THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 31, pp. 25917–25926, July 27, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Identification of a Novel Cryptochrome Differentiating
Domain Required for Feedback Repression in Circadian Clock
Function*□
S

Received for publication, March 30, 2012, and in revised form, June 4, 2012 Published, JBC Papers in Press, June 12, 2012, DOI 10.1074/jbc.M112.368001

Sanjoy K. Khan‡, Haiyan Xu‡, Maki Ukai-Tadenuma§, Brittany Burton¶, Yongmei Wang¶, Hiroki R. Ueda§储**,and Andrew C. Liu‡1
From the Departments of ‡Biological Sciences and ¶Chemistry, University of Memphis, Memphis, Tennessee 38152-0001 and the§Laboratory for Systems Biology, 储Functional Genomics Unit, RIKEN Center for Developmental Biology, and **Laboratory for
Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

Circadian clocks in mammals are based on a negative feedback loop in which transcriptional repression by the cryptochromes, CRY1 and CRY2, lies at the heart of the mechanism.
Despite similarities in sequence, domain structure, and biochemical activity, they play distinct roles in clock function.
However, detailed biochemical studies have not been straightforward and Cry function has not been examined in real clock
cells using kinetic measurements. In this study, we demonstrate, through cell-based genetic complementation and realtime molecular recording, that Cry1 alone is able to maintaincell-autonomous circadian rhythms, whereas Cry2 cannot.
Using this novel functional assay, we identify a cryptochromedifferentiating ␣-helical domain within the photolyase
homology region (PHR) of CRY1, designated as CRY1PHR(313– 426), that is required for clock function and distinguishes CRY1 from CRY2. Contrary to speculation, the divergent carboxyl-terminal tail domain (CTD) is dispensable, but
serves to modulate rhythm amplitude and period length.
Finally, we identify the biochemical basis of their distinct
function; CRY1 is a much more potent transcriptional repressor than CRY2, and the strength of repression by various
forms of CRY proteins significantly correlates with rhythm
amplitude. Taken together, our results demonstrate that
CRY1-PHR(313– 426), not the divergent CTD, is critical forclock function. These findings provide novel insights into theevolution of the diverse functions of the photolyase/cryptochrome family of flavoproteins and offer new opportunities
for mechanistic studies of CRY function.

* This work was supported by National Science Foundation Grant IOS0920417 (to A. C. L.), an intramural Grant-in-aid from the RIKEN Center for
Developmental Biology (CDB) (to H. R. U.), and a Grant-in-aid for Scientific
Research on Innovative Areas “Spying Minority in Biological Phenomena
number 3306” (23115006) of MEXT, Japan (to H. R. U.).
□S
This article contains supplemental Table S1 and Figs. S1–S5.
1
To whom correspondence should be addressed. Tel.: 901-678-2985; Fax:
901-678-4457; E-mail: acliu@memphis.edu.

JULY 27, 2012 • VOLUME 287 • NUMBER 31

In mammals, many aspects of behavior and physiology, mostnotably the sleep-wake cycle, are regulated by endogenous circadian clocks and are subject to daily oscillations (1, 2). The
mammalian circadian time-keeping system is a hierarchical,
multioscillator network with the central clock in the suprachiasmatic nucleus (SCN)2 synchronizing and coordinating
peripheral oscillators elsewhere in the body (3). Although virtually all cells in the body have circadian clocks (4 – 6), the SCN
clocks are qualitatively more robust because of functional intercellular coupling mechanisms that are present in the SCN, butabsent in most, if not all, peripheral oscillators (7, 8). As a result,
peripheral tissues or cells, when cultured in vitro, display cellautonomous circadian rhythms.
The various clock cells in different tissues share a remarkably
similar biochemical mechanism, the autoregulatory negative
feedback loop, consisting of negative and positive molecularcomponents (1, 9, 10). The positive components include thetwo basic helix-loop-helix/PAS domain-containing transcription factors, BMAL1 and CLOCK, that form a heterodimerictranscriptional complex to activate target gene expression via
E/E⬘-box enhancer elements. Periods (Per1, -2, and -3) andcryptochromes (Cry1 and -2) constitute the negative components of the loop. The PERs and CRYs repress transcription oftarget genes, by directly interacting with and inhibiting
BMAL1-CLOCK complex activity. In particular, the Per and
Cry genes themselves are targets of the BMAL1-CLOCK and inturn repress their own transcription, thereby forming the autoregulatory negative feedback loop (11–13). Genetic studies
established that CRYs are essential clock components (8, 14,15). This observation, together with the finding that CRYs aremuch more potent repressors than PERs for BMAL1-CLOCK
complex activity (16, 17), placed the CRYs at the heart of thecore clock mechanism.

2

The abbreviations used are: SCN, suprachiasmatic nucleus; PHR, photolyase
homology region; CTD, C-terminal tail domain; RRE, ROR/REV-ERB-binding
element; CC2, coiled-coil 2.

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Background: Mammalian CRY1 and CRY2 have distinct functions in circadian clock mechanisms.
Results: A core domain within the photolyase homology region of CRY1 differentiates CRY1 from CRY2 in clock function.
Conclusion: The CRY1/2 differentiating domain is required for strong transcriptional repression and rhythm generation,
whereas the divergent tail domain fine tunes clock function.
Significance: This study provides novel insights into functional evolution of photolyase/cryptochrome flavoproteins.

