3

4

0

5

1

2

3

4

5

D

S243A

30

S243A:S261A

S261A

T249A
WT
S247A
S252A
Y254A
25

S243A:S252AS243A:Y254A

20

S243A:S247A
S243A:T249A

15

S243A:S285A

S158D:S285A

Period
(hr)

S158D

S285A

C

35

40

E
S243A:S261A
10

1.0
S243A:S261A (37.9 ± 1.0 hr)

S243A:Y254AS158D:S285A (19.1 ± 1.1 hr)
0.5

5

S243A:S247A

S243A:S252AS243A:T249A

0.0

0
S243A:S285A-0.5
R2 = 0.99
p < 0.01

S158D:S285A

0

5

10

-1.0
0

1

2

3

4

5

(legend on next page)

4 Molecular Cell 65, 1–15, January 5, 2017

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

asparagine N393C (corresponds to insect-specific cryptochromes) (28.7 ± 0.5 hr) support robust circadian oscillations.
Many of phosphorylation-mimetic mutation on the phosphorylation cluster near the P loop domain greatly shorten the circadianrhythmicity (e.g., S243D, S247D, T249D, S252D, and S285D).
Among all analyzed phosphorylation residues, S243 appears tobe the most critical residue for period determination because
this site is the only residue that has bidirectional effects on
circadian period when mutated to A or D.
Multisite Phosphorylation of CRY1 Can Serve as a
Cumulative Timer
We then asked the logic behind the period determination, especially focusing on the phosphorylation cluster near the P loop
domain (Figure 2A). If the serial phosphorylation events are triggered by priming phosphorylation at the specific site, then thepriming site works as a ‘‘switch’’ and governs the others. In
this case, alanine mutation on the priming phosphorylation site
is epistatic to the alanine mutation of the other residues. In
contrast, if the accumulation of local negative charge additively
evokes the response, then the effects of alanine mutations canbe cumulative, working as a ‘‘timer.’’ To distinguish these scenarios, we introduced two alanine mutations for a various pairof phosphorylation sites indicated in Figure 2A. We also included
one phosphorylation-mimetic mutant (S158D) outside of theregion near the P loop domain.
The period of every analyzed CRY1 with mutations in two
phosphorylation residues revealed clear additive relationshipon the period determination (Figure 2B). The additive rule wasapplicable not only to the distant pairs of residues (i.e., S158Dand S285A) but also to local pairs within the restricted area
near the P loop (Figures 2C and 2D; Table S3). This assay also
revealed the highly flexible nature of CRY1-dependent period
determination at nearly 2-fold dynamic range (Figure 2E). These
results suggest that multisite phosphorylation of CRY1 serves as
a cumulative timer in mammalian circadian clock.
Because S243 was the only residue that had both shortening
and lengthening effects on period, we investigated what kinase
is responsible for the phosphorylation at S243. Several algorithms for kinase prediction listed casein kinase I (CKI) as apotential kinase to phosphorylate S243 or downstream S247
(Table S4). Furthermore, we found that one of the responsible kinase for S243 phosphorylation, at least in 293T cells, is CKId/ε, amajor kinase that accelerates the pace of circadian oscillation(Figure S2A; Table S5) (Mohawk et al., 2012).

S243 is conserved in mammalian CRY proteins and plant 6-4
photolyases but is rarely conserved among the other proteins incryptochrome superfamily (Figure S2B). Phylogeny analysis revealed that serine/threonine and negatively charged aspartic
acid and glutamic acid reciprocally emerge among the areasurrounding S243 position (Figures S2B–S2D). A phylogenetic
theory suggests that phosphorylation activating the protein function tends to be aspartic acid or glutamic acid in the ancestralform (Pearlman et al., 2011). Applying this rationale to mammalian CRY proteins, mammalian cryptochromes for the circadianclock might interchange the static negative charge of D/E witha dynamic phosphorylation site for controlling circadian period.
The Development of a KO-Rescue ES Mouse Method
To directly confirm the in vivo significance of period-determining
residues identified in Cry1-rescue assay in Cry1 / :Cry2 /MEF, we next conducted Cry1-rescue assay under Cry1 / :Cry2 / mice. The coat color of Cry1 / :Cry2 / ES mice wasalmost identical with the original Cry1 / :Cry2 / mice, suggesting the contamination of host embryo cells, which would resultin white coat color, was negligible (Figure 3A). PCR-based genotyping further confirmed that the contamination of cells from thehost embryo having a wild-type Cry2 allele was no more than
0.001% (Figure 3A). Both original Cry1 / :Cry2 / mice andthe double-knockout ES mice had arrhythmic behavior (Figure 3B). We then knocked in Cry1 under the control of Cry1 promoter including an intronic RRE element (Ukai-Tadenuma et al.,2011) into the ROSA26 locus (Figure 3C; Table S6). During theseries of knockin targeting, we used not only a conventional targeting method with on-feeder ESC culture but also amore efficient targeting method with feeder-free ESC culture
and TALENs (transcription activator-like effector nucleases)(Figures S3A–S3F) (Sung et al., 2013). These gene-targeting
conditions are specified in Table S7.
The Cry1 cassette introduced in ROSA26 locus rescued circadian rhythmicity in their behavior with slightly different periods
depending on the direction of insertion (Figures 3D and 3F; Table
S7). We chose the cassette designed to be inserted to ROSA26
locus in antisense direction for subsequent analyses, because
their behavioral period in ES mice with a single antisense
cassette (24.2-24.3 hr) was closer to the free-running periodsof Cry1+/–;Cry2–/– mice (24.29 hr) (van der Horst et al., 1999).
The stringency of the Cry1 KO-rescue ES mouse method wasconfirmed by the following two experiments. First, circadianrhythmicity was not observed if Cry1 gene is driven by

