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, 176–190
January 5, 2017 ª 2017 Elsevier Inc.
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

176 Molecular Cell 65, 176–190, January 5, 2017 ª 2017 Elsevier Inc.

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)

Molecular Cell 65, 176–190, January 5, 2017 177

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.

178 Molecular Cell 65, 176–190, January 5, 2017

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

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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

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S158D:S285A
WT

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S243A:S285A
WT

0.9
0.6

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0.3

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0.3

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1

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3

4

2

3

4

5

S243A:S261A
WT

0.0
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5

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0.9

0.6

0

S261A

0.9

1

2

3

4

0

5

1

2

3

4

5

D

S243A:S261A

30

S243A:S252AS243A:Y254A

S243A

S261A

T249A
WT
S247A
S252A
Y254A
25

S243A:S247A
S243A:T249A

20

S243A:S285A

15

S158D

Period(hr)

S158D:S285A

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)

Molecular Cell 65, 176–190, January 5, 2017 179

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.

180 Molecular Cell 65, 176–190, January 5, 2017

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

WT
mated pup
(hr)

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ES mouse (clone #1)
mouse ID
6 7

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(bp)

#7 #6
E1

XbaI
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(hr)

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E3

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Cry1 cassette Puro#3
#4

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(hr)

0

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

48

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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

Arrhythmic

W

W

T

21

ry

DD

(Days)(Days)
21

7

Arrhythmic

23

LD

LD

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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

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

DD

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-/-

LD

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Standard(% of WT)

Marker-/-Arrhythmic

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

Molecular Cell 65, 176–190, January 5, 2017 181

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.

182 Molecular Cell 65, 176–190, January 5, 2017

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.

Molecular Cell 65, 176–190, January 5, 2017 183

A

36

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

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 rescueLong

184 Molecular Cell 65, 176–190, January 5, 2017

Hyperrepression

D
W
W
W

D
61

32
Y4

S2

N

39

3A

D

Y

04
S4

D

H
H

S4

92

A

A
43

61
S2

S2

D
85

D

Dominant-short

S2

D
47

49
T2

D
43

S2

S2

D
58

A

Short

S1

85
S2

CRY1 mutant

W
T

0.0

Hyporepression(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.

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

-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

Molecular Cell 65, 176–190, January 5, 2017 185

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

186 Molecular Cell 65, 176–190, January 5, 2017

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-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).

Molecular Cell 65, 176–190, January 5, 2017 187

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 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.

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.

AUTHOR CONTRIBUTIONSEXPERIMENTAL PROCEDURES
Detailed information
Procedures.

wasdescribed

inSupplemental

188 Molecular Cell 65, 176–190, January 5, 2017

Experimental

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.

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

(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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Molecular Cell, Volume 65

Supplemental Information

Knockout-Rescue Embryonic Stem Cell-Derived
Mouse Reveals Circadian-Period Control
by Quality and Quantity of CRY1
Koji L. Ode, Hideki Ukai, Etsuo A. Susaki, Ryohei Narumi, Katsuhiko Matsumoto, Junko
Hara, Naoshi Koide, Takaya Abe, Masato T. Kanemaki, Hiroshi Kiyonari, and Hiroki R.
Ueda

A

B

293T cell + P(CMV)-Cry1(+ P(CMV)-Per2)(+ MG-132 or +TPA)
Cell lysis

W320+ Phosphatase inhibitors

Y413

W374

Protease treatment
Phospho-peptide enrichment by IMAC

H359

W397

H355

Pyrimidine dimer

N393

LC-IT-FT MS analysis

5.0
2.5
0.0

0.0
S243D
WT

0.9

12.5

S243D
WT

10.0
7.5

0.6

5.0

0.3

2.5
0.0
S247D
WT

0.9
0.6

S247D
WT

5.0
4.0

Normalized luc. signal

0.0

3.0
2.0

0.3

1.0
0.0

0.0
T249D
WT

5.0

T249D
WT

0.9

4.0
3.0

0.6

2.0

0.3

1.0

0.0

0.0

0.6

4.0

0.3

2.0

0.0

0.0
0

1

2
3
Time (days)

4

5

S285D
WT

6.0

0

1

2
3
Time (days)

4

Overexpressed in NIH 3T3

N393A
WT

0.9

1.5

N393A
WT

1.0

0.6
0.5

0.3
0.0

0.0
S404D
WT

0.9

1.5

S404D
WT

1.0

0.6
0.5

0.3

0.0

0.0

S261D
WT

0.9

4.0

0.3

2.0

0.0

0.0

Y432D
WT

0.9

4.0

0.3

2.0

0.0

0.0
W374A; W397A
WT

Y432D
WT

6.0

0.6

0.9

S261D
WT

6.0

0.6

5.0

W374A; W397A
WT

4.0
3.0

0.6

2.0
0.3

1.0

0.0

0.0

5
Normalized luc. signal

S285D
WT

0.9

Normalized luc. signal

0.3

Normalized luc. signal

7.5

0.6

Normalized luc. signal

no CRY1
WT

10.0

Normalized luc. signal

12.5

Cry1-rescued Cry1-/-:Cry2-/- MEF

Arrythmic hypo-repression

Normalized luc. signal
Normalized luc. signal
Normalized luc. signal

Arrhythmic dominant-shortOverexpressed in NIH 3T3

Empty vector
WT

0.9

Normalized luc. signal

Normalized luc. signal

Cry1-rescued Cry1-/-:Cry2-/- MEF

Arrhythmic hyper-repression

C

FAD

W374A; W397A
WT

0.9

W320A; W374A; W397A
WT

4.0
3.0

0.6

2.0

0.3
0.0

5.0

1.0
0.0
0

1

Figure S1: Ode et al.

2
3Time (days)

4

5

0

1

2
3
Time (days)

4

5

Figure S1 related to Figure 1. Phosphorylation sites and conserved canonicalelectron-transfer residues in CRY1.
(A) Scheme for the LC-MS based identification of phosphorylation residues in CRY1.
CRY1 was over-expressed under the CMV promoter in 293T cells. Assay condition
and identified peptide list are summarized in Table S1 and Supplemental
Experimental Procedures. TPA: 12-O-tetradecanoylphorbol 13-acetate. IMAC:
immobilized metal affinity chromatography.
(B) A canonical electron-transfer pathway in 6-4 photolyase of Drosophilamelanogaster (PDB 3CVY) (Maul et al., 2008). Green; damaged DNA substrate. Red;
Co-factor FAD. Cyan; residues involved in a canonical-transfer pathway. During thephotoexcitation of flavin chromophore at 6-4 photolyase, tryptophan triplet (W320,
W374, and W397 as corresponding residues in mice CRY1) and histidine/tyrosine
triplet (H355, H359, and Y413 in mice CRY1) are thought to mediate electron transfer
to excite the chromophore and damaged DNA substrate (Aubert et al., 2000; Hitomi
et al., 2001; Maul et al., 2008; Sadeghian et al., 2010). The photoactivatedchromophore may be stabilized through the interaction with asparagine reside (N393
in mice CRY1) (Hitomi et al., 2009; Li et al., 2010; Liu et al., 2011). Note that all
residues are conserved in mouse CRY1, and the amino acid numbers are shown
based on mouse CRY1.
(C) Examples of arrhythmic cellular phenotype in CRY1-rescued Cry1-/-:Cry2-/- MEF(left). The arrhythmic phenotype was further classified based on the phenotype when
the same CRY1-rescue and P(Per2)-dLuc plasmids were co-transfected into NIH 3T3cells (right). For the experiments using NIH 3T3 cells in this and the other figures, theluciferase signal was normalized such that the maximum value of wild-type CRY1’stime course data in each experiment is to be one, and shown as average (solid line) ±SD (shade) (n > 4, taken from at least two independent experiments).