Differential Functions of Cryptochromes 1 and 2

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level may be higher than CRY2, or CRY1 may be a stronger
repressor than CRY2. In this study, we examined CRY1 andCRY2 function in a genetic complementation assay in which
their transcription is under control of the same promoter andproteins are expressed to similar levels (see below).
A hallmark of circadian clock function is the rhythmic
expression of clock genes, the functional importance of which
has been revealed by recent studies. For example, whereas theBmal1 gene is essential, its rhythmic expression is dispensable
for core clock function (35). In contrast, rhythmic expression of
Cry1 is required for cell-autonomous circadian oscillation (29).
In addition to the E/E⬘-box (responsible for morning-timephase of gene expression, e.g. Rev-erb␣) at the core of the clock
mechanism, at least two other circadian cis-elements are
involved: the DBP/E4BP4 binding element (D-box; daytime
phase, e.g. Per3) and the ROR/REV-ERB-binding element (RRE;nighttime phase, e.g. Bmal1). In a recent study, we showed that
Cry1 transcription is mediated by all three circadian elements(i.e. E/E⬘-box and D-box elements in the promoter and RREs in
the first intron of the Cry1 gene), giving rise to the distinct Cry1
evening time phase. Furthermore, through genetic complementation, we showed that this distinctive delayed phase of
Cry1 expression is required to restore circadian rhythmicity inarrhythmic Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts (29).
In the present study, we took advantage of the Cry1 rescue
assay to dissect the differential functions of Cry1 and Cry2.
First, we confirmed that Cry1 is required for cell-autonomous
circadian rhythms, whereas Cry2 is dispensable. Through systematic analyses of protein domain structure-function relationships, we identified a highly conserved ␣-helical domain
within the PHR that distinguishes CRY1 from CRY2. Contrary
to previous speculation, the least conserved CTD is dispensable
for circadian oscillation, but serves to modulate rhythm amplitude and period length. Finally, we demonstrated that CRY1 is amuch stronger repressor than CRY2, and that repression
strength positively correlates with rhythm amplitude. Thus,
our data demonstrate that CRY1-specific repression is necessary for normal clock function.

MATERIALS AND METHODS
Plasmid Construction—The Cry1 expression vector, pMU2P(Cry1)-intron-Cry1, was made in a previous study (29). To
generate pMU2-P(Cry1)-intron-Cry2, the full-length coding
region of mouse Cry2 was amplified using HiFi-DNA polymerase (Invitrogen) with forward primer (5⬘-TCTAGATGGCAAACAGCTATTATGGGTATTATGGGTGCGGCGGCTGCTGTGGTG-3⬘; underline, XbaI restriction site) and reverse
primer (5⬘-GTCGACTGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTCAGGAGTCCTTGCT-3⬘; underline, SalI). The PCR product was cloned into pCR2.1-TOPO
vector (Invitrogen) and the digested XbaI/SalI fragment wasthen subcloned into pMU2 vector (36) in place of the Cry1 gene.
Domain swap constructs were generated by overlapping
PCR. The primers (supplemental Table S1) were designed sothat swap junctions reside in highly conserved or identicalsequences, so as to minimize major structural changes and protein folding problems. Site-directed mutagenesis using overlapping PCR was performed to generate single mutations within
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The CRYs belong to the photolyase/cryptochrome (PHL/
CRY) superfamily of flavoproteins. All CRYs from different species share a highly conserved core domain at the N terminus,
the photolyase homology region (PHR), whereas the C-terminal tail domain (CTD), on the other hand, has diverged during
evolution (18, 19). Although photolyases lack this tail region,
CRYs from plants to animals contain an extended CTD, but ofvariable length and amino acid composition. Despite similarity
in sequence and domain structures, these flavoproteins play
diverse biological roles. Bacterial photolyases, upon activation
by light, are DNA repair enzymes that revert UV-induced photoproducts to normal bases to maintain genetic integrity (20).
In eukaryotes, however, the CRYs do not exhibit photolyase
activity, and the CRYs in plants and Drosophila are photoreceptors. Although CRYs in plants function to mediate phototropism, growth, and development (21, 22), Drosophila dCry is
directly involved in the light input pathway for circadian clock
entrainment (23, 24). In contrast, the mammalian CRYs areneither photolyases nor photoreceptors; rather, they function
as light-independent transcriptional repressors (16, 17).
Although less well characterized, Drosophila CRY was shown
to exhibit repressor function in certain peripheral tissues (25,26). Functional evolution of this superfamily of flavoproteinsremains one of the most intriguing questions in circadian biology. However, detailed biochemical studies have not beenstraightforward and Cry function has not been examined in real
clock cells using kinetic measurements.
Experimental data suggest that Cry1 and Cry2 have overlapping but differential functions in the clock mechanism.
Although they are both repressors, Cry1 and Cry2 play opposite
roles in regulating animal behavior: Cry1⫺/⫺ and Cry2⫺/⫺ micedisplay shorter and longer free-running period lengths of locomotor activity rhythms, respectively, compared with wild typemice (14, 15). Similarly, SCN explants from Cry1⫺/⫺ mice
exhibit shorter period length than wild type, whereas Cry2⫺/⫺
SCN explants exhibit longer periods (8). Interestingly, Cry1 andCry2 play distinct roles in generating and maintaining cell-autonomous circadian rhythms. For example, dissociated individual SCN neurons derived from Cry1⫺/⫺ mice are arrhythmic or
only transiently rhythmic, whereas neurons from Cry2⫺/⫺ SCN
show persistent rhythms of higher amplitude with longer
period lengths than in wild type (8). Similarly, peripheral tissue
explants and cells from Cry1⫺/⫺ mice are arrhythmic (8, 27).
The more essential role of Cry1 is also supported by behavioral
phenotypes of compound knockouts; Cry1⫹/⫺:Cry2⫺/⫺ miceshow more persistent rhythms than Cry1⫺/⫺:Cry2⫹/⫺ mice,
and whereas Per2⫺/⫺:Cry2⫺/⫺ mice are rhythmic, Per2⫺/⫺:Cry1⫺/⫺ mice are arrhythmic (14, 28). Taken together, thesestudies indicate that Cry1 is required for cellular rhythmicity
and plays a more prominent role than Cry2 in the clock
mechanism.
However, molecular details underlying the functional distinction between the two are not well understood. It is known
that Cry1 is regulated by a combinatorial transcription mechanism and strongly rhythmic in most tissues including the SCN,
whereas Cry2 has only weak rhythms (16, 29 –34). Their differential expression patterns may partially explain the differential
roles in clock function in vivo. Alternatively, the CRY1 proteinDifferential Functions of Cryptochromes 1 and 2