Figure 2. Multisite Phosphorylation of CRY1 Can Serve as a Cumulative Timer(A) Sequence alignment of P loop and downstream sequence. Indicated residues were used in double mutants in this figure. Mm: Mus musculus. Hs: Homosapiens.
(B) Example of circadian period of wild-type (black), or mutant CRY1 with single (green) or double (blue) phosphorylation residues. The experiment was carried out
as in Figure 1. Note that the data of wild-type and of single site mutants (green) were identical data shown in Figures 1C and 1D.
(C) The rescued period lengths for indicated combination of dual-sites mutants were calculated. Note that the periods of single site mutants (green) were identical
data shown in Figure 1D.
(D) The effect of double mutants to circadian period is the sum of the effect of individual single mutations. The x axis is the period of rescued Cry1 / :Cry2 / MEF
rescued by indicated double mutants as shown in (C). The y axis is the estimated period by adding the period difference between wild-type and individual point
mutants. The period lengths are shown as average ± SD and summarized in Table S3.
(E) Circadian oscillation of CRY1 double mutant showing shortest period (S158D:S285A) and longest period (S243A:S261A). The experiment was carried out as
Figure 1, and detrended luciferase signal are shown as average ± SD.
See also Tables S3, S4, and S5 and Figure S2.

Molecular Cell 65, 1–15, January 5, 2017 5

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

A

B
Cry1-/-:Cry2-/ES mouse (clone #1)

WT
mated pup
(hr)

0

48

0

WT
ES mouse
(hr)

48
1

LD

LD

1

7

7

DD

DD

(Days)(Days)

21

22
Cry1 :Cry2
ES mouse (clone #1)
mouse ID
6 7

0

8

- 300

DD

- 400
- 200

Cry2 specific(control)

(bp)

#7 #6
E1

XbaI
E2

WT-rescued (S)ES mouse (clone #1)
(hr)

0

E3

0

DD

Cry1 cassette Puro#3
#4

Mut-rescued (AS)ES mouse (clone #1)
(hr)

0

0

48

Mut-rescued (AS)ES mouse (clone #2)
(hr)

48

1

1
LD

pA

LD

FLAG Intron
+RRE

22

22

Cry1

P(Cry1)

7

FRT

Cry1 rescue cassette
WT rescue1(Days)#1

48

7

Cry1 cassette Puro#8
#1

Rescue (AS) DT

WT-rescued (AS)ES mouse (clone #1)(hr)1(Days)

Rescue (S) DT

18

48

LD

#2

7

18

D

ROSA26

1

LDC
1 kbp #9
#5

48

7

21

F

DD

21

Arrhythmic

C

21(Days)(Days)

7

Arrhythmic

23

LD

LD

1

7

DD

48

Arrhythmic

0
1

W
W Tm
1 -/T
C
:C
E ate
ry
ry
C
1 -/- S m d
ry
2 -/:
1
ou
C
/W
ES
ry
T- :Cr2 -/- se
m
y2
re
o
m
/us
at
W scu
e
ed
e ES
T(c
re d (
m
l
o
S
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s
W
ne
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u
)
u
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Ted ESe#rem (clo 1)
W scu (S)oTed
ES use nere(c #2)msc (ASMue) E ous lone
ut
d
e
-re
S
#
(c
m
sc (ASlo 1)
M) E ou
ue
ut
se ne
S
-re d (
#2
(c
m
A
sc
)
ue S) E ous lone
W
e
d
S
#1
T(c
(
m
A
)
re
lo
S)
ou
s
se ne #
W cue ES
2)
T(c
d
m
lo
re
(
ne
sc AS ous
)(
e
ue
#1
cl
(c
d
)
o
l
(A
ne on
S)
#2 e #
(c
2
)
)
lo
ne F1
#2 RC
+
)F
1
R
C
-

48

24

ry

0

Rescue cassette (-)(hr)

Arrhythmic

Period (hr)F1 littermates

21

25

Cry1-/-:Cry2-/- × WT-rescued (AS) ES mouse (clone #2)

ArrhythmicE

Rescue cassette (+)
(hr)

DD

pA

DD

FLAG Intron+RRE(mutated)(Days)Cry1

P(SV40)7(Days)

Mut rescue(Days)

7

- 100
-/-

0

(Days)

WT specific

48
1

9

LD

4 5

Cry1-/-:Cry2-/ES mouse (clone #1)
(hr)

DD

2 3

Cry1-/-:Cry2-/mated pup
(hr)

DD

1

-/-

LD

1

0.
1
0.
01
0.
00
0 1

Standard(% of WT)

Marker-/-Arrhythmic

Cry1-/-:Cry2-/mated pup(legend on next page)