1
0.8

**

0.6
0.4
0.2

7
24

KI
δ

+

SP

+

C

01
60

on

tro
l

25

0

C

KI
δ

0

S252

1.2

+
3
24

C

3.0

2.0
Bits

Mm CRY1 238 R M N A N S L L A S
Mm CRY2 256 R M N A N S L L A S
Hs CRY1 238 R M N A N S L L A S
Hs CRY2 257 R M N A N S L L A S
At 64PHR 265 K G D P S A F L K P
Os 64PHR 257 K G D P S A F L K P
At CRY1 238 R R - - - K A D S A
Dm CRY1 253 N Q A L P N I H D S
Ag CRY1 253 T Q A K P E I L G P
Cq CRY1 206 T Q A R P D F L A P
Os CRY-DASH 338 R N G M L G P D Y S
Xl CRY-DASH 244 R N G L I G L D Y S
Vp CRY-DASH 226 R N G L D G M A Y S
Ec CPD-PHR 227 R D - F P A V E G T
Nc CPD-PHR 356 R N - I P A M Q G T

DNormalized signal intesity ofphosphorylated / non-phosphorylated peptide

C
8
23

B

*

0.2

+

SP

+

25
01
60

on

tro
l

0

KI
δ

**

0.4

C

1

0.6

+

2

0.8

25

3

1

01

4

1.2

60

5

1.4

tro
l

6

SP

7

S247

1.6

on

**

C

Normalized signal intesity ofphosphorylated / non-phosphorylated peptide

S243

8

C

Normalized signal intesity ofphosphorylated / non-phosphorylated peptide

A

1.0

R

G P
N
DN L

N

0.0

ST

DA

G
M
SLLG
L
K
YL
FNA
TM
A
D
P
Q
A

D

P

A
P
E
F
K VNS

A
C
NIG

D
D
K
QG
NI
E
E
S
S
K
M
R

E
H
R

I

S

238

S

I

A
N

D
Q

F
R

K
H
V
G

K
V
F
RT
Y V

243
Residue in Mm CRY1

P

H

G

V

247

Mus musculus
S/T and D/E presence
Rattus norvegicus
around S243 (Mm CRY1) position
Spalax judaei
Homo sapiens
S/T without D/E
CRY1
Macaca fascicularis
S/T with D/E
Gallus gallus
Without S/T without D/EErithacus rubecula
Without S/T with D/E
Sylvia borin
Gallus gallus
Homo sapiens
CRY2
Mus musculus
Rattus norvegicus
Oryza sativa subsp. japonica(6-4) PHR
Arabidopsis thaliana
Drosophila melanogaster
Drosophila pseudoobscura pseudoobscura
Anopheles gambiae
CRY1
Aedes aegypti
Culex quinquefasciatus
Vibrio cholerae serotype O1
CRY2
Vibrio cholerae serotype O1
Vibrio parahaemolyticus serotype O3:K6
Idiomarina loihiensis
Neurospora crassa
Gibberella zeae
Natronomonas pharaonis
Rhodopirellula baltica
Gloeobacter violaceus
CRY-DASH
Synechocystis sp.
Ostreococcus tauri
Danio rerio
Xenopus laevis
Oryza sativa subsp. japonica
Arabidopsis thaliana
Solanum lycopersicum
Streptomyces griseus
CPD PHR
Arabidopsis thaliana
CRY2
Sinapis alba
CRY1
Arabidopsis thaliana
Agrobacterium fabrum
Thermus thermophilus
Thermus thermophilus
Synechocystis sp.
Synechococcus sp.
Halobacterium salinarum
Bacillus pseudofirmus
CPD PHR
Saccharomyces cerevisiae
Neurospora crassa
Vibrio cholerae serotype O1
Buchnera aphidicola subsp. Baizongia pistaciae
Buchnera aphidicola subsp. Acyrthosiphon pisum
Escherichia coli
Salmonella typhimurium(6-4) PHR
Agrobacterium fabrum
Methanothermobacter marburgensis
Methanothermobacter thermautotrophicus
Arabidopsis thaliana
Oryza sativa subsp. japonica
CPD PHR
Carassius auratus
0.50
Potorous tridactylus(Number of substitutions per site)

Figure S2: Ode et al.

Figure S2 related to Figure 2. Evolutional conservation of S243 and neighboring
phosphorylation residues in CRY1.
(A) Quantification of phosphorylated peptides derived from CRY1. CRY1 were
overexpressed in 293T cells in the absence or presence of 100 µM SP600125, or cooverexpression of CKIδ. The ratio of peptide with phosphorylation at indicatedresidues to unphosphorylated peptide was quantified by triple quadrupole mass
spectrometer using isotopically-labelled synthetic peptides as internal controls. The
ratio of phosphorylated S243 to non-phosphorylated one was decreased by theaddition of potent CKIδ/ε inhibitor SP600125 (Isojima et al., 2009) and increased by
the co-expression of CKIδ. SP600125 treatment also decreased the phosphorylationlevel of S247 and T249, residues adjacent to S243 and responsible for arrhythmic
dominant-short phenotype although the effects of CKIδ co-expression were notsignificant. The quantified values are shown as average ± SD (n = 4). ** p < 0.05, * p< 0.1, n.s.; p > 0.1; Student’s t-test compared with “Control”. Quantified sequencesand quantification method are summarized in Supplemental Experimental
Procedures and Table S5.
(B) Sequence alignment of S243 surrounding region of cryptochrome super family.
The alignment was conducted against all protein listed in (C). Arginine/lysine (R/K) at
R238 position and serine/threonine (S/T) at S247 position are well conserved across
many phyla. The area between these two conserved residues (shaded), S/T residue
are colored in blue and aspartic acid/glutamic acid are colored in red. Mm: Mus
musculus. Hs: Homo sapiens. At: Arabidopsis thaliana. Os: Oryza sativa. At:
Arabidopsis thaliana. Dm: Drosophila melanogaster. Ag: Anopheles gambiae. Cq:
Culex quinquefasciatus. Xl: Xenopus laevis. Vp: Vibrio parahaemolyticus. Ec:
Escherichia coli. Nc. Neurospora crassa.
(C) Amino acid conservation of S243 surrounding region displayed in the Sequence
Logo. S243 is located in the middle of evolutionally well-conserved R/K at R238
position of CRY1 and also well-conserved S/T at S247 position of CRY1. Between

these well-conserved residues, negatively-charged aspartic acid and glutamic acid(D/E) often appear.
(D) Phylogenetic tree of the cryptochrome superfamily. For each protein, residuesaligned within the 239 - 246 position in Mm CRY1 (brown shaded in (B)) were analysed.
Blue; at least one S or T residue is involved in the 239 - 246 region, and no D, or E isinvolved in the region. Green; at least one S or T residue and at least one D, or E
residue are involved in the region. Purple; the region does not involve any S, T, D, andE residues. Red; at least one D, or E residue is involved in the 239 - 246 region, andno S or T is involved in the region. The phylogenetic tree shows that mammalian
cryptochromes have S but no D/E within the 239 - 246 position in Mm CRY1. Oh theother hand, most of the other cryptochrome superfamily proteins have D/E but no S/Twithin this area. If the role of phosphorylation for regulating the protein function largely
relies on its negative charge rather than specific surface structure of specific resideposition, then phosphorylation within local area similarly plays a role regardless of theexact position (Nash et al., 2001; Orlicky et al., 2003). Therefore, it the view ofelectrostatic condition, phoshorylation of S243 might mimic the protein surface of othercryptochrome superfamily proteins.

A
XbaI

1 kb

chr6 :ROSA26E1

E2

E3

tctgcaactccagtctttctagaagatgggcgggagtcttctgggcaggcttaa
XbaI
5’ homology armCB
5’ homology arm
8 kbp

PuroR

3’ homology arm
4 kbp

4 kbp

4 kbp

1 kbp

1 kbp

Linearization site

Back-bone vector

Efficiency of correct targeting (%)

3’ homology arm

100
80
60
40
20

0
Targeting Linear
vector
TALEN

E
Efficiency of correct targeting (%)

D-

On feeder

LinearCircular++Feeder free

100
80
60
40
20
0

1 - 1 kbp

4 - 4 kbp

8 - 4 kbp

Length of homology arms

F
Line of ESC

Culture condition

No. of embryos

No. of pups born

No. of 100% chimeras

Ratio of 100% chimeras
to pups born

#1

On feeder

96

17

16

94.1%

#2

On feeder

97

14

11

78.6%

#3

Feeder free

83

10

8

80.0%

#4

Feeder free

96

21

17

81.0%

#5

Feeder free

83

19

16

84.2%

#6

Feeder free

75

30

15

50.0%

Figure S3: Ode et al.

Figure S3 related to Figure 3. TALEN-based high-efficient establishment ofknock-in ES mouse(A) Schematic of the TALEN-targeting site at the ROSA26. The nucleotide sequenceof the target site containing the TALEN recognition sites (labelled in magenta) is shown.
XbaI-digestion site sequence is underlined. Corresponding regions for 3’-terminal of 5’
homology arm and 5’-terminal of 3’ homology arm of targeting vector are indicated as
green lines.
(B) Schematic of three different targeting vector constructs for assays shown in C and
D. Each vector includes 5’ and 3’ homology arm (green lines) of the different length
each. Arm length is given in kilo base pairs. Gene for targeting is shown as a whitebox. The back-bone vector is illustrated as a loop. A linearization site at the flanking
site of 5’ homology arm is indicated by an arrow. Puromycin-resistant gene was
knocked-into the ROSA26 locus so that the efficiency of targeting was quantified as
the number of survived colonies in the presence of puromycin.
(C)

Efficiency

ofcorrect

targetingoflinearized/circulartargeting

vector.