JULY 27, 2012 • VOLUME 287 • NUMBER 31

Bmal1 and Clock plasmid DNA (40) was also included. Empty
vector was used to make up the total amount of DNA to 4.1
␮g/well. As an internal control, 50 ng of a phRL-SV40 plasmid
expressing Renilla luciferase (RLuc) (Promega) was added ineach transfection. Forty-eight hours after transfection, cells
were harvested and assayed with the Dual Luciferase Reporter
Assay System (Promega). Luciferase activity was normalized by
RLuc activity.
For evaluation of correlation between rhythm amplitude andrepression activity, linear fit of a first-order polynomial was
performed by the least square method. Statistical significancewas evaluated by Pearson’s correlation. Analysis was performed
using Microsoft Excel or R version 2.8.1.
Protein Structure Homology Modeling—Homology models
for full-length mCRY1 and mCRY2 were generated using the
I-TASSER protein structure prediction server (41– 43). This
server first threads fragments of the target sequence to representative PBD structure templates with matched sequence
identity greater than 70%. The fragments are then assembled
into a full-length model, whereas the unmatched regions arebuilt via ab initio modeling. Hence, unlike other homology
modeling software, this server predicts the structure even when
there are no matched sequences in known PBD structures. The
quality of predicted structure was assessed with a scoring
method, and five atomistic models with the highest scores wereobtained for each input protein sequence. Images of predictedstructures were created using PyMOL software, version
1.2r3pre (Schrödinger, LLC).

RESULTS
Cry1, But Not Cry2, Can Restore Circadian Clock Function in
Cry1⫺/⫺:Cry2⫺/⫺ Fibroblasts—To confirm the differentialfunctions of Cry1 and Cry2 in clock function, we first tested
their ability to restore circadian rhythms in otherwise arrhythmic Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts through genetic complementation and kinetic bioluminescence recording. In this assay,
expression of Cry is under control of a composite Cry1-phasepromoter containing E/E⬘-box and D-box elements in the promoter and RREs in the first intron of the Cry1 gene (Fig. 1A).
As expected, Cry1 was able to restore rhythms in these cells(Fig. 1B), consistent with previous results (29), and the rescued
cells showed longer period lengths than wild type, characteristic of Cry2⫺/⫺ cells (8). In contrast, however, Cry2 was unable
to restore circadian oscillation to Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts,
confirming results found for cells from Cry1⫺/⫺ mice (8) (Fig.
1B). As the Cry expression level in these fibroblasts was below
the detection limit, the ability of P(Cry1)-Intron-Cry constructs
to express CRY proteins was tested by Western blot in transfected 293T cells (supplemental Fig. S1A). Additionally, tocompare their relative expression in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts, we determined that 3xFlag-Cry1 and 3xFlag-Cry2 (functionally comparable with Cry1 and Cry2, respectively, in the
rescue assay; supplemental Fig. S1B, left panel) are expressed tosimilar levels in these cells (supplemental Fig. S1B, right panel).
Interestingly, rescue of rhythmicity is largely independent of
the dose of Cry1, ranging from nanograms to micrograms of
DNA used in the transfection (Fig. 1C, left panel). On the other
hand, Cry2 of any amount failed to rescue circadian rhythmicity
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the CRY1-PHR(313– 426). Similarly, the PCR products werecloned into pCR2.1-TOPO and subsequently into the pMU2
vector, as described above. For construction of pMU2P(CMV)-Cry2, the full-length coding sequence of Cry2 wasdigested from pMU2-P(SV40)-Cry2 (29) with PI-PspI and PISceI, and the Cry2 fragment was cloned into the PI-PspI-PISceI sites immediately downstream of the CMV promoter.
Each Cry construct (1 ␮g) was co-transfected with either
empty vector (0.4 ␮g) or Bmal1/Clock (0.2 ␮g each) in 293T
cells or in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts in a 12-well plate. Fortyeight hours after transfection, cells were lysed in RIPA buffer
containing protease inhibitors. The lysates were cleared by centrifugation and supernatants were used for Western blot analysis with guinea pig polyclonal antibodies against CRY1 orCRY2 as described previously (16, 30, 31, 37) or against FLAG
tag according to the manufacturer’s protocol (Sigma).
Kinetic Bioluminescence Recording and Data Analysis—
Real-time circadian reporter assays were performed using a
Lumicycle luminometer (Actimetrics, Inc.) as previously
described (8, 29). Briefly, Cry1⫺/⫺:Cry2⫺/⫺ mouse embryonic
fibroblasts were maintained in DMEM supplemented with 10%
fetal bovine serum (FBS) and antibiotics (100 units/ml of penicillin and 100 ␮g/ml of streptomycin). One day prior to transfection, 4 ⫻ 105 cells were plated onto 35-mm culture dishes.
Cells were cotransfected using FuGENE 6 (29) with 3.95 ␮g ofpGL3-P(Per2)-dLuc reporter plasmid (38) and 0.075 ␮g of a Cry
expression plasmid. For the Cry1 dose-response experiment,
the amount of plasmid was adjusted to 5.45 ␮g with empty
vector. Three days post-transfection, the medium was replacedwith HEPES-buffered recording medium supplemented with
B-27 and containing 0.1 mM luciferin and 10 ␮M forskolin aspreviously described (29). Bioluminescence from each dish wascontinuously recorded with a photomultiplier tube for ⬃70 s atintervals of 10 min at 36 °C. Raw data (counts/sec) were plottedagainst time (days) in culture and are presented in the figures.
For analysis of rhythm parameters, we used the LumiCycle
Analysis program (version 2.31, Actimetrics, Inc.). Raw data
were baseline fitted, and the baseline-subtracted data were fitted to a sine wave (damped), from which the period was determined. For samples that showed persistent rhythms, goodnessof-fit of ⬎80% was usually achieved. Due to high transient
luminescence upon medium change, the first cycle was usually
excluded from rhythm analysis.
Amplitude of bioluminescence rhythms was determined as
described previously (29). First, a moving average of the linearly
detrended bioluminescence was calculated. The window size ofthe moving average was set to half of the estimated period. The
moving average was smoothed by the smoothing spline
method, resulting in an amplitude trend, which was thenremoved by dividing by the trend curve of the original timeseries.
Transcription Repression Assay—Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts were grown and transfected as described above with thefollowing modifications. In transfection, 1 ␮g of reporter plasmid, pGL3-P(Per2)-dLuc (38), pGL3–3xE⬘-box-P(SV40)-dLuc,pGL3–3xE-box-P(SV40)-dLuc, or pGL3-P(SV40)-dLuc (39)was used together with 2 ␮g of a Cry expression plasmid. In
some assays as presented in supplemental Fig. S5, 0.5 ␮g each of