6 Molecular Cell 65, 1–15, January 5, 2017

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

non-circadian promoter (Figures 3C and 3D, Mut rescue) (UkaiTadenuma et al., 2011). Second, F1 littermates of rescued
mice and Cry1 / :Cry2 / mice showed circadian rhythmicity
only if the offspring had Cry1-knocked-in allele in the ROSA26
locus (Figure 3E). These results indicate that the behavioral
rhythmicity is induced by the Cry1-rescue cassette. Note that,for the ‘‘Mut rescue’’ condition, we did not analyze the exact
reason of arrhythmicity; it may be due to the altered periodicity
for the expression timing of Cry1 or the altered expression levelof Cry1. In the following experiments, we used the same (wildtype [WT]) promoter to compare the phenotypes of different
Cry1 mutants.
The Phenotype Analysis of KO-Rescue ES MiceSeventeen Cry1 mutants (see Figure 1D, red colored mutants)
were selected for the assay of Cry1-rescue ES mice. The
normal-period mutants in MEFs (N393C and S404A) had almost
identical or shorter period in ES mice (24.2 ± 0.1 and 23.9 ±0.1 hr, respectively) compared with the circadian period of
WT-rescued ES mice (24.2 ± 0.1 hr) (Figure 4A). The short-periodmutants in MEFs (S158D and S285A) had shorter circadianperiod in ES mice (23.9 ± 0.1 hr and 23.8 ± 0.1 hr, respectively)(Figure 4B). In contrast, long-period mutants in MEFs (S243A,
S492D, and HHY) had significantly longer circadian period in
ES mice (24.6 ± 0.05, 25.4 ± 0.1, and 26.6 ± 0.3 hr) (Figure 4C).
The arrhythmic dominant-short mutants in MEFs (S243D,
S247D, T249D, and S285D) rescued the circadian rhythmicity in
KO-rescue ES mice and exhibited markedly shorter period(21.1 ± 0.1, 22.4 ± 0.1, 22.7 ± 0.1, and 22.7 ± 0.1 hr, respectively)(Figure 4D). These results strongly suggest that the intrinsic periods of arrhythmic dominant-short mutants are markedly shorter
than that of any other rhythmic mutants in MEFs. In addition, allthree arrhythmic hyporepression mutants (S261D, Y432D, andWWW) in MEFs also had arrhythmic phenotype in mice (Figure 4E).
These results further strengthen the conclusion that arrhythmic hyporepression mutants are the loss-of-function form of protein andare qualitatively different from arrhythmic dominant-short mutants.
Correlation between the Circadian Phenotype of
KO-Rescue MEFs and ES Mice
Overall, KO-rescue ES mice results summarized in Figure 5A
demonstrates a striking correlation between circadian periods

observed in MEFs and those observed in KO-rescue ES mice.
Note that the magnitude of circadian-period alteration in each
CRY1 mutant ES mice from the wild-type was smaller than thatof CRY1 mutant MEFs, converging close to 24 hr in KO-rescue
ES mice (Figure 5B).
There are several mutants, the phenotypes of which in ES
mice are not in line with those of MEFs; S261A mutant had
long-period phenotype in MEFs whereas the mutant ES mice
had an arrhythmic phenotype (Figure 5C). In addition, the phenotypes of N393A and S404D mutant both of which had arrhythmic
hyperrepression phenotypes in MEFs were not reproduced in ES
mice: N393A mutant ES mice had a long-period phenotype(25.0 ± 0.4 hr) (Figure 5D), whereas the period of S404D tends
to be short in the first 2 weeks after the entry to constant darkness condition and then became longer in the next 2 weeks(Figure 5E). Interestingly, we found that all CRY1 mutants witharrhythmic (S261A, S261D, Y432D, and WWW) or unstableperiod (S404D) phenotypes in ES mice had a significant reduction in the interaction with PER2 when these CRY1 mutants
were expressed in 293T cells and quantified for the amount of
PER2 co-immunoprecipitated with CRY1 (Figure 5F). These
results highlight the importance of CRY1-PER2 interaction instable rhythmicity in vivo.
Correlation of CRY1 Turnover Rate and Circadian Period
To investigate what biochemical properties of CRY1 protein candetermine the circadian period and amplitude, we analyzed
CRY1’s degradation rate (Figure S4A) and transcription-repression activity (Figure S4B). As a result, significant correlation
was observed between CRY1 half-life and circadian period
and between CRY1 repression activity and circadian amplitude
but not for other combinations (Figure S4C). These results are
consistent with the canonical relationship between CRY1 degradation and circadian period (Forger, 2011) and between CRY1
repression activity and circadian amplitude (Khan et al., 2012).
To our surprise, a number of mutants had altered circadianperiod without significant changes in protein half-life (Figure 6A,
red circles). Of note, a group of long period mutants (HHY,
S492D, S492A, and S243A) had no significant change in protein
stability, but their period was significantly longer than the moststable mutant S588D (Gao et al., 2013). Also, it is notablethat the arrhythmic dominant-short mutants (S243D, S247D,

Figure 3. The Development of a KO-Rescue ES Mouse Method(A) 3i-cultured Cry1 / :Cry2 / ESCs were derived from B6-backcrossed Cry1 / :Cry2 / strain (van der Horst et al., 1999). ESC clones producing male ES
mouse with black coat color were selected. Contamination of host embryo cells was evaluated by semiquantitative genomic PCR for Cry2 locus with primers
specific to wild-type or Cry2 / allele.
(B) Representative actograms of mated pups (wild-type or Cry1 / :Cry2 / C57BL/6) and ES mice derived from ESCs of wild-type or Cry1 / :Cry2 / C57BL/6.
Shaded regions indicate dark phase. Time windows marked as blue line indicate system shutdown due to the electric outage of the mouse facility.
(C) A structure of Cry1-rescue cassette and targeted ROSA26 locus. ‘‘WT rescue’’ cassette drives Cry1 expression by promoter of Cry1 gene and intronic RRE
element (Ukai-Tadenuma et al., 2011). In the ‘‘Mut rescue’’ cassette, the promoter is swapped to SV40 promoter and the sequences of intronic RRE are mutated.
Arrows with numbers indicate PCR primers for ESC screenings and genome integrity confirmations, summarized in Table S6. AS, antisense, pA, poly-A tail; S, sense.
(D) Representative actograms of Cry1 / :Cry2 / ES mice rescued with WT rescue cassette in sense (S) or antisense (AS) direction, and Mut rescue cassette inantisense (AS) direction.
(E) Representative actograms of F1 offspring obtained by crossing the rescued ES mice with Cry1 / :Cry2 / strain. The offspring harboring the Cry1-rescue
cassette (indicated as ‘‘+’’) at the ROSA26 locus showed circadian behavioral rhythmicity.
(F) Summary of the quantified period of circadian behavioral rhythmicity shown as average ± SD (during DD condition). The period lengths and gene-targeting
conditions are summarized in Table S7.
See also Tables S6 and S7 and Figure S3.