Linearized/circular targeting vector were transfected with/without TALEN. Length of 5’
and 3’ homology arm of targeting vector is 4 kbp. Targeted integration at the ROSA26
were assayed by PCR for over twelve puromycin-resistant colonies (each experimentcontains 2 samples; n = 2). TALEN-based targeting on the ROSA26 locus significantly
increased the efficiency of genome editing up to almost 90% with circular vector asreported in zinc finger nuclease based targeting (Orlando et al., 2010).
(D) Efficiency of correct targeting of three different targeting vectors. Circular targeting
vectors (illustrated in (B)) were transfected with TALEN. Targeted integration at the
ROSA26 assayed by PCR for over 12 puromycin-resistant colonies for eachexperiments. Average ± SEM (error bar) of three independent experiments are shown.
We concluded that the optimal length was from 4 kbp to 8 kbp. Thus, in the KO-rescueof ES mice experiments, we chose 8 kbp length for the 5’ homology arm of ROSA26

locus and 4 kbp for the 3’ homology arm of ROSA26 locus as reported previously (Abeet al., 2011).
(E) Three-days cultured HK3i ESCs (Kiyonari et al., 2010) under with (left) or without(right) feeder cell condition. Scale bar, 200 µm.
(F) Efficiency of contribution for chimeric embryos of ESCs cultured under with orwithout feeder cells condition. HK3i ESCs were injected into 8-cell embryos after tenpassages. The efficiency of contribution was judged with a coat color. That feeder-free
culture condition can gain ESCs that kept comparable efficiency to contribute chimericembryos with ESCs in classical culture condition with feeder cells.

D

1

1.0

0.5

Normalized luc. signal

D

0.0

200

160

120
CRY1 half-life (min)

35
1.0

200

160

120

0.0

0

1

2

3
4
5
Time (days)

0.2
0.4
0.6
Relative amplitude

6

p > 0.1 (not significant); R2 = -0.07

0.0

E

1.5

1.0

+ NAA

0.5

0.0
20

F

**

7

Tir1

Figure S4: Ode et al.

0

*

25
30
Period (hr)

**
**

0.5

35

*
**

p > 0.1 (not significant); R2 = 0.05

0.0

5
10
NAA (µg/mL)

**

0.0

1.25

1.00

0.75

0.00

15

Y254A

**

1.00
**

S261A
Y432D
S97D

**

S588D

**
**
**

Empty vector

**

S261D

300

S261D

Y254D

**

WW

**

S71D

**

WWW

**

WWW

**

S97D

S582D

**

Y432D

0.75

S71D

T131D

**

WW

S582D
N393A

**

S583D

*
**

T30D

**

S404D

S252A

S492D

S158D

T131A

S270D

WT

T180D

S243A

S243D

S280D

S492A

**

Y254D

T180A
T155D

**
**

S261A

*

T180D

*

T30D

0.50

S243D

T131A

*

Y254A

T249D
HHY

S102D

200

S285D

**

T131D

T180A

S492A

S102D

T249D

S247D

S285A

T270D

WT

S280D

S492D

S252D

0.25

Relative P(Per2)-dLuc. expression

Relative P(Per2)-dLuc. expression

p < 0.05; R2 = 0.35
S404A

T155D

*

S158D

S583D

S285A

**

Normalized luc. signal

25
30
Period (hr)
S243A

S252A

S285D

**

S404A

S247D

**

S252D

**

1m
AI

ry

C

**

S588D

HHY

N393A

CRY1 half-life (min)
100
**

ry

*

C

r

20

to

Relative P(Per2)-dLuc. expression

**

S404D

0.00

ve
c

1

ry

C

C

CRY1 half-life (min)

B

pt
y

r

to

ve
c

Normalized luc. / Rluc. signal

0

Em

pt
y

Em

A
**

400

**

p < 0.05; R2 = 0.33

1.0

0.5

0.2
0.4
0.6
Relative amplitude

1.50

CRY1, - TIR1

CRY1, + TIR1

CRY1-mAID, + TIR1

CRY1-mAID, - TIR1

Figure S4 related to Figure 6. Correlation between CRY1 half-life and circadianperiod length.
(A) Protein half-life of indicated mutant/wild-type CRY1 were plotted. A vector plasmid
expressing CRY1::luciferase fusion protein was transfected into Cry1-/-:Cry2-/- MEFs.
The luciferase signal was then chased upon addition of 400 µg/mL cycloheximide.
Data are shown as average ± SD (n = 4). ** p < 0.05, * p < 0.1, n.s.; p > 0.1; Student’s
t-test compared with WT. Dark-blue bars; mutants with significantly reduced protein
half-life. Ligh-blue bars; mutants with protein half-life similar to that of wild type. Green
bars; mutants with significantly increased protein half-life. WW; W374A:W397A,
WWW; W320A:W374A:W397A, HHY; H355A:H359A:Y413A.
(B) E-box repression activity of indicated mutant/wild-type CRY1 are plotted. A vector
plasmid expressing CRY1, P(Per2)-dLuc reporter, and a plasmid expressing Renillaluciferase (RLuc) were co-transfected to Cry1-/-:Cry2-/- MEFs. The signal of luciferase
normalized to the signal of RLuc are plotted. Data are shown as average ± SD (n =4~8). ** p < 0.05, * p < 0.1, n.s.; p > 0.1; Student’s t-test compared with WT. Dark-bluebars; mutants with significantly increased transcription-repression activity. Ligh-bluebars; mutants with transcription-repression activity similar to that of wild type. Green
bars; mutants with significantly increased transcription-repression activity.
(C) Simple regression analysis for protein half-life (A), repression activity (B), circadianoscillation period and amplitude (shown in Figure 1D). For CRY1 mutants that havesignificant change in their half-life, the correlation of CRY1 half-life with circadianperiod or circadian amplitude observed in the assay of KO-rescue MEFs wascalculated. Similarly, for CRY1 mutants with significantly altered transcriptionrepression activity, the correlation of CRY1 repression activity with circadian period or
circadian amplitude was calculated. Note that arrhythmic mutants were excluded fromthe analysis including oscillation period because the exact period lengths wereundetermined, and S588D mutant was excluded from the analysis including protein
half-life because this mutant shows exceptionally long half-life, though including theS588D for this correlation analysis does not change our conclusion (i.e. significant
correlation between period and half-life). Solid line: regression line with two-sided 95%
confidence interval. P-value of F-test and adjusted R-squared value are shown inupper right.
(D) E-box repression activity of AID-tagged CRY1. The experiment was carried out
same as in (B) but using a plasmid expressing CRY1-mAID, and a plasmid expressing
TIR1 for the indicated assay conditions.
(E) Baseline response of P(Per2)-dLuc reporter signal to the addition of NAA. The
reporter plasmid, plasmid expressing CRY1-mAID and TIR1 were transfected into
Cry1-/-:Cry2-/- MEFs as indicated in Figure 6C. To monitor the baseline level ofluciferase reporter, forskolin stimulation was not applied in this experiment (i.e. thecells were circadian-asynchronous). Four days after the medium exchange, NAA wasadded; and then signal at the first trough after NAA addition was quantified (red dottedline) and normalized to the signal at the NAA addition (black dotted line). The plotshows a representative result with the addition of 15 μg/mL NAA.
(F) The normalized baseline signal as analysed in (E) in various conditions. The
baseline signal responded to NAA in a dose-dependent manner in the presence of
TIR1 and AID-tagged CRY1.

25
21

17
KL001 (µM)

0

0.9
WT

0

0.9

0

S243A

S243A
0 µM KL001
0.9 µM KL001

1.0

0.5

0.0
0

0.9

1

2
3
(Days)

4

HHY

0 20 40 60

P-loop

420

4

0.0

5

0

1

2
3
(Days)

4

R238

KL001

R236

S243
S247

R236
21
120
Mm CRY2 (4I6E)

5P-loop

C-lid

S243
S247

220

320

420

R227

T249

R227
S285

S285
75°

21
124
Mm CRY2-FAD (4I6G)

227

339

439
S243

R238

KL001

S247

R236

T249
21

120

220

334

R238

434

S243
S247

R236

R227

T249

R227

S285

S285
120

220

324

424

524

Mm CRY1-Mm PER2 (4CT0)

D

FAD-bound Mm CRY2
(4I6G)KL001-bound Mm CRY2
(4MLP)
F409

F409
202

310

R367

R367

410

F410
F410

E

222
Residue

326

426

F

R227K
R227A
WT

0.5

0

1

2
3
(Days)

4

5

R367K
R367A
WT

1.0

0.5

0

1

2
3
(Days)

4

5

Normalized luc. signal

1.0

Normalized luc. signal

Cry1-rescued Cry1-/-:Cry2-/- MEF
R238A
R236A
WT

1.0

0.5

0.0

0

1

2
3
(Days)

4

5

F410A
F409A
WT

1.0

0.5

0.0
0

1

2
3
(Days)

4

Overexpressed in NIH 3T3

5

3

R227A
WT

2
1
0

0

1

2
3
(Days)

4

5

F409A
WT

1

0
0

1

2
3
(Days)

Figure S5: Ode et al.