Differential Functions of Cryptochromes 1 and 2

in these cells (Fig. 1C, right panel). Thus, our data establish that
Cry1 and -2 play differential roles at the level of core clockfunction: whereas Cry1 is essential for generation of cell-autonomous circadian clock function, Cry2 is dispensable.
Unlike the high-amplitude rhythmic expression of Cry1 invarious tissues and cells, Cry2 expression is either not rhythmicor rhythmic at very low amplitude (16, 30, 31, 37). It is thus
possible that this differential rhythmic expression contributes
to functional differences in vivo. In our in vitro rescue assay, thesame Cry1-phase promoter is used to control both Cry1 andCry2 expression, so this strategy eliminates confounding effects
of differential transcriptional regulation. Thus, our data showing that Cry1 (but not Cry2) restores circadian rhythms in
Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts suggest that CRY1 and -2 possess
different intrinsic biochemical properties at the protein levelthat call for further investigation.
CRY1-PHR(313– 426) Is Critical for CRY1 Function—To
probe the biochemical origin of the differential functions of
CRY1 and -2, we set out to identify the critical structural region
that differentiates the two. Based on known structure anddomain functions of PHL/CRY proteins (18, 19, 44), we divided
CRY1 and -2 proteins into four regions, namely A, B, C, and D(Fig. 2A; supplemental Fig. S2). Using an overlapping PCR strategy, we generated a series of Cry swapping chimeras by systematically substituting different regions of Cry1 with the corresponding sequences from Cry2 (Fig. 2A). To minimize major
structural changes and protein folding problems, we selected
highly conserved or identical sequences at swap junctions (supplemental Fig. S2). The ability of these chimeras and the mutant
Cry constructs to express CRY proteins was tested by Western
blot (supplemental Fig. S1). These chimeras were then tested
for their ability to restore circadian rhythms in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts. Cry1 chimeras that harbor A, C, or D
regions of Cry2 were able to generate cellular rhythms, suggesting that these regions of Cry1 and -2 have comparable clock

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function (Fig. 2B). However, when the B region in Cry1
(Cry1-B) is replaced with the corresponding Cry2-B, theA1B2C1D1 chimera failed to restore rhythms, suggesting that
Cry1-B is required for clock function (Fig. 2B).
To further confirm the role of Cry1-B, we generated a Cry2
chimera, A2B1C2D2, in which the B region of Cry2 is replacedby the corresponding Cry1-B, designated as Cry*. Similar to
Cry1, Cry* was also able to generate rhythms, indicating thatthe B region of Cry1 is sufficient to render Cry2 able to performthe role of Cry1 in clock function (Fig. 2C). In fact, all chimerasthat harbor Cry1-B were able to sustain circadian oscillation,
whereas those containing Cry2-B failed to do so (Fig. 2A). Interestingly, a previous mutagenesis study also hinted that thisregion likely differentiates CRY1 and CRY2 (45). Thus, we haveidentified a critical region within the highly conserved ␣-helicaldomain of CRY1 PHR (from amino acid 313 to 426) that candifferentiate CRY1 from CRY2 and is critically required forCry1 function. We name this region as CRY1-PHR(313– 426).
Identification of Critical Amino Acid Residues within the
CRY1-PHR(313– 426)—Because the CRY1-PHR(313– 426)
underlies functional divergence of CRY1 and CRY2, we performed site-directed mutagenesis to identify the critical aminoacid residues. Among the ⬃100 residues within the CRY1PHR(313– 426), 12 are divergent between CRY1 and -2 (supplemental Fig. S2). Each of the 12 amino acids in Cry* was mutatedto the corresponding residue in Cry2, one or two at a time.
Because these amino acid residues exist naturally in Cry2, major
structural changes are unlikely to occur. We then tested individual mutants for their ability to rescue rhythms in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts. Among 12 mutants, six restored circadianrhythms in these cells, similar to Cry1 and Cry*, whereas theother 6 failed to do so: Cry*-V316I, K322R, I372V, I392V,
S404A, and N425S (Fig. 2D), indicating that these six residues
within the CRY1-PHR(313– 426) are critical for CRY functionin the clock mechanism.
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FIGURE 1. Cry1, but not Cry2, restores circadian rhythmicity in arrhythmic Cry1ⴚ/ⴚ:Cry2ⴚ/ⴚ fibroblasts. A, schematic representation of expression vectors
and general experimental design. In the Cry expression vector, Cry is under control of a composite Cry1-phase promoter that contains all three circadianelements: E⬘-box, D-box from the Cry1 promoter, and RRE from a Cry1 intron. The reporter vector contains the destabilized Luciferase (dLuc) gene driven by the
Per2 promoter. Transfected Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts are either harvested for a transcription repression assay, or synchronized for kinetic bioluminescencerecording. B, representative bioluminescence records from Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts expressing Cry1 or Cry2. Genetic complementation of Cry1 (red), but not
Cry2 (blue), restored circadian rhythms in these cells. Each expression construct was cotransfected with the P(Per2)-dLuc into the cells. Three days posttransfection, the cells were synchronized by forskolin treatment and followed by bioluminescence recording for 5– 6 days. C, Cry1 of different amounts ofplasmid DNA restored circadian rhythms in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts. Experiments were done as in B.