Molecular Cell 65, 1–15, January 5, 2017 7

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

B

Normal period in KO-rescue MEF
Cry1 (N393C)-rescued ES mouse(hr)

Cry1 (S404A)-rescued ES mouse

48

0

(hr)

1
LD
DD

DD

DD

28

28

C

14

(Days)

14(Days)

14(Days)(Days)

14

1

1

LD

LD

1

Cry1 (S285A)-rescued ES mouse
0
(hr)
48

Cry1 (S158D)-rescued ES mouse
0
(hr)
48

48

LD

0

Short period in KO-rescue MEF

DD

A

28

28

Long period in KO-rescue MEF
Cry1 (S492D)-rescued ES mouse

Cry1 (S243A)-rescued ES mouse
0

(hr)

48

0

(hr)

48

1

0

Cry1 (H355A:H359A:Y432A)-rescued ES mouse
48
(hr)

LD

1

LD

LD

1

DD

DD

DD

14

(Days)

14(Days)(Days)

14

28

28

D

28

Arrhythmic dominant-short in KO-rescue MEF
Cry1 (S247D)-rescued ES mouse
0
(hr)
48

Cry1 (S243D)-rescued ES mouse
0
(hr)
48

Cry1 (T249D)-rescued ES mouse
0
(hr)
48
1

LD

LD
DD

DD

DD

LD
DD

28

28

E

14

(Days)

14(Days)

14(Days)(Days)

14

1

1

LD

1

Cry1 (S285D)-rescued ES mouse
0
(hr)
48

28

28

Arrhythmic hypo-represion in KO-rescue MEF
Cry1 (S261D)-rescued ES mouse

Cry1 (Y432D)-rescued ES mouse
48

0

(hr)

48

0
1

DD

DD

28

14

(Days)

14(Days)(Days)

14

48
1

LD

LD

1

Cry1 (W320A:W374A:W397A)-rescued ES mouse
(hr)

LD

(hr)

DD

0

28

28

Figure 4. The Phenotype Analysis of KO-Rescue ES Mice
Representative actograms of a Cry1 / :Cry2 / mouse rescued with indicated mutant Cry1 in anti-sense direction, exhibiting near-wild-type period length (A),shorter period length (B), longer period (C), arrhythmic dominant-short phenotype (D), or arrhythmic hyporepression phenotype (E) in Cry1 / :Cry2 / MEF
rescue experiment shown in Figure 1. All mutant CRY1-rescued ES mouse lines analyzed in this study except for mutants shown in Figures 5C–5E are shown. The
period lengths and gene-targeting conditions are summarized in Table S7.
See also Table S7.

8 Molecular Cell 65, 1–15, January 5, 2017

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

A36

Arrhythmic
HHY

S261A
S261D

27

HHY
S492D
S243A

Y432D
W320A:W374A:W397A

26

32

Unstable

S492D

N393A

S404D
25
WT
S158D

Period (hr)Period in ES mice (hr)Figure 5. The Circadian Phenotype of KORescue ES Mice

B

S243A
N393C

24
S404A

S285A

N393C
28
WT
S404A

23
T249D
S285D
S247D

24 S158D

22

S285A

S243D

20
25
30
Period in MEFs (hr)

C

MEFs

35

H
re yp
pr er
es si
on

20

D
sh om
or in
t a

nt

-

21

E

Long period in KO-rescue MEF

ES mice

Arrhythmic hyper-repression in KO-rescue MEF

Cry1 (S261A)-rescued ES mouse
(hr)
0
48

0

Cry1 (S404D)-rescued ES mouse
(hr)
48

1
LD

LD

1

(Days)

14

14

DD(Days)

28

28

DD

D

(A) Summary of period in behavioral rhythmicity. The
representative behavioral plots are shown in Figure 4
and Figures 5B–5D. The behavioral period in constantdarkness condition was calculated and shown as
average ± SD. The period (or, categories in arrhythmic
mutants) of molecular oscillation recorded in rescued
MEF (i.e., results shown in Figure 1D) is plotted against
the period of behavioral rhythmicity in rescued mice.
Mutants shown in red are arrhythmic or show unstable
period in rescued mice.
(B) For each mutant that was rhythmic both in rescued
MEF and rescued mice, the period lengths of MEF andmice were compared.
(C) A representative actogram of a Cry1 / :Cry2 /mouse rescued with S261A mutant CRY1.
(D) A representative actogram of a Cry1 / :Cry2 /mouse rescued with N393A mutant CRY1.
(E) Top: a representative actogram of a Cry1 / :Cry2 /mouse rescued with S404D mutant CRY1. Because theperiod lengths of behavioral rhythmicity are unstablein this mutant line, the recoding was extended over
4 weeks in constant darkness. Bottom: the period was
calculated for the first 2 weeks and the second 2 weeks
of constant darkness condition for each S404D-rescued
mouse.
(F) Interaction of CRY1 mutant and PER2 was quantified
by co-immunoprecipitation (IP) and mass spectrometrybased quantification. Three different PER2-derived
peptides were quantified in two independent experiments. Data are shown as box-and-whisker plot andindividual quantified values; each circle indicates thequantified value of peptide derived from PER2 protein
normalized by the amount of CRY1 protein.
**p < 0.05; p > 0.1, Student’s t test compared with WT.
See also Table S7.