4Normalized luc. signal

109

5Normalized luc. signal

3

Normalized luc. signal

100 200
0

50 100 150

B−factor (/Ca)

023(Days)

C-lid

0 100 200320

0 40 80
0 50 100 150

1

R238

3
102
Mm CRY1 (4K0R)
0 20 40 60 80

0

0.5

T249

21

Normalized luc. signal

0.0

50 µM KL001
0.9 µM KL001

1.0

C-lid

Mm CRY2-Mm FBXL3 (4I6J)

Normalized luc. signal

0.5P-loop21
120
220
Mm CRY2-Mm PER2 (4U8H)

0.0

1.0

C

Mm CRY2-KL001 (4MLP)

0.0

H355A; H359A; Y412A
0 µM KL001
0.9 µM KL001

Normalized luc. signal

Normalized luc. signal

29

Arrhythmic

Period (hr)

WT

**

33

B

Cry1-rescued Cry1-/-:Cry2-/- MEF

n.s.

Normalized luc. signal

37

Normalized luc. signal

A

R367A
WT

1

0

0

1

2
3
(Days)

4

5

F410A
WT

1

0
0

1

2
3
(Days)

4

5

Figure S5 related to Figure 7. Effect on circadian period by the modulation ofresidues around co-factor binding pocket.
(A) The HHY mutant CRY1 has reduced sensitivity to KL001. Circadian rhythmicityof Cry1-/-:Cry2-/- MEF rescued by indicated wild-type/mutant CRY1 was monitored in
the presence of indicated concentration of KL001. KL001 significantly affectedcircadian period of wild type and long-period S243A mutant. In contrast, KL001 had
no detectable effect on HHY mutant. Quantified period plot (left) and circadianoscillation (right) were shown as average ± SD (n = 4). ** p < 0.05, n.s. p > 0.1;
Student’s t-test compared with the period in the absence of KL001.
(B) The B-factor (temperature factor) of alpha carbon atoms in published mammalian
CRY1 crystal structures (Czarna et al., 2013; Nangle et al., 2013; Nangle et al., 2014;
Schmalen et al., 2014; Xing et al., 2013). Green bars indicate p-loop (R227 – S247 in
Mm CRY1; R245 – S265 in Mm CRY2) and orange bars indicate C-lid (S404 – Y413
in Mm CRY1; A422 – Y431 in Mm CRY2). The B-factor of these loops are higher thanother domains of CRY protein, or the structure of P-loop domain was partially notdetermined (shaded in blue) in several structures.
(C) Upper half represents Mm CRY2 structure colored based on p-loop (green), C-lid(orange) and phosphorylated residues responsible for the arrhythmic dominant-shortphenotype (cyan). Arginine triplet on p-loop are also indicated. Bottom half represents
the Mm CRY2 surface colored based on electrostatic potential in a linear color ramp
from -6.0 kT (red) to 6.0 kT (blue). The protein surface is based on Mm CRY2-KL001
structure PDB: 4MLP (Nangle et al., 2013). Note that all amino acid residue number
are based on Mm CRY1 in this and following panels.
(D) Structural alteration of C-lid upon co-factor binding. C-lid (orange), R367 residue(cyan) and their up/down stream helices are shown. Compared with the FAD boundform of Mm CRY2 (PDB: 4I6G) (Xing et al., 2013), F409 and F410 residues on c-loopchange their position to be close to R367 residue upon KL001 binding (PDB: 4MLP)(Nangle et al., 2013).

(E) Circadian rhythmicity of wild-type/mutant CRY1 rescued Cry1-/-:Cry2-/- MEF. The
quantified periods are shown in Figure 7D. The experiments were carried out sameas in Figure 1. Note that the data for wild type on each panel are identical.
(F) The arrhythmic mutants were expressed in NIH 3T3 for the classification ofarrhythmic dominant-short, hyper-repression, and hypo-repression phenotypes. The
experiments were carried out same as in Figure 1. R227A was classified asarrhythmic dominant-short, whereas the other three mutants were classified asarrhythmic hyper-repression. The classified results were also shown in Figure 7D.
Note that the data for wild type on each panel are identical.

A

C412
C-lid
C363

C412
C414

N393

C414

C363

N393

KL001

Y413

45°

H359

Y413

H359

KL001

H355
H355
D387
R358

D387

B

Normalized luc. signal

Cry1-rescued Cry1-/-:Cry2-/- MEF

**
32

27

Normalized luc. signal

C414A

C412A

C363A

D387A Dominant-short

WT

17

**

R358K

22

D387N Dominant-short**

R358A Dominant-short

Period (hr)

**

R358K
R358A
WT

1.0

0.0

0.5

0

1

2
3
Time (days)

4

5

C363A
WT

1.0

0.0

0

1

2
3
Time (days)

4

5

C412A
WT

1.0

0.5

0.0

D387N
D387A
WT

1.0

0.5

0.5

0

1

2
3
Time (days)

C

4

0.0

5

C414A
WT

1.0

0.5

0

1

2
3
Time (days)

4

5

0.0

0

1

2
3
Time (days)

4

5

Overexpressed in NIH 3T3

Normalized luc. signal

10

D

R358

Empty vector
WT

5

R358A
WT

4

2

0

0

1

2
3
Time (days)

4

5

0

R387A
WT

10

5

0

FBXL3

1

2
3
Time (days)

4

5

0

5

0

1

2
3
Time (days)

E

FBXL3

R387N
WT

10

4

5

0

0

1

2
3
Time (days)

4

CRY2-FBXL3
F410
F409

P-loopC-lid

P-loop45°

C-lid

W397 Zn

W320

Zn
CRY2-PER2

PER2
Y432

Y432
P-loop

W320
W374

W374
S404

C-lid

W397

PER2

R367

45°

S404

P-loop

S261

Figure S6: Ode et al.

R367 F410

F409
C-lid

5

Figure S6 related to Figure 7. Disulfide bond and hydrogen bond regulating thecircadian period length.
(A) A close-up view of FAD-binding pocket. The salt-bridge between R358 and D387
has been shown to affect the repression of BMAL1/CLOCK activity in Mm CRY1
(Hitomi et al., 2009) and photoreception activity of Drosophila CRY (cryb) (Stanewsky
et al., 1998). The disulfide bridge between C363 and C412, and zinc binding of C414
have been shown to affect Mm CRY1-Mm PER2 interaction (Schmalen et al., 2014).
(B) Circadian rhythmicity of Cry1-/-:Cry2-/- MEF rescued by indicated wild-type/mutant
CRY1. The quantified periods are shown in bar plot on the left. Panels on the rightindicate circadian oscillation of rescued cells. The experiments were carried out sameas in Figure 1. Note that the data for wild type is the same as shown in Figure S5E.
Arrhythmic mutants were classified based on the experiments shown in (C). ** p <0.05; Student’s t-test compared with “WT”. The period lengths are shown as average± SD (n = 4) and summarized in Table S8.
(C) The arrhythmic mutants shown in (B) were expressed in NIH 3T3 for theclassification of arrhythmic dominant-short, hyper-repression, and hypo-repression
phenotypes. The experiments were carried out same as in Figure 1. R358A, D387A,
and D387N were classified as arrhythmic dominant-short. Note that the data for wild
type is the same as shown in Figure S5F.
(D) Crystal structure of Mm CRY2 in complex with FBXL3 (PDB 4I6J) (Xing et al.,2013). and Mm CRY2 in complex with PER2 (PDB 4U8H) (Nangle et al., 2014). In the
CRY2-PER2 complex, residues important for the CRY1-PER2 interaction found in
Figure 5F are shown.
(E) A close-up view of the C-lid and surrounding regions of CRY2 in complex withFBXL3 (PDB 4I6J) or PER2 (PDB 4U8H).

LIST OF SUPPLEMENTAL TABLES
Table S1 related to Figure 1:
Phosphorylated amino acid residues in CRY1
Table S2 related to Figure 1:
Circadian period and amplitude of CRY1 mutants
Table S3 related to Figure 2:
Circadian period of CRY1 double mutants
Table S4 related to Figures 2 and S2:
Prediction of kinases responsible for analyzed phosphorylation sites in CRY1
Table S5 related to Figures 2 and S2:
SRM method for the quantification of phospho-CRY1 peptides
Table S6 related to Figure 3:
Primers for ESC screening and genome integrity check
Table S7 related to Figures 3-5:
Circadian phenotype of Cry1 (mutant)-rescued ES mouse
Table S8 related to Figures 7 and S6:
Period of CRY1 mutants around the P-loop, C-lid, and co-factor binding pocket