Differential Functions of Cryptochromes 1 and 2

We further performed protein homology modeling to determine the locations of the 6 critical residues in the modeled
CRY1 structure. CRY1 and CRY2 have conserved structures forregions A–C, with a root mean square deviation less than 2.0 Åamong structures predicted by different programs using different templates. Most homology modeling programs failed to
predict a structure for the CTD, except for I-TASSER, whichplaced it in many different orientations, implying intrinsic
flexibility for this region. In a model excluding the CTD, theidentified critical residues are all solvent exposed with the
exception of Ile-392 (Fig. 2E), which is located near the FADbinding cavity. Asn-425 is localized within a loop motif
between helix ␣18 in region B and ␣19 in region C, and ispotentially involved in protein-protein interactions. The
other four residues (i.e. Val-316, Lys-322, Ile-372, and SerJULY 27, 2012 • VOLUME 287 • NUMBER 31

404) are readily available for potential interaction with the
CTD (see below), CC2, or other clock factors. Ser-404 is
localized within a recognition loop between ␣17 and ␣18,
which is recently implicated in interaction with the CTD of
Drosophila CRY (46).
The CTD Is Dispensable for Clock Function, but Modulates
Rhythm Amplitude and Period Length—The CTD represents
the least conserved region among the CRYs. It is generally
accepted that the CTD is critical for CRY function (47). To test
the functional importance of the CTD, we generated a Cry1
CTD-deletion construct, Cry1(⌬CTD) (Fig. 3A). To our surprise, Cry1(⌬CTD) was able to rescue circadian rhythms in
Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts. Thus, contrary to expectation,
our data suggest that the CTD is not absolutely essential for
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FIGURE 2. CRY1-PHR(313– 426) is critical for Cry1 function. A, Cry expression constructs. Cry1 (red) and Cry2 (blue) were divided into four regions: 1) region
A includes the N-terminal ␣/␤ domain and the inter-loop domain of the PHR; 2) region B is the CRY1-PHR(313– 426) and includes the core ␣-helical domain of
the PHR (from ␣13 to ␣18); 3) region C contains the rest of the ␣-helical domain, including sequences immediately after ␣18 and before the CTD where CC2
resides; and 4) region D is the CTD. For sequence details, see supplemental Fig. S2. The chimeras were made by swapping these regions between CRY1 and
CRY2. The schematics are drawn to scale; CRY2 has an extended N terminus, whereas CRY1 has an extended C terminus. Circadian phenotypes are shown on
the right: R, rhythmic; AR, arrhythmic. B and C, representative bioluminescence records from Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts expressing different Cry chimeras.
Domain-swapped chimeras (in B and C) were tested for their ability to rescue rhythms in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts as described in the legend to Fig. 1. All thechimeras that contained the B region from Cry1 (B1, red) were able to restore rhythms (B), implicating the B domain from Cry1 in rhythm generation. The B
region of Cry1 is sufficient to render Cry2 able to generate rhythms. Chimera A2B1C2D2 (Cry*, green) restored rhythms, but A1B2C1D1 (light blue) failed to doso (in C), confirming a required role of CRY1-PHR(313– 426) in Cry1 function. D, representative bioluminescence records from Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts
expressing Cry mutants. Within the CRY1-PHR(313– 426), there exist 12 distinct amino acid residues that diverge between CRY1 and CRY2. Cry* was mutated tothe corresponding residues in CRY2 as indicated. Six of the CRY* mutants failed to rescue rhythms, indicating the critical role of the CRY1-PHR(313– 426) in CRY1
function. E, three-dimensional homology model structure of CRY1 without the CTD. The modeling was based on crystal structures of bacterial photolyase andArabidopsis (6-4) PHR (UVR3). Region A, blue; B, CRY1-PHR(313– 426), green; and C, cyan. The CTD is not shown. With the exception of Ile-392 (purple sphere), theother 5 critical residues identified within the CRY1-PHR(313– 426), namely Val-316, Lys-322, Ile-372, Ser-404 (red spheres), and Asn-425 (pink sphere), are largely
solvent exposed. FAD, black (O, red; N, blue; P, orange).