Arrhythmic hyper-repression in KO-rescue MEF

0

Cry1 (N393A)-rescued ES mouse
(hr)

48

42
1

24

DD

(Days)

14

Period (hr)

LD

24.5

23.5
(15 - 28 days)

28(29 - 42 days)

F
Co-IP PER2 / IP CRY1 (AU)

2.5
2.0
1.5
1.0

**

**

**

**

0.5

**

Phenotype in
MEF rescue

Long

Hyperrepression

D
W
W
W

D
61

32
Y4

S2

N

39

3A

D

Y

04
S4

D

H

92
S4

H

A

A
43

61
S2

S2

D
85

D

Dominant-short

S2

D

49
T2

D

47
S2

D

43
S2

58

A

Short

S1

85
S2

CRY1 mutant

W
T

0.0

Hyporepression

Molecular Cell 65, 1–15, January 5, 2017 9

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

A

Figure 6. Causality of Increased CRY1 Turnover Rate to Circadian Period Shortening

B
1.00
S588D

Relative P(Per2)-dLuc. expression

CRY1 half-life (min)

450

200

150
S243D

0.75

0.50

S492D

S243D
0.25
S285D
S492A
S247D

S243A
S492A

T249D

100

17.5

20

25
Period (hr)30

N393A S404D S252D

0.00
0.0

35

0.2

0.4
Relative amplitude

0.6

D

om

in
sh antor t

H355A:H359A:Y413A

CD
0 μg/mL NAA
7.5 μg/mL NAA

Normalized luc. signal

Cry1-/-:Cry2-/- MEF
Tir1

P(CMV)

Cry1

P(Cry1)mAID

FLAG Intron
+RRE

pA

SCF-TIR1

0.9

0.6

0.3

0.0
0

CRY1-mAID

1

2
3
Time (days)

1.0
Detrended luc. signal+Ub
NAA
SCF-TIR1

0.5

0.0

-0.5
-1.0

Proteasome-mediated CRY1-mAID degradation

0

E

5

0 μg/mL NAA
7.5 μg/mL NAA+ NAA

CRY1-mAID

4

F

1

2
3
Time (days)

4

5

G
0.1

1

0.1
0.0

-2
-3

∆Amplitude

0.0∆Amplitude∆Period (hr)

0
-1

-0.1

-0.2

-0.2

- TIR1+ TIR1-4- TIR1+ TIR1R2 = 0.83
p < 0.05
-0.3

-0.3
0

1
NAA (μg/mL)

10

-0.1

0

1
NAA (μg/mL)

10

T249D, and S285D) had decreased protein stability, if any,similar to the other short mutants. Hence, it is difficult to quantitatively explain the remarkable period shortening observed in
ES mice only by decreased CRY1 protein stability.
Causality of Increased CRY1 Turnover Rate to Circadian
Period Shortening
The finding of mutants that alter the circadian period with modest
effect on the CRY1 stability challenges the canonical relationshipbetween CRY1 degradation and period shortening. To ask the

10 Molecular Cell 65, 1–15, January 5, 2017

-4

-3

-2
-1
∆Period (hr)01(A) Circadian period of each mutant CRY1 inrescued MEF (data shown in Figure 1D) was
plotted against protein half-life (average ± SD).
Mutants with significantly shorter or longer half-life(p < 0.1, Student’s t test compared with wild-type)are shown in blue, and mutants with similar halflife compared with wild-type are shown in red.
‘‘Arrhythmic hyper-repression’’ mutants were notplotted. See Figure S4A for the half-life of eachmutants and Figure S4C for the linear regression.
(B) Relative amplitude of circadian rhythmicity
in rescued MEF (data shown in Figure 1D) was
plotted against relative activity of P(Per2)-dLuc
co-transfected with each mutant CRY1 (average ±SD). Mutants with significantly higher or lower
repression activity (p < 0.1, Student’s t testcompared with wild-type) are shown in blue, andmutants with similar repression activity comparedwith wild-type are shown in red. See Figure S4B
for the half-life of each mutants and Figure S4C
for the linear regression.
(C) Scheme of the construction of CRY1-mAID
system. Tir1 was expressed under constitutive(non-circadian) promoter. A fragment of the original AID/IAA17 tag called mini-AID (mAID) (Kubota
et al., 2013) was fused with the C-terminal of
CRY1. AID-tagged Cry1 was expressed under
the Cry1-rescue promoter. Addition of NAA induces the degradation of AID-tagged CRY1. Ub,
ubiquitin.
(D) Circadian oscillation of rescued MEF with AIDtagged CRY1 in the presence of TIR1. The experiments were carried out as in Figure 1 except forthat indicated amount of NAA was added to themedium. The upper half shows the normalized
signal of P(Per2)-dLuc signal, and the lower half isthe detrended oscillation signal.
(E) Changes in the oscillation period of CRY1mAID rescued MEF. The period length of eachexperiment was compared with the average
period of CRY1-mAID rescued MEF in the presence of TIR and 0 mg/mL of NAA.
(F) Changes in the oscillation amplitude of CRY1mAID rescued MEF. The relative amplitude ofeach experiment was compared with the average
amplitude of CRY1-mAID rescued MEF in thepresence of TIR and 0 mg/mL of NAA.
(G) Correlation of amplitude reduction and period
shortening by induction of CRY1-mAID degradation.
See also Figure S4.

causality between them, we tried to induce targeted and artificial
proteolysis of CRY1 by using Auxin-induced degradation (AID)
system (Figure 6C) (Kubota et al., 2013; Nishimura et al., 2009):
the AID-tag is recognized by TIR1 protein, a subunit of
SCF ubiquitin ligase, in the presence of auxin, leading to theproteasome-mediated degradation of AID-tagged protein in an
auxin’s dose-dependent manner. The tagged CRY1 successfully
repressed the E-box-mediated transcription and that activity is
reduced by the addition of synthetic auxin (naphthaleneacetic
acid [NAA]) in a dose-dependent and TIR-dependent manner