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Plasmid construction
Cry1, Per2, and CKIδ used in this study were subcloned in the pMU2 vector (Ukai etal., 2007) as described previously (Narumi et al., 2016) and expressed under the CMV
promoter as a FLAG-tag fusion in their N terminus (pMU2-Cry1, pMU2-Per2, andpMU2-CKIδ). pMU2-Cry1::luciferase was constructed by inserting the luciferase
sequence of pGL3-basic (Promega) into the C-terminus of Cry1. A circadian reporter
plasmid pGL3-P(Per2)-dLuc was constructed as described previously (Sato et al.,2006). The expression vector for codon-optimized OsTir1 (Oryza sativa) wasconstructed based on pAID1.2-N vector (Kubota et al., 2013) by removing the IRES
and AID-tag-MCS sequences from the plasmid: the pAID1.2-N was digested by using
restriction enzymes BamHI and PvuI. The product was treated with T4 DNApolymerase for creating blunt ends and self-ligated to obtain pAID1.2str-P(CMV)OsTir1.
For the vector of Cry1-rescue experiment, pMU2-P(Cry1)-FLAG-(Cry1 intron336)-Cry1 vectors used in the original study (Ukai-Tadenuma et al., 2011) was slightly
modified to enhance the efficiency of splicing at intronic RRE (i.e. Cry1 intron 336). To
this end, the sequence of (Cry1 intron 336) was designed to be flanked by the donor
and acceptor of a chimeric intron derived from pCMV-TNT (Promega), which iscomposed of 5 ́-donor site from the first intron of the human β-globin gene and thebranch and 3 ́-acceptor site from the intron of an immunoglobulin gene heavy chain
variable region (Bothwell et al., 1981). First, a fragment flanked by two AflII sites in thepMU2-MCS (Ukai et al., 2007) vector was removed. Second, the chimeric intron ofpCMV-TNT was amplified with PCR with primers harboring I-SceI or PI-PspI sites(primers:

5’-

ATCGATAAGCTTGATAATTACCCTGTTATCCCTAATAAGTTGGTCGTGAGGCAC-3’
and

5’-

CTGCAGGAATTCGATACCCATAATACCCATAATAGCTGTTTGCCAAGAGCTGTAATTGAACTGGG-3’) and cloned into EcoRV site of pBluescript vector. Then, inverted
PCR fragment of this vector (primers: 5’-TAGGTGCCTATCAGAAACGCA-3’ and 5’TTGGTCTTACTGACATCCACT-3’) was ligated with the Cry1 intron 336 fragment (WTor R1,2 mutation) amplified from pMU2-P(Cry1)-FLAG-(Cry1 intron 336)-Luc/Cry1
vectors(PCR

primers:

5’-TTATGACACAGTGTAGAAACTATG-3’

and

5’-

ACCTTTTACTACTATAAAAACGTACATG-3’). Finally, P(Cry1) and Cry1 coding
sequences (from pMU2-P(Cry1)-FLAG-(Cry1 intron 336)-Cry1), P(SV40) (from pGL3basic, Promega), and the complete Cry1 intron (5’ donor - Cry1 intron 336 - 3’
acceptor) were ligated into KpnI/HindIII sites, PI-PspI/PI-SceI sites, KpnI/HindIII sites
and I-SceI/PI-PspI sites of the modified pMU2-MCS vector, respectively. The final
composition of the Cry1 rescue constructs were as follow: P(Cry1) or P(SV40) - leader
sequence (from pCMV-TNT) - ATG - FLAG - intron (WT or R1,2 mutation) - Cry1 (WT)
- SV40 late poly(A) (from pCMV-TNT). These plasmids were then named as pMU2P(Cry1)-FLAG-I/RRE-Cry1 or pMU2-P(SV40)-FLAG-I/RRE(Mut)-Cry1, respectively.
Site-directed mutagenesis with circular PCR followed by self-ligation of bluntend product was performed using Mighty Cloning Reagent Set (Blunt End) (Takara bio.
inc., Japan). pMU2-Cry1, pMU2-Cry1::luciferase, and pMU2-P(Cry1)-FLAG-I/RRECry1 were used for the template plasmids of mutagenesis. AID-tagged Cry1 vector
was constructed by inserting the 66-132 a.a. sequence of AID tag (from pAID1.2-N)
into the C-terminus of Cry1 of pMU2-P(Cry1)-FLAG-I/RRE-Cry1, and called pMU2P(Cry1)-FLAG-I/RRE-Cry1-mAID.
Targeting vectors of ROSA26 locus were prepared as follows: pENTR-1A(Invitrogen) with Puro-resistance gene cassette (pPGK-PuroR-poly A: PuroR) flanked
by FRT sequences was prepared by the insertion of PuroR at the EcoRV site (forsense direction) or at the BamHI-KpnI site (for antisense direction) of the vector. Then,the Cry1 rescue cassettes (cut with MluI from the pMU2-P(Cry1)-FLAG-I/RRE-Cry1
vector) were cloned into the pENTR-1A-PuroR vector at the BamHI-NotI site (for sense
direction) or NotI-KpnI site (for antisense direction). The resulting expression vectors

(pENTR-1A_attL1-Cry1 cassette-PuroR-attL2 for sense or pENTR-1A_attL1-reversed[Cry1 cassette-PuroR]-attL2 for antisense direction) were then mixed with the
ROSA26 targeting vector, which contains the Reading Frame Cassette B from theGateway Conversion System (Invitrogen) at the blunted KpnI and AscI sites, toperform the LR recombination reaction using the Gateway system (Abe et al., 2011;
Susaki et al., 2014). Plasmids for a series of mutated Cry1 were constructed byswapping the Cry1 coding sequence between pENTR-1A_attL1-reversed [Cry1cassette-PuroR]-attL2 and pMU2-P(Cry1)-FLAG-I/RRE-Cry1 (mutant) using EcoRI
and NotI.
Targeting vectors for comparison of the effect of the arm-length of the targeting
vector to the targeting efficiency, illustrated in Figure S3B, were constructed asfollows: 3’ homology arms (4 kbp and 1 kbp) were amplified by PCR with forward
primers(5’-GGCTCGATCCCTCGAGTCTAGAAGATGGGCGGGAGTCTT-3’)

andreverseprimer(5’or

CGGGAATTCGATGATATCCTCTATAGGTAGGGTTACTAGGTCA-3’

5’-

CGGGAATTCGATGATATCAGTAAGCAGTAATCAATACCATGTGG-3’) and inserted
into the XhoI and EcoRV digested back-bone vector, pTVCI3 (Tainaka et al., 2014),
by using In-Fusion HD Cloning Kit (Clontech Laboratories, Inc. A Takara Bio Company).
5’ homology arms (8 kbp, 4 kbp and 1kbp) were amplified by PCR with forward primers(5’-TAGGGATAACAGGGTAATATAGCTGGGATTACAAGCAGCACACACA-3’,

5’-

TAGGGATAACAGGGTAATATAGATTAAAAGATGGAGGAATTCATGTCAC-3’ or 5’TAGGGATAACAGGGTAATATAGGTACGAGTCGTGACGCTGGAAG-3’)

andreverse(5’-

primerGCGGAGAAAGAGGTAATGAAATGGTCTAGAAAGACTGGAGTTGCAGATC-3’).
Then those products of 1st PCR for 5’ homology arms were amplified with primers (5’GCAGTTACGCTAGGGATAACAGGGTAATATAG-3’

5’-

and

GATCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGG-3’)

andinserted

into the I-SceI and PI-SceI digested pTVCI3-Rarm by using In-Fusion HD Cloning Kit.

The Puromycin resistance gene cassette (P(PGK)-PuroR-polyA) was inserted into ICeuI and PI-PspI digested pTVCI3-LRarm.

Identification of phosphorylation sites in CRY1
Cry1 was expressed in 293T cells as previously described with several modifications(Narumi et al., 2016). At 24 hr before transfection, 293T cells were plated at 10 6 cells
per 90-mm dishes. A total of 12 μg of pMU2-Cry1, or a mixture of 6 μg pMU2-Cry1 andpMU2-Per2 was transfected to the 293T cells on a 90-mm dish. After 68 hr, cells weretreated with 10 µM MG-132 or 20 nM TPA at indicated condition in Table S1. Four hrafter the addition of the reagents, cells were collected by 0.25% trypsin–EDTA (LifeTechnologies) treatment. The cell pellets were lysed in 1 mL PTS buffer (12 mM
sodium deoxycholate, 12 mM sodium N-lauroylsarcosinate, 50 mM NH4HCO3)
containing Phosphatase Inhibitor Cocktail (Nacalai tesque) followed by extensive
sonication. The cell lysates were then flash-frozen in liquid nitrogen and stored at 80°C. For the immunoprecipitation sample, cells were collected 72 hr after thetransfection and cell pellets were lysed in 1 mL M-PER solution (Thermo Fisher
Scientific) containing Phosphatase Inhibitor Cocktail (Nacalai tesque)followed byextensive sonication. The lysate was mixed with 50 µL of anti-FLAG M2 agarose beads(Sigma) by rotating for 4 hr at 4°C. The beads were rinsed with 1 mL M-PER solution
three times and then incubated with 0.1 mL of 0.1 M glycine-HCl (pH 3.5) for 10 min.
After the gentle centrifugation, the supernatant (IP sample) was collected and mixed
with 250 μL of modified PTS buffer.
Cell lysates or IP samples were enzymatically digested according to a phasetransfer surfactant (PTS) protocol (Masuda et al., 2008). The IP sample was reduced(10 mM dithiothreitol at RT for 30 min) and alkylated (50 mM 2-iodoacetamide at RTfor 30 min), and then it was diluted to 5 folds by adding 50 mM NH 4HCO3 solution,
followed by the digestion with 1 μg Lysyl Endopeptidase (LysC; Wako Pure Chemical
Industries, Osaka, Japan). The samples were incubated for overnight at RT. The