Differential Functions of Cryptochromes 1 and 2

ous study, which found that CTD is not absolutely required for
repression (45). A coiled-coil 2 (CC2) motif within the C region,
which is immediately downstream of the CRY1-PHR(313– 426)
and upstream of the CTD, was previously implicated in mediating interactions with other clock proteins (47). Here we showthat a larger C-terminal deletion, Cry1(⌬CC2-CTD), whichlacks both CC2 and CTD, eliminated the ability of Cry1 tomaintain rhythmicity (supplemental Fig. S3), indicating animportant role for CC2 in clock function.
Although the CTD of CRY1 is dispensable, rhythms rescued
by Cry1(⌬CTD) showed decreased rhythm amplitude compared with rhythms rescued by full-length Cry1 (see later
results), suggesting that CTD modulates rhythm amplitude.
Also, interestingly, although Cry1-rescued cells displayed along period (⬃27 h), characteristic of Cry2⫺/⫺ cells, whenCry1-CTD is replaced by Cry2-CTD (chimera A1B1C1D2), therescued cells displayed shorter period lengths that are comparable with wild type cells (⬃24 h) (Fig. 3A and supplemental Fig.
S3B). In fact, among all Cry chimeras containing the B region of
CRY1 (and therefore conferring circadian rhythmicity), thosethat contain Cry2-CTD showed a period of ⬃24 h, whereas
those that contain Cry1-CTD showed a longer period (⬃27 h).
Taken together, our data suggest that although the CTD is dis-

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FIGURE 3. CTD is dispensable for CRY function, but modulates period
length. A, schematic diagram of various Cry constructs, including the truncation construct Cry1(⌬CTD) in which the CTD is deleted. Period length corresponding to each construct is shown on the right. Mean ⫾ S.D. (error bar) of
two independent experiments are shown. Raw data are presented in Figs. 2, B
and C, 3B, and supplemental Fig. S3. B, representative bioluminescencerecords from Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts expressing different Cry constructs.
Deletion of CTD did not render Cry1 unable to generate circadian rhythms,
suggesting that the CTD is dispensable for CRY1 function. Cry1(⌬CTD), green.
C, three-dimensional homology model structure of full-length CRY1. The
model was generated using the I-TASSER protein structure prediction server.
Color scheme: region A, blue; B, CRY1-PHR(313– 426), green; C, cyan; and D,
CTD, orange. The CTD assumes a flexible structural configuration, and one ofthe predicted orientations is shown. In this configuration, the CTD resides inclose proximity with the core CRY1-PHR(313– 426), particularly with the 4
critical residues (red spheres).

pensable for Cry1 function, it plays important roles in modulating rhythm amplitude and period length.
Our homology models for full-length CRY1 and CYR2 suggested plausible interactions between the CTD and the identified cryptochrome differentiating domain involving the above
identified critical residues. Consistent with previous observations, the CTD assumes flexible structural configurations (48).
Among possible arrangements of the CTD, those involving
interactions with CRY1-PHR(313– 426) are energeticallyfavored, especially interactions with the side chains of Val-316,
Lys-322, Ile-372, and Ser-404 (Figs. 2E and 3C), each shown tobe critical for CRY function. The observation that these residues reside in critical regions (e.g. Ile-392 and Ser-404) and/or
at an interface (e.g. Val-316, Lys-322, Ile-372, and Ser-404)
available for potential protein-protein interaction explains why
mutating them impairs normal clock function.
CRY1 and CRY2 Display Differential Transcription Repression Activity—In kinetic rhythm assay experiments, we noticed
low expression levels of the P(Per2)-dLuc reporter in rhythmic
cells and high levels in arrhythmic cells, suggesting that rhythm
amplitude may be related to potency of repression of BMAL1CLOCK transcriptional activity. To examine this correlation
more quantitatively, we measured P(Per2)-dLuc expression in
the presence of Cry1 or Cry2 in transiently transfected, nonsynchronized cells. When assayed under nonrhythmic conditions
in which Cry expression is controlled by a strong, constitutive
promoter such as CMV or SV40, Cry1 and -2 both displayedslightly different but strong levels of repression (Fig. 4A andsupplemental Fig. S4), consistent with previous studies (16, 17,
38, 45). To test for differences in repression activity of CRY1
and CRY2, we measured Cry repression under our conditions ofgenetic complementation in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts, inwhich Cry is regulated by the Cry1-phase promoter. Under
these conditions, CRY1 still displayed strong repression on theP(Per2)-dLuc reporter. CRY2, however, did not repress transcription to the same extent as CRY1, showing a repression
activity 10 times weaker than CRY1 (Fig. 4A). This difference in
repression by CRY1 and CRY2 was independent of the reporter
used in the assay, as similar results were obtained with 3xE-boxP(SV40)-dLuc or 3xE⬘-box-P(SV40)-dLuc (supplemental Fig.
S4). Similar differential repression was also observed whenBmal1 and Clock were co-transfected in these cells (supplemental Fig. S4). Therefore, we conclude that CRY1 is a much
more potent transcriptional repressor than CRY2 when
expressed under control of a Cry1-phase promoter.
CRY Transcriptional Repression Positively Correlates with
Rhythm Amplitude—These differential repression data
prompted us to analyze the dependence of rhythm generation
on transcriptional repression. To do this, we determined therepression activity of a subset of Cry chimeras and mutants
used in our rescue studies. Under control of the Cry1-phasepromoter, these Cry constructs showed various strengths ofrepression activity (Fig. 4B). Importantly, we observed that all
the constructs that were able to rescue the rhythms exhibitedstronger repression activities, similar to Cry1, whereas thosethat failed to rescue have much weaker repression, similar to Cry2
(Fig. 4C). For example, Cry1 (A1B1C1D1) and chimera
A2B1C1D1 exhibited low but similar P(Per2)-dLuc expression,Differential Functions of Cryptochromes 1 and 2