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

Figure 7. P Loop and C-lid as PeriodDetermining Domains of CRY1 Protein

A
C-lid

C-lid

Y413
S243

S492

H359

S247

P-loop

H355

KL001

S404
KL001

N393

45°

T249

P-loop

S285

B

C
KL001

P-loop

S492

R238

F409

S243
R367
S247
R236

(A) Crystal structure of Mm CRY2 bound to FAD
analog KL001 (PDB: 4MLP) (Nangle et al., 2013).
Note that S404 is not conserved in Mm CRY2
(substituted with alanine).
(B) A close-up view of the P loop and surrounding
regions.
(C) A close-up view of the C-lid and surrounding
regions. Two phenylalanine residues (F409, F410)
on the C-lid are sterically close to an R367 residue
that penetrates into the C-lid loop structure.
(D) Circadian period lengths of indicated CRY1
mutants. The arrhythmic mutants were classified
based on the phenotype of NIH 3T3 overexpressedwith the mutant (shown in Figure S5F). The experiments were carried out as in Figure 1.
**p < 0.05; Student’s t test compared with WT. The
period lengths are shown as average ± SD andsummarized in Table S8.
(E) Protein half-life of indicated CRY1 mutants
shown as average ± SD. The experiments werecarried out as in Figure 6. **p < 0.05, n.s. p > 0.1;
Student’s t test compared with WT.
(F) Proteolysis-dependent and -independent circadian-period determination by CRY1.
See also Table S8 and Figures S5 and S6.

F410
T249
R227

C-lid

S285

S404

KL001

E

500

300
250
200
150

n. s.

n. s.

R367K

Hyper-repression
F410A

350

R227A

Hyper-repression
F409A

R367A

Hyper-repression

**

R238A

R236A

WT

18

**

R227K

23

Dominant-short

**

**

400

100
50
0
WT

28

CRY1 half-life (min)

450

S588D

**

R227A

Period (hr)

33

R367K

D

decreased by the induction of CRY1 proteolysis (Figure 6F), resulting in a linear correlation between period and amplitude(Figure 6G). The result of induced CRY1
degradation suggest that the canonicalrelationships between circadian period
and CRY1’s half-life is applicable tocircadian phenotype several CRY1 mutants, although such relationships cannot
explain some exceptional mutants withdrastic effects on the circadian period
and rhythmicity.

P Loop and C-lid as PeriodDetermining Domains of CRY1
ProteinF
Quality dynamics
Quantity dynamics
We then mapped the positions of suchexceptional mutation on the reported
Cumulative phospho-timer & flexible loop structure
Transcription-translation
Proteolysis
CRY structures. Note that CRY2 structure
FAD
CRY1
bound with KL001 was used in this strucCRY1
P
P-loopC-lidtural analysis because P loop structure
CRY1
P
P
CRY1
was determined only in CRY2 but not
P
CRY1
CRY1
in CRY1 (Nangle et al., 2013). Residue
position number corresponding to CRY1
was used because of the consistency(Figures S4D–S4F). Furthermore, tagged CRY1 rescued the with our mutagenesis assay. We focused on residues that,circadian rhythmicity in the Cry1 / :Cry2 / MEF (Figure 6D). when mutated, result in markedly long-period phenotypes
The period of rescued rhythmicity became shorter in a NAA- (HHY, S492D, S492A, and S243A), arrhythmic dominant-shortdose-dependent manner (Figures 6D and 6E), confirming the phenotypes (S243D, S247D, T249D, and S285D) or arrhythmic
causal effect of CRY1 degradation on circadian period short- hyperrepression phenotypes (S404D and N393A). Figure 7A
ening. The amplitude of oscillating reporter signal was also indicates that all the above-listed residues are located aroundP-loopC-lid

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Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
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the co-factor (FAD/KL001) binding pocket of the CRY protein,especially the two loop domain called the P loop and C-lid.
H355, H359, and Y413 are located near the co-factor (FAD/
KL001) binding pocket and can interact with a potential co-factor. Consistently, KL001 had no detectable effect on HHY mutant(Figure S5A). Although S492 has no direct interaction with C-lid
domain or co-factor binding pocket, phosphorylation of S492
residue might affect the structure of downstream, non-crystalized C-terminal domain. On the basis of these arrangementof non-canonical period-determining residues, we anticipatedthat the P loop and C-lid, both of which are suggested to be flexible domain (Figure S5B) (Czarna et al., 2013; Nangle et al., 2013,2014; Schmalen et al., 2014; Xing et al., 2013), are the core domains of CRY1 in circadian-period determination. If these domains are responsible for period determination without changing
CRY1 protein stability, it may be possible that structure-guided
engineering of CRY1 mutation on these domain structures
recapitulates proteolysis-independent period modulation.
To test this, we focused on the arginine triplet on the P loop
domain (i.e., R227, R236, and R238) (Figures 7B and S5C)
because the positively charged and surface-oriented arginine
triplet may be involved in the interaction between CRY1 and otherfactors. We also focused on R367 and F409/F410 for C-lid
structure because the interaction of these residues may restrict
the flexibility of the C-lid (Figures 7C and S5D). As expected,
mutation on each residue altered circadian oscillation of CRY1rescued Cry1 / :Cry2 / MEF (Figure 7D). The alanine substitution of each arginine triplet resulted in a short-period phenotype
with most drastic effect for the R227A mutation. The arrhythmic
dominant-short phenotype of R227A was partially attenuatedwhen the positively charged residue lysine substituted the position (R227K). In contrast, alanine substitution of the F409 andF410 at C-lid and the same substitution of their contact partner
R367 resulted in arrhythmic hyper-repression phenotype (Figures
7D, S5E, and S5F; Table S8). When lysine substituted the R367
position, the R367K mutant had a significantly longer phenotype.
We then picked up R227A and R367K mutants for P loop and C-lid
domain, respectively, and investigated the protein stability ofthese mutants. As expected, Figure 7E shows that the protein stability of these mutants was not significantly changed comparedwith wild-type. The longer half-life of S588D mutant as observedin Figure 6A confirmed the robustness of this assay. The involvement of FAD-binding pocket and surrounding regions in the period
determination are also supported by the result showing thatcysteine residues involved in the disulfide bond at C-lid terminal(Schmalen et al., 2014) and residues mutated in Drosophila CRY(cryb) (Stanewsky et al., 1998) are also important for the circadian-period determination in mammalian CRY1 (Figures S6B
and S6C). Taken together, these data suggest that structural/electrostatic properties of P loop and C-lid are important for proteolysis-independent circadian-period determination (Figure 7F).
DISCUSSION
Multisite Phosphorylation of CRY1 Can Serve as a
Cumulative Timer
The linear and additive effect of phosphorylation mutants (Figure 2) implies that CRY1 plays a role as a cumulative timer. Pre-