sample was further digested with 1 μg trypsin for 6 hr at RT. After the digestion, an
equal volume of ethyl acetate was added to the sample, which was acidified with 0.5%
TFA and well mixed to transfer the detergents into an organic phase. The sample wasthen centrifuged at 10,000 x g for 10 min at room temperature, and an aqueous phase
containing peptides was collected and dried with SpeedVac (Thermo Fisher Scientific).
The dried peptides were solubilized in 100 μL of 2% acetonitrile and 0.1% TFA, andapplied to Fe-IMAC (Immobilized Metal Ion Affinity Chromatography) according to theprevious report (Narumi and Tomonaga, 2016) to enrich the phophopeptides.
The LC-MS analyses were performed by data-dependent MS/MS with a IT-FT
mass spectrometer (Orbitrap velos mass spectrometer, Thermo Fisher Scientific)
equipped with an HPLC system containing a nano HPLC equipment (Advance UHPLC,
Bruker Daltonics) and an HTC-PAL autosampler (CTC Analytics) with a trap column(0.3 x 5 mm, L-column, ODS, Chemicals Evaluation and Research Institute, Japan).
An analytical sample solubilized in 5 μL of 2% acetonitrile and 0.1% TFA was loaded
to the LC-MS system to be separated by a gradient using mobile phases A (0.5%acetic acid) and B (0.5% acetic acid and 80% acetonitrile) at a flow late 200 nL/min(Cell lysate; 5% to 35% B in 190 min, 35% to 95% B in 1 min, 95% B for 1 min, 95%
to 5% B in 1 min and 5% B for 7 min. IP sample; 5% to 40% B in 40 min, 40% to 95%
B in 2 min, 95% to 5% B in 1 min and 5% B for 16 min) with a home-made capillarycolumn (length of 200 mm and inner diameter of 100 μm) packed with 3 μm C18 resin(L-column2, Chemicals Evaluation and Research Institute, Japan). The eluted
peptides were electrosprayed (1.8-2.3 kV) and introduced into the MS equipment(positive ion mode, data-dependent MS/MS). The obtained raw data was subjected todatabase search with MASCOT (Matrix Science) algorithm running on Proteome
Discoverer (Thermo Fisher Scientific).

Circadian reporter assay using Cry1-/-:Cry2-/- MEF and NIH 3T3 cells
Real time circadian reporter assays were performed as described previously (UkaiTadenuma et al., 2011) with following modifications. For the assay with Cry1-/-:Cry2-/MEF, the cells in 35-mm dish were transfected with 4 µg of pGL3-P(Per2)-dLuc
reporter plasmid and 0.03 µg of pMU2-P(Cry1)-FLAG-I/RRE-Cry1 expression vector.
For the AID-tagged CRY1 rescue assay, the plasmid mixture of 3µg pGL3-P(Per2)dLuc, 0.03 µg pMU2-P(Cry1)-FLAG-I/RRE-Cry1-mAID, 1 µg pAID1.2str-P(CMV)OsTir1 was transfected. For the assay with NIH 3T3 cells, 0.4 µg of pGL3-P(Per2)dLuc reporter plasmid, 0.4 µg of pMU2-P(Cry1)-FLAG-I/RRE-Cry1 expression vector,
and 1.2 µg of pMU2-MCS vector were transfected. After 72 hr, cells were stimulated
by exchanging media for DMEM containing 30 µM (Cry1-/-:Cry2-/- MEF) or 10 µM (NIH
3T3) forskolin (nacalai tesque). In the AID-tagged CRY1 rescue assay, indicatedconcertation of NAA (N1641, Sigma-Aldrich) was added to the medium.
Bioluminescence was then measured with photomultiplier detector systems (PMTTron for Cry1-/-:Cry2-/- MEF experiment shown in Figure 1; LM-2400, Hamamatsu
Photonics, for the other experiments) at 30°C.
From the obtained bioluminescence time series, significance of rhythmicity,
period length, detrended signal and relative amplitude were calculated as described
previously (Ukai-Tadenuma et al., 2011). In this study, data resulting in the statisticalsignificance (p = 0, most significant; p = 1, least significant) of circadian oscillation p >0.05, or the data resulting in the relative amplitude below the 30% of averaged wild
type’s relative amplitude (i.e. cutoff = 0.18 for PMT-Tron assay, 0.20 for LM-2400
assay) was classified as “arrhythmic”. Note that period length of the exceptionally-longperiod S243A:S261A mutant was calculated as the distance between the second to
the third peak points. The peak points were defined using the wavelet method
implemented in R software by wmtsa package, and the results were manuallyconfirmed.

Prediction of kinases responsible for analyzed phosphorylation sites in CRY1
Prediction of kinases that catalyze the phosphorylation sites analyzed in our Cry1/-:Cry2-/- MEF rescue assay was conducted by using GPS3.0, an updated version of

GPS2.0

(Xue

et

al.,

2008),(http://gps.biocuckoo.org/),

runningonlocal

NetPhos

windows
3.1

machineserver(http://www.cbs.dtu.dk/services/NetPhos/) (Blom et al., 2004), KinasePhos2.0 server(http://kinasephos2.mbc.nctu.edu.tw/) (Wong et al., 2007), and Scansite 3 server(http://scansite3.mit.edu/), an updated version of Scansite 2 (Obenauer et al., 2003).

LC-MS quantification of the target CRY1 phosphorylation.
A

CRY1

peptide(239-256),

MNANSLLASPTGLSPYLR

and

the

peptidesphosphorylated at S243, S247, T249 or S252 were synthesized with a peptide
synthesizer Syro Wave (Biotage) using Fmoc solid-phase chemistry. Concentrationsof the synthesized peptide were measured by an amino group determination method(Fields, 1972). In order to use the peptide as heavy-labeled internal standard in an MS
analysis, the peptides were dimethyl-labeled with isotope-labeled formaldehyde(13CD2O, ISOTEC) according to the previous report (Boersema et al., 2009). The
heavy labeled peptide, which was solved in 2% acetonitrile and 0.1% TFA at 200
pmol/μL, was stored at -80°C.
For the expression of CRY1, 6 μg of Cry1 vector or a mixture of 6 μg Cry1
vector and 6 μg CKIδ vector was transfected by using 20 uL of X-tremeGENE HP(Roche) to the 5 x 106 293T cells on 90-mm dishes according to the manufacturer’s
instruction. The amount of transfected plasmid was adjusted to 18 μg with empty
vector pMU2-MCS. After 24 hr, culture medium (DMEM/10% FBS) was replaced with
5 mL of culture medium supplemented with or without 100 µM SP600125. After 20 hr,
medium in each dish was replaced with 5 mL of culture medium supplemented with
200 µM MG-132. After 4 hr, cells were collected using cell scraper and were washedwith D-PBS(-).