indicative of high repression. In contrast, A1B2C1D1 displayed
significantly elevated reporter activity, similar to Cry2. In addition,
mutation at each of the 6 critical residues within the CRY1PHR(313–426) impaired repression (supplemental Fig. S5). These
results are consistent with reporter activities observed in kinetic
recordings (Figs. 1–3). Thus, strong repression activity is highly
correlated with the capacity for rhythm generation.
Finally, we asked if repression activity is also correlated withrhythm amplitude. Using a previously described algorithm (29),
we determined the rhythm amplitude of Cry-rescued circadianoscillations in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts (Fig. 4D). We
observed that rhythm amplitudes were low when the repression
activities were relatively low; and conversely, rhythm amplitudes were high when the repression activities were relatively
high. For example, compared with Cry1, Cry1(⌬CTD) showedattenuated transcriptional repression and accordingly lower
rhythm amplitude. Overall, repression activity and rhythm
amplitude bear a highly significant positive correlation, with
82% of the variance in rhythm amplitude explained by strengthof Cry transcriptional repression (r2 ⫽ 0.82, p ⬍ 0.001) (Fig. 4E).
Thus, our data suggest that the strong repression conferred by
CRY1, but not CRY2, on BMAL1-CLOCK-mediated transcription is the key to generating cell-autonomous circadian rhythms.
JULY 27, 2012 • VOLUME 287 • NUMBER 31

DISCUSSION
Unlike hourglass-type timers, oscillator-type timers such as
the circadian clock regulate cyclic processes that repeat uponcompletion of a cycle. The mechanism underpinning this circadian oscillation in mammals is an autoregulatory transcriptional-translational negative feedback loop (1, 10), in whichtranscriptional repression by the CRYs lies at the heart of thismechanism (16, 38, 39). To gain basic understanding of thisbiochemical mechanism, we sought to investigate the uniquebiochemical and structural aspects of the CRYs. Through a systematic analysis of protein structure-function relationships, we
identified the distinct sequences that distinguish Cry1 function
from Cry2, and demonstrated that Cry1-specific transcriptional (strong) repression is required for mammalian clockfunction. This study provides insights into the unique biochemical and structural properties of CRY1, and presents new opportunities for future dissection of its precise role in the circadianclock mechanism.
Genetic Complementation of Cry1 in Cry-deficient Cells Provides a Functional Clock Model for Mechanistic Studies—In arecent study, we identified the full set of cis-elements responsible for the circadian expression pattern of Cry1, including priJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 4. Transcriptional repression positively correlates with rhythm amplitude. A, dual luciferase reporter assay in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts. For Cry1
expression, three different promoters were tested. Each Cry construct was cotransfected with P(SV40)-dLuc (control) or P(Per2)-dLuc reporter. A Renillaluciferase (RLuc) was added in each transfection to normalize transfection efficiency. Under the control of the Cry1-phase promoter, CRY1 acted as a much morepotent repressor than CRY2. Mean ⫾ S.D. (error bars) of two independent experiments are shown (n ⫽ 3 for each experiment). B, repression activities of various
Cry chimeras and mutants. Dual luciferase reporter assay was done as in A. The constructs that rescued rhythms exhibited stronger repression, similar to Cry1,
whereas those that failed to rescue rhythms exhibited much weaker repression, similar to Cry2. Mean ⫾ S.D. (error bars) of two independent experiments areshown (n ⫽ 3). C, representative bioluminescence records from Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts expressing various Cry chimeras and mutants. The Cry rescue assay
was performed as described in the legend to Fig. 1B. D, relative amplitudes of rescued rhythms in C. Mean ⫾ S.D. (error bar) of two independent experiments
are shown (n ⫽ 3). E, relative rhythm amplitude (x axis) is plotted against relative repression activity (y axis). Rhythm amplitude bears a positive correlation withtranscriptional repression by various CRYs. Mean ⫾ S.D. (error bar) of two independent experiments are shown (n ⫽ 3).