12 Molecular Cell 65, 1–15, January 5, 2017

vious studies as well as database/algorithm-based prediction
provide (Table S4) various possible kinases responsible forthe phosphorylation of CRY1. The multisite phosphorylation
may integrate internal information (e.g., spent time) andexternal information (e.g., environmental signals) and converts
them into circadian-period modulation. Indeed, we identified
dual phosphorylated peptide near the flexible P loop (Table
S1, S243/S247). Multisite phosphorylation often occurs andregulates flexible structures or intrinsically disordered regions.
This type of regulation was found in FRQ in Neurospora andPER in mammals (Wright and Dyson, 2015) and may be ashared design principle for the control of circadian time keeping mechanism.
The role of phosphorylation may be different dependingon the target site; in our assay, S243 is the only residue,of which phosphorylation-mimetic and non-phosphorylationmimetic mutants have opposite effect in period length, suggesting that proper level of phosphorylation on this site and/or atimely phosphorylation along with the circadian cycle is important for the circadian time keeping. There are other classes ofphosphorylation site, in which only either the phosphorylationmimetic or the non-phosphorylation-mimetic mutant had a significant effect on the circadian period (e.g., S71). This may berelated to the phosphorylation level of each site. If one site isconstitutively phosphorylated during the circadian cycle, thennon-phosphorylation-mimetic mutation rather than phosphorylation-mimetic one would cause a severe effect. Contrary, if
one site is rarely phosphorylated, then phosphorylation-mimetic
mutation may cause a greater effect. For the mutants showingthat both of alanine and aspartic acid substitution cause thesimilar effect on period length (e.g., T131), it is likely that theexact composition of side chain as well as negative charge
might be important for the circadian phenotype. To validate
these predictions in future studies, quantitative measurementof phosphorylation at each site, efficient detection of multiphosphorylated peptides, and identification of responsible
kinases/phosphatases will be important.
CRY1-PER2 Interaction Confers a Robust Circadian
Rhythmicity in Mice
Previous study indicated that disulfide bond between C363
and C412 residues in CRY1 controls CRY1’s affinity to PER2
protein, and mutation on either one of two cysteine residues
increased the CRY1-PER2 interaction (Schmalen et al.,2014). However, our study showed that C363A mutation resulted in period lengthening while C412A mutation resulted
in period shortening (Figure S6B). Thus, the phenotype of
circadian period appears to be not matched with the phenotype of CRY1-PER2 interaction. This suggests that the phenotype of circadian period is caused by altered structure ofmutated residues not simply by the breakage of disulfide
bond. This result also implies that the importance of CRY1PER2 interaction for circadian clockworks may lie in aspects
other than period-determination processes. Notably, our KOrescue ES mouse analyses revealed the role of CRY1-PER2
interaction for the robust circadian rhythmicity in vivo rather
than period-determination and CRY1’s repression activity(Figure 5).

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022

Convergence of Period Length toward 24 hr in Mice
It has been reported that period variance of SCN and organism
behavior is smaller than that of MEFs and dispersed SCN cells(Liu et al., 2007; Welsh et al., 2004). Our KO-rescue ES mouse
analyses further revealed that period difference of CRY1 mutants
observed in MEFs converged toward 24 hr in ES mice (Figure 5B).
It is difficult to attribute the conversion effect to Cry1-driven feedback loop because we created long and short period mutants, allof which directly targeted the CRY1 molecule. Despite this, theperiods of short mutants measured in MEFs were lengthened
in ES mice, and the periods of long mutants measured in MEFs
were shortened in ES mice. Thus, it is suggested that the molecular/cellular nature of convergence-to-24 hr-effect, not just the
reduction of variance, is not related to Cry1 gene. Interestingly,
Ono et al. demonstrated that circadian oscillation can beobserved in Cry1 / :Cry2 / mice in a limited developmental
condition (Ono et al., 2013), suggesting the presence of Cry1/
2-independent circadian oscillator. Our CRY1 mutagenesis
assay revealed that CRY1-PER2 interaction is particularly important for robust rhythmicity in mice. Phosphorylation of PER2 by
CKId/ε have several intriguing features such as temperaturecompensation (Isojima et al., 2009) and reciprocal regulation of
CKIε (Qin et al., 2015) that have a potential to create phosphorylation-based autonomous oscillators (Jolley et al., 2012). Thesefeatures imply that part of the mammalian circadian rhythmicity
might rely upon Per2-driven oscillators. Therefore, one possible
perspective is that Per2-driven oscillator couples with Cry1driven oscillator to confer a near 24-hr rhythmicity.
Circadian-Period Determination by CRY1 DegradationDependent and -Independent Mechanisms
Our study suggests that the P loop and C-lid are responsible forthe protein-stability-independent period determination. Previous
studies revealed that C-lid forms an interface between CRY1/2
and PER2 (Figure S6D) (Nangle et al., 2014; Schmalen et al.,2014) or FBXL3 (Xing et al., 2013), and the C-lid structure isaffected by the binding of these proteins (Figure S6E). P loop is
not directly involved in the interface between PER2 or FBXL3,
but it may affect the interaction through creating the co-factor
binding pocket. Indeed, residues of CRY1 affecting the interaction
with PER2 identified in this study locate around co-factor binding
pocket (hence surrounding C-lid or P loop) (Figure S6D). However,
our study suggests that the period-determination mechanism canbe independent from CRY1 stability that is regulated throughFBXL3 and PER2 competition. Thus, it is also possible that the
C-lid and P loop may regulate circadian period through the interaction with other proteins because CRY proteins function in a 2-MDacomplex composed of tens of proteins (Brown et al., 2005; Duonget al., 2011; Kim et al., 2015). In summary, this study proposes thatmammalian circadian period is controlled not only by the turnoverrate of CRY1, a behavior determined by the quantity of molecules,
but also by the quality of each molecule characterized by the
structure of flexible loops and phosphorylation status.
EXPERIMENTAL PROCEDURES
Detailed information
Procedures.