The cell pellets were lysed in 1 mL of RIPA buffer (50 mM Tris-HCl pH 7.5, 0.3 M
NaCl, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, 0.1% (v/v) NP-40, phosphataseinhibitor (Nacalai tesque, Japan) and protease inhibitor (Nacalai tesque, Japan)) byextensive sonication. The cell lysates were incubated by rotating for 20 min at 4°C,
followed by centrifugation at 7,300 g for 15 min at 4°C. The supernatant wereincubated with 0.1 mL of anti-FLAG M2 agarose beads (Sigma-Aldrich) with rotationfor 1 h at 4°C. The beads collected by a gentle centrifugation (500 g for 2 min) were
washed three times by rotating in 1.2 mL of wash buffer (20 mM Tris-HCl pH 7.5, 0.15
M NaCl, 0.25% (v/v) NP-40). The beads were rinsed with 50 mM NH4HCO3 solution
and then incubated with 0.1 mL of 0.1 mg / mL 3X FLAG peptide (Sigma-Aldrich)solved in PTS buffer for 1 h at 4°C. After the gentle centrifugation, the supernatant (IPsample) was collected and stored at -80°C.
Immunoprecipitated CRY1 protein was enzymatically digested according to aphase-transfer surfactant (PTS) protocol (Masuda et al., 2008). The IP sample was
reduced (10 mM TCEP at 37°C for 15 min) and alkylated (50 mM 2-iodoacetamide at
37°C for 15 min), and then it was diluted 5 fold by adding 50 mM NH 4HCO3 solution
followed by the digestion with 1 μg Lysyl Endopeptidase (LysC; Wako Pure Chemical
Industries, Osaka, Japan) of which the reaction was performed in REDS (AMR Inc.)
400 W at 37°C for 20 min. The sample was further digested with 1 μg trypsin in thesame manner. After the digestion, an equal volume of ethyl acetate was added to thesample, which was acidified with 0.5% TFA, and well mixed to transfer the detergents
into an organic phase. After the centrifugation at 10,000 x g for 10 min at RT, anaqueous phase containing peptides was collected and dried with SpeedVac (Thermo
Fisher Scientific). The dried peptides were solubilized in 100 μL of 2% acetonitrile and0.1% TFA, and the peptide mixture was trapped on a C18 pipette tip prepared aspreviously reported (Rappsilber et al., 2007), The trapped peptides were subjected todimethyl-labeling procedure with formaldehyde (CH2O, Sigma) according to theprevious report (Boersema et al., 2009). After the labeling, 150 μL of 15% acetonitrileand 0.1% TFA was passed through the tip to remove the excess of 3X FLAG peptide
and then the dimethyl-labeled peptides left on the tip were eluted by 150 μL of 80%
acetonitrile and 0.1% TFA. The eluent was mixed with heavy dimethyl-labeled peptidesof the non-phosphorylated MNANSLLASPTGLSPYLR peptide (5 pmol) and the 4phosphorylated versions (100 fmol each) as the internal standard for LC-MS analysis.
A fiftieth of the mixture was subjected to LC-MS analysis. The remaining mixture wasapplied to Fe-IMAC (Immobilized Metal Ion Affinity Chromatography) according to aprevious report (Narumi and Tomonaga, 2016) to enrich for phophopeptides and thensubjected to LC-MS analysis.
The LC-MS analyses were performed by selected reaction monitoring (SRM)with a triple quadruple mass spectrometer (TSQ Vantage EMR mass spectrometer,
Thermo Fisher Scientific) equipped with a captive spray ionization source (Michrom
Bioresources) and an HPLC system containing a nano HPLC equipment (Advance
UHPLC, Bruker Daltonics) and an HTC-PAL autosampler (CTC Analytics) with a trap
column (0.3 x 5 mm, L-column, ODS, Chemicals Evaluation and Research Institute,Japan). An analytical sample solubilized in 5 μL of 2% acetonitrile and 0.1% TFA wasloaded to the LC-MS system to be separated by a gradient using mobile phases A(0.5% acetic acid) and B (0.5% acetic acid and 80% acetonitrile) at a flow late 300
nL/min (5% to 31% B in 3 min, 31% to 35% B in 42 min, 35% to 95% B for 1 min, 95%
B for 2 min, 95% to 5% B in 1 min and 5% B for 11 min) with a home-made capillarycolumn (length of 200 mm and inner diameter of 100 μm) packed with 2 μm C18 resin(L-column2, Chemicals Evaluation and Research Institute, Japan) and then the eluted
peptides were electrosprayed (1.6 kV) and introduced into the MS equipment (positivemode, scan width of 0.002 m/z, Q1 and Q3 resolutions of 0.7 FWHM, a cycle time of
1 sec, a gas pressure of 1.8 mTorr). SRM transitions to monitor the target peptides areshown in a Table S5. From the obtained raw data, SRM chromatograms for target
peptides were extracted using Qual Browser of Xcaliber 2.2 (Thermo Fisher Scientific)
to obtain peak areas corresponding to target peptides. The amounts of the targetpeptides in a sample were determined by dividing the peak areas of the peptides
derived from CRY1 by that of the corresponding internal standards.

Culture of ESCs
A conventional on-feeder culture of ESCs are as described previously (Abe et al.,2011; Kiyonari et al., 2010). The Cry1-/-:Cry2-/- mouse ESCs were established fromblastocyst of Cry1-/-:Cry2-/- mouse (van der Horst et al., 1999) in 3i medium culture
condition as described previously (Kiyonari et al., 2010), and their ES mouseproduction ability were qualified by injection into 8-cell-stage ICR embryos to selectclones for further analysis. Optimized feeder-free culture condition is as below; before
beginning cultivation, surface of BD PURECoatTM amine dishes were exposed tomedium which contain LIF plus 6-bromoindirubin-30-oxime (BIO) (Sato et al., 2009)
for more than 5 hr at 37°C with 5% CO2. ESCs were cultured in 3i medium (Clontech
Laboratories, Inc. A Takara Bio Company) on the BIO/amine-coated dish without
feeder cells at 37°C with 5% CO2.

Generation of targeted ESCs
For the conventional targeting method, the targeting vector was purified andintroduced into the Cry1-/-:Cry2-/- mouse ESCs as described previously (Abe et al.,2011; Kiyonari et al., 2010; Susaki et al., 2014). For the TALEN-mediated targeting, Cterminal-truncated (+63) TALENs (Miller et al., 2011) that bind to target sequence (5'CTGCAACTCCAGTCTTTCTAGAAGATGGGCGGGAGTCTTCTGGGCAGGCTTA-3',
TALEN binding sequences are indicated with italics) at the ROSA26 locus were
designed using TALE-NT (Doyle et al., 2012). The Cry1-/-:Cry2-/- mouse ESCs (4×105
cells) were co-transfected with 0.9 µg of circular targeting vectors and 1.2 µg each of
TALEN-L and TALEN-R expression vector using Xfect Transfection Reagent(Clontech Laboratories, Inc. A Takara Bio Company).

The homologously recombined, puro-resistant ESC clones were isolated forfurther culture and expansion. For the first screening, an aliquot of the cells was lysedand successful homologous recombination was detected by PCR of the 3’ homology
arm region with a pair of primers of #1 to #4 in Figure 3C. After expansion and stock,
the ESC genome was collected and used for further confirmation of genome integrityin the targeted region. For this purpose, genomic PCR was performed by using aseries of primers annealing outside the homologous recombination arms and within
the inserted cassettes (Figure 3C). Successful targeting in the single allele wasconfirmed by the detection of all PCR bands from WT 5’ (primers #5, 6, 9), 3’ (primers#2 and #7), targeted 5’ (primers #1, 5, 8, 9) and 3’ homology arm regions. Primer
sequences used for these screenings were summarized in Table S6.
The copy number of the inserted cassette was confirmed with a quantitative
PCR assay using the extracted ESC genome and primers annealing to the coding
sequencesofthepuro-resistance

CTCGACATCGGCAAGGTGTG-3’

,

gene

(forward

reverseprimer:primer:

5’5’-

GGCCTTCCATCTGTTGCTGC-3’) , normalized to the amount of TATA-box binding
protein gene amplification (forward primer: 5’- CCCCCTCTGCACTGAAATCA-3’,
reverse primer: 5’-GTAGCAGCACAGAGCAAGCAA-3’) using the SYBR Premix Ex
Taq GC (Takara #RR071A) and the ABI PRISM 7900 (Applied Biosystems) orLightCycler 480 II (Roche) (Susaki et al., 2014). The Puro value normalized by Tbp
value should be one if the single copy of inserted cassette was harbored in the genome.
Note that the PuroR cassette flanked by FRT (Figure 3C) was not removed in thisstudy. The selected ESC clones were injected into 8-cell-stage ICR embryos togenerate ES mice (Kiyonari et al., 2010).

Estimation of host-cell contamination and chimerism of ES mouse
For the initial estimation of ES mouse production ability of the Cry1-/-:Cry2-/- ESCs,

semi-quantitative genomic PCR was performed (Figure 3B). The brain was dissectedfrom sacrificed animals, frozen on a dry ice, and the hypothalamic region including the
SCN was collected by cutting the frozen brain. The collected brain region was
homogenized and lysed in 600 ul of lysis buffer with Proteinase K (100 mM of NaCl,
50 mM of Tris-HCl (pH 8.0), 10 mM of EDTA, 1% of SDS, 0.6~0.7 mg/mL of ProteinaseK) at 55°C for 1-2 hr, and the genomic DNA was purified and collected by phenolchloroform purification (treated with RNase in the procedure) followed by ethanol
precipitation. To detect contaminated wild-type cells in Cry1-/-:Cry2-/- ES mice, PCR for
Cry2

locus

genotyping(wild-typeallele-specificforwardprimer:5’-

TACCTGCGCTTTGGATGCCTCTCC-3’, Cry2-/- allele-specific forward primer: 5’TCCCACTTTGTGTTCTAAGTACTGTGGT-3’,

commonreverseprimer:5’-

AAACCACCCATCTCTGGCTTCTCT-3’) were performed in a highly sensitive
condition with 200 ng of the total genomic DNA and 0.7 U of KOD-FX Neo (TOYOBO).
The expected band sizes were 210 bp for wild type amplicon and 310 bp for mutant
amplicon, respectively. Template genome for standard were prepared by mixing
genome of wild type ICR mouse, a strain for host embryos, and a Cry1-/-:Cry2-/- mouse
genome, purified as above, in the indicated ratios (Figure 3A). In this condition, thedetection limit of wild-type cell contamination was estimated as ~0.001%. Coat color
was used for brief estimation of the chimerism in the following ES mouse production.