Differential Functions of Cryptochromes 1 and 2

25924 JOURNAL OF BIOLOGICAL CHEMISTRY

remain unknown. Current structural data on the CTD are confined to limited proteolysis and qualitatively interpreted solution NMR spectra (48), confirming predictions that CTD islargely disordered. A recent study described the crystal structure of full-length Drosophila CRY in which the CTD is foundto interact with the FAD binding core domain (i.e. region B in
our study). The CTD of dCRY contains only 20 residues,
whereas CTDs of mCRYs are much longer (80 –100 residues)
and diverge from dCRY, and thus, structurally more flexible.
Our homology models of mCRYs confirmed the potential forinteractions between CTD and the cryptochrome differentiating domain. However, future structural and functional studies
are required to elucidate the mechanism of coordinated function of CTD and the cryptochrome differentiating domain of
CRY proteins.
CRY1-specific Transcriptional Repression Is Required for Circadian Clock Function—The basic concept of a circadian negative feedback loop in mammals was established in the late
1990s (1, 9, 10), and feedback repression is mediated primarily
by CRYs, not PERs (16, 17). Through studies of Bmal1 andClock mutants that interfere with CRY interaction, it waslater demonstrated that CRY-mediated repression of BMAL1CLOCK activity is required for clock function and maintenanceof circadian rhythmicity (38). A hallmark of circadian clockfunction is the rhythmic expression of clock genes. Recently, wedemonstrated that Cry1 expression at the evening time phase(i.e. not morning or day time) and therefore proper phasing in
feedback repression by Cry1 is important for normal circadianclock function (29). Here we further demonstrate that Cry1specific repression is the key to generating circadian rhythms;
Cry1 was able to rescue the rhythms in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts, but Cry2 failed to do so. In addition, Cry1⫺/⫺ cells arelargely arrhythmic, suggesting that endogenous Cry2 alone isunable to support clock function (8, 27). Thus, experimental
data from both gain-of-function (this study) and loss-of-function studies in cellular clock models (8, 51), as well as in circadian behavior of composite knock-out mice (14, 28), establishthat Cry1 plays a more prominent role in clock function thanCry2. Despite the essential role of Cry1 in cell-autonomousmodels, Cry1⫺/⫺ mice, nevertheless, display persistent freerunning rhythms (14, 15, 28). Therefore, there exists a gap inknowledge as to how transient rhythms in individual Cry1⫺/⫺neurons are organized into coherent rhythms in the SCN.
Future Perspective—Importantly, the mechanism by which
CRY1 represses BMAL1-CLOCK complex activity remains elusive. Our findings that CRY1, but not CRY2, plays an essential
role in clock function, and that CRY1 possesses unique biochemical features, especially within the key CRY1-PHR(313–426) domain, suggest that Cry1 holds the key to our understanding of the feedback repression mechanism. A recent study
showed that CRY1 and CRY2 bind to the CLOCK-BMAL1-Ebox complex with the same affinity (52). Thus, it is possible that
their functional difference lies at their different intrinsic repression activities or differential post-translational mechanisms,
and future studies need to focus on the precise biochemicalmechanism by which CRYs repress BMAL1-CLOCK transcriptional activity. The functional assay established in this study
provides new opportunities for future investigations into CRY1
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marily the E/E⬘-box and D-box elements in the promoter and
RREs in the first intron of the Cry1 gene. This allowed us toengineer a synthetic composite promoter that is both necessary
and sufficient for establishing the Cry1-phase. Importantly, wedemonstrated, through genetic complementation in Cry1⫺/⫺:Cry2⫺/⫺ fibroblasts, that Cry1 expression at the evening phaseis required for generation and maintenance of cell-autonomous
circadian rhythms.
This Cry rescue assay provided us with a unique opportunity
to study CRY function in clock cells and confirmed that Cry1
and Cry2 indeed have differential functions in clock regulation.
This assay also enabled us to uncover for the first time thedifferent potency in transcriptional repression exhibited byCry1 and Cry2, which underlie their differential roles in clockfunction. In several prominent structure-function studies inwhich Cry expression was under a strong constitutive promoter(45, 47, 49, 50), CRY protein (likely saturated) was assayed atsteady-state levels, masking differences in repression activity
between CRY1 and CRY2. Consistent with this notion, we show
that, compared with the stronger CMV promoter, SV40-driven
Cry1 and Cry2 exhibited a more noticeable difference in transcriptional repression (Fig. 4A). In our study, Cry expression,under the control of the Cry1-phase promoter, is properly connected to the negative feedback loop involving both the E/E⬘box and D-box elements and the RREs; under this condition,
CRY expression levels would not reach saturation.
Sequence and Domain Structural Features that Distinguish
CRY1 from CRY2—In this study we demonstrated that thefunctional difference between CRY1 and CRY2 lies primarily at
the CRY1-PHR(313– 426) and secondarily at the CTD. Mechanistically, the level of appropriately timed CRY1 repression is
the key to generating robust rhythms. The CRY1-PHR(313–426) is critical for potent transcriptional repression. We
observed a significant positive correlation between CRY repression activity and amplitude of the rhythms (Fig. 4). As therepression activity goes up, so does the amplitude of therhythms. Thus, from the evolutionary point of view, it isthe elaboration of a new function for the conserved core
domain of CRY that rendered it a core clock component.
Although the CTD is not absolutely required for circadianclock function, it participates in modulating basic clock function. Compared with wild type CRY1, the CRY1 chimera harboring the CTD of CRY2 (A1B1C1D2) shortened the period
length (Fig. 3), indicating its role in period length regulation.
Compared with CRY1, CRY* (A2B1C2D2) displayed slightly
reduced, but by and large similar repression activity. Interestingly, however, compared with the full-length CRY1,
CRY1(⌬CTD) displayed less transcriptional repression andgenerated lower amplitude rhythms, whereas CRY*(⌬CTD)
exhibited dramatically reduced repression activity and failed togenerate rhythms, similar to CRY2. Thus, our data suggest that
CTD1 and CTD2 (from CRY1 and CRY2, respectively) playdifferential roles in fine-tuning the clock function, and that
there might be a mechanism for signal transduction from theidentified cryptochrome differentiating domain to CTD toaccomplish the fine-tuning.
However, the mechanism of repression by CRY and potential
signal transduction from the CRY1-PHR(313– 426) to the CTD

Differential Functions of Cryptochromes 1 and 2
structure-function relationships. Our findings shed new lighton the functional importance of the CRY1-PHR(313– 426) andthe CTD in the clock mechanism. Several previous studies
identified a subset of common motifs and sites, including
nuclear localization sequences, coiled-coils, phosphorylationsites of CK1⑀, GSK3␤, MAP kinase, and AMP-activated protein
kinase (44, 45, 47, 49, 50, 53–56), and surely additional motifs
remain yet to be identified. The functional significance of thesevarious sequences and structural features in CRY function willneed to be tested using the assays developed in this study. Thesefuture studies will ultimately provide important insights into
the biology of CRYs and their role in the negative feedback
mechanism, as well as the functional evolution of the PHL/CRY
family of flavoproteins.

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C. B., and Evans, R. M. (2009) AMP-activated protein kinase regulates thecircadian clock by cryptochrome phosphorylation and degradation. Science 326, 437– 440

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Kondo, T., Yagita, K., Shigeyoshi, Y., and Ueda, H. R. (2007) Melanopsindependent photo-perturbation reveals desynchronization underlying the
singularity of mammalian circadian clocks. Nat. Cell. Biol. 9, 1