wasdescribed

inSupplemental

Experimental

Circadian Reporter Assay using Cry1–/–:Cry2–/– MEFs and NIH 3T3
Cells
Real-time monitoring of circadian reporter using culture cells was performed
as previously described (Ukai-Tadenuma et al., 2011). In brief, cells were transfected with pGL3-P(Per2)-dLuc reporter plasmid and each Cry1 gene expression vector. The cells were synchronized by adding forskolin to the medium.
The bioluminescence was monitored at 30 C.
Generation of ES Mice and Behavior Analysis
An ESC clone was injected into 8-cell-stage ICR embryos to generate
ES mice (Kiyonari et al., 2010). C57BL/6 wild-type mice (9 weeksold), Cry1 / :Cry2 / mice (12 weeks old), ES mice (7-10 weeks old), F1
animals of WT rescue (AS) (10 weeks old) were entrained to a light12 hr:dark-12 hr cycle for at least 2 weeks and then the locomotor activity
was collected in the light-dark condition for 1-2 weeks and then in thedark-dark (DD) condition for another 2-4 weeks. All experimental procedures and housing conditions involving animals and their care wereapproved the Institutional Animal Care and Use Committee of RIKEN
Kobe Branch.
Analysis of CRY1 Half-Life and Transcription-Repression Activity
Vector construct that express Cry1::luciferase under the non-circadian CMV
promoter (pMU2-Cry1::luciferase) was transfected to Cry1 / :Cry2 / MEFs.
Luciferase signal was chased after the addition of 400 mg/mL of cycloheximide
to the medium. Transcription repression assay was performed as described
previously (Khan et al., 2012).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and eight tables and can be found with this article online at
http://dx.doi.org/10.1016/j.molcel.2016.11.022.
AUTHOR CONTRIBUTIONS
H.R.U., K.L.O., H.U., and E.A.S. designed the study. K.L.O performed most of
the cellular/biochemical experiments, and data analysis. H.U., E.A.S., N.K.,
T.A., and H.K. established a KO-rescue ES mouse method. E.A.S and J.H. performed animal experiments. R.N. and K.M. performed phospho-peptide quantification. M.T.K. and K.L.O. established the AID system in circadian assay.
H.R.U., K.L.O., H.U., and E.A.S. wrote the manuscript. All authors discussed
the results and commented on the manuscript.
ACKNOWLEDGMENTS
We thank all the lab members at RIKEN QBiC and the University of Tokyo,
in particular, Dr. Masahiro Hayashi and Dr. Genshiro A. Sunagawa for helping with animal experiments; Chikako Imai, Ayumi Kishimoto, Satoko Morino, and Marie Muramatsu for their effort to maintain ESCs; Junko Garçon,
Kaori Yamanaka, and Shino Hirahara for their effort with the production of
ES mice; Maki Ukai-Tadenuma for helping with the cellular assay; Dr. Rikuhiro G. Yamada for assisting mice behavior analysis; and Yoshiki Nagashima for supporting analyses with a mass spectrometer. We also thankthe LARGE, RIKEN CLST for housing the mice. We thank Dr. Arthur Millius
for critical reading and valuable comments on the manuscript. We are
grateful to Dr. Eisuke Nishida for providing pcDNA3-Myc-mPer2 andDr. Haruhiko Takisawa for sharing unpublished data. This work was
supported by a grant from AMED-CREST (H.R.U.), CREST (H.R.U.),
Brain/MINDS (H.R.U.), Basic Science and Platform Technology Programfor Innovative Biological Medicine (H.R.U.), the Cell Innovation Program(H.R.U), KAKENHI Grants-in-Aid from JSPS (Scientific Research S,
25221004, H.R.U.; Scientific Research on Innovative Areas, 23115006,
H.R.U.; Young Scientists B, 25830146, K.L.O.; Challenging Exploratory
Research, 16K14653, K.L.O.; Young Scientists A, 15H05650, E.A.S.), thestrategic programs for R&D of RIKEN (H.R.U.), an intramural Grant-in-Aid
from the RIKEN QBiC (H.R.U.), grants from Takeda Science Foundation

Molecular Cell 65, 1–15, January 5, 2017 13

Please cite this article in press as: Ode et al., Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and
Quantity of CRY1, Molecular Cell (2016), http://dx.doi.org/10.1016/j.molcel.2016.11.022(H.R.U. and E.A.S.), the RIKEN Special Postdoctoral Research Program(E.A.S.), a Grant-in-Aid by PRESTO from JST (E.A.S.), a Grant-in-Aid
from the Japan Foundation for Applied Enzymology (E.A.S.), and a grantof the Brain Sciences Project of the Center for Novel Science Initiativesof the National Institutes of Natural Sciences (BS281004, E.A.S.).
Received: September 14, 2016
Revised: October 24, 2016
Accepted: November 15, 2016
Published: December 22, 2016
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