Measurement of locomotor activity
For monitoring locomotor activity, C57BL/6 wild-type mice (9 weeks old), Cry1-/-:Cry2/- mice (12 weeks old) (van der Horst et al., 1999), ES mice (7-10 weeks old), F1

animals of WT rescue (AS) (10 weeks old) were housed in a light-dark controlling rack
(Nippon Medical & Chemical instruments Co., LTD., Japan) combined with an infrared
monitoring system (NS-AS01, Neuroscience inc.) (Minami et al., 2009). They wereentrained to a light-12 h: dark-12 h cycle for at least two weeks and then the locomotor
activity was collected in the light-dark condition for 1-2 weeks and then in the dark-

dark condition for another 2-4 weeks.
Double-plot visualization of the collected data and calculation of chi-square(Refinetti et al., 2007; Sokolove and Bushell, 1978) were performed with ClockLab
software (ActiMerics) or our own scripts implemented in Mathematica 10 software(Wolfram Research). For calculation of chi-square, the first two weeks in the dark-dark
condition was used expect for the S404D mutant (Figure 5E).
All experimental procedures and housing conditions were approved by theInstitutional Animal Care and Use Committee of RIKEN Kobe Branch, and all of theanimals were cared for and treated humanely in accordance with the institutionalguidelines for experiments using animals. All mouse strains were maintained bycrossing onto a C57BL/6 background.

Co-immunoprecipitation and quantification of CRY1-PER2 complex
At 24 hr before transfection, 293T cells were plated at 1 x 105 cells per 35-mm dishes.
A mixture of 1 μg of pMU2-Cry1 (wild type or mutant series shown in Figure 5F) and1 μg of pcDNA3-Myc-Per2 was then transfected to the 293T cells. After 48 hr, cells
were collected by scraping in D-PBS(-). The cell pellet was lysed in M-PER solution(Thermo Fisher Scientific) followed by extensive sonication. The lysate was mixed with
20 µL of anti-FLAG M2 agarose beads (Sigma-Aldrich) by rotating for 1 hr at 4°C. The
beads were rinsed with 1 mL M-PER and mixed with 100 uL of PTS buffer. The
immuno-precipitants including beads were enzymatically digested as written in above
section “Identification of phosphorylation sites in CRY1”. The dried peptides weresolubilized in 200 μL of 2% acetonitrile and 0.1% TFA. 100 μL of peptide mixture wastaken from each wild-type/mutant CRY1 condition and mixed (“mixture” sample). The
remaining 100 μL of peptide mixture for each wild-type/mutant CRY1 condition was
called “individual” sample. The peptide samples were trapped on a C18 pipette tip
prepared as previously reported (Rappsilber et al., 2007), followed by dimethyl-labeling according to the previous report (Boersema et al., 2009). Formaldehyde(CH2O, Sigma) was used to label the mixture sample (“light-label”), and isotopelabeled formaldehyde (13CD2O, ISOTEC) was used to label the individual samples(“heavy-label”). The dimethyl-labeled peptides on the tip were eluted by 80%
acetonitrile and 0.1% TFA. Then, equal amount of eluted solution of light-labeled
mixture sample was added to each heavy-labeled individual sample (wild-type ormutant CRY1 series). Thus, the relative amount of immuno-precipitants in theindividual samples can be compared to each other by using the equally-added lightlabeled samples as a standard.
The samples were analyzed by data-dependent MS/MS with a Q-FT mass
spectrometer (Q-Exactive mass spectrometer, Thermo Fisher Scientific) equippedwith an HPLC system containing a nano HPLC equipment (Advance UHPLC, Bruker
Daltonics) and an HTC-PAL autosampler (CTC Analytics) with a trap column (0.3 x 5mm, L-column, ODS, Chemicals Evaluation and Research Institute, Japan). An
analytical sample solubilized in 20 μL of 2% acetonitrile and 0.1% TFA was loaded to
the LC-MS system to be separated by a gradient using mobile phases A (0.5% aceticacid) and B (0.5% acetic acid and 80% acetonitrile) at a flow late 300 nL/min (4% to
36% B in 55 min, 36% to 95% B in 1 min, 95% B for 5 min, 95% to 4% B in 1 min and
5% B for 8 min) with a home-made capillary column (length of 200 mm and innerdiameter of 100 μm) packed with 2 μm C18 resin (L-column2, Chemicals Evaluation
and Research Institute, Japan) and then the eluted peptides were electrosprayed (1.82.3 kV) and introduced into the MS equipment (positive ion mode, data-dependent
MS/MS). The obtained raw data was subjected to database search with SequestHTalgorithm running on Proteome Discoverer (Thermo Fisher Scientific).
The area of precursor ions corresponding to the following four peptides
derived from CRY1 were then manually quantified by chromatogram generated with
Xcalibur 2.2 software (Thermo Fisher Scientific): lATEAGVEVIVR (z = 2),
YIYDPWNAPEGIQk (z = 2), AWVANFERPR (z = 2), and KLATEAGVEVIVR (z = 2).

For each peptide, the area of heavy-labeled peptide (H) and light-labeled peptide (L)were quantified, and the averaged H/L ratio is used as the “relative amount of CRY1”
involved in each “individual” sample. Similarly, the area of precursor ionscorresponding to following three peptides derived from PER2 were also manuallyquantified: DLQAVLK (z = 2), FVEFLAPHDVSVFHSYTTPYK (z = 3), and
ILQAGGQPFDYSPIR (z = 2). The H/L ratio of each PER2-derived peptide wascalculated and then divided by the relative amount of CRY1. Thus, the relative valueof (co-immonoprecipitated PER2-derived peptide) / (immunoprecipitated CRY1) could
be obtained for each PER2 derived peptide and plotted in Figure 5F.

CRY1 half-life assay
Cry1-/-:Cry2-/- MEF were plated at 4 x 105 cells per 35-mm dish. After 24 hr, 1 µg of
pMU2-Cry1::luciferase and 1 µg of pMU2-MCS were transfected with FuGene6(Promega) according to the manufacture’s instruction. After 72 hr, the medium was
replaced with DMEM (21063-029, Life technologies) supplemented with 0.1 mM
luciferin (Promega). Bioluminescence was measured in LM-2400 (Hamamatsu
Photonics) at 30°C. After 96 hr, 400 µg/mL cycloheximide (Sigma-Aldrich) was added.
Half-life of luciferase signal was calculated by fitting the time series of 0 – 300 min
after the cycloheximide addition to the equation,
X(𝑡) = 𝑎 ∙ 𝑒 −𝑘∙𝑡 + (1 − 𝑎), where X(t) is the time series of luciferase signal, and a and k are the fitting parameters.
The half-life time is given as
0.5

𝑡ℎ𝑎𝑙𝑓−𝑡𝑖𝑚𝑒 =ln (1 − 𝑎 )𝑘E-box repression assay
Transcription repression assay was performed as described previously (Khan et al.,2012) except for that 1 x 105 Cry1-/-:Cry2-/- MEFs were plated onto one well of a 24well plate, and total plasmid amount was adjusted to 810 ng per well (a mixture of 400
ng pMU2-P(Cry1)-FLAG-I/RRE-Cry1 vector, 400 ng pGL3-P(Per2)-dLuc, and 10 ng
phRL-SV40 plasmid). In the assay shown in Figure S4D (+TIR condition), a mixtureof 200 ng pMU2-P(Cry1)-FLAG-I/RRE-Cry1(-mAID) vector, 200ng of pAID1.2strP(CMV)-OsTir1, 400 ng pGL3-P(Per2)-dLuc, and 10 ng phRL-SV40 plasmid wastransfected.

Sequence alignment and molecular phylogenetic analysis
The amino acid sequences for cryptochrome superfamily were collected from SwissProt database with search keywords “cryptochrome”, “6 4 photolyase”, or “CPD
photolyase”. Proteins that are not involved in the cryptochrome superfamily weremanually excluded. The sequence dataset was aligned by MUSCLE algorithm (Edgar,
2004) running on MEGA7 software (Kumar et al., 2016). A phylogenetic tree forcryptochrome superfamily proteins were calculated by the MEGA7 using Maximum
Likelihood method based on the JTT matrix-based model (Jones et al., 1992). A
sequence logo is generated through WebLogo 3.5.0 website (Crooks et al., 2004).

Protein structural analyses
All images for protein structures were built by using PyMOL software (Schrodinger,
LLC). For the calculation of electrostatic potential, PDB file was converted to PQR fileby the PDB2PQR web service with PARSE forcefiled (Dolinsky et al., 2007; Dolinsky
et al., 2004). pH 7 was chosen for pKa calculation. The PQR file was then used for thegeneration of electrostatic potential map by using APBS software (Baker et al., 2001)
running on PyMOL software.

Statistical analysis and data processing
All statistical analysis and data processing were done in R 3.2.0, Mathematica 10(Wolfram Research), or Excel 2013 (Microsoft).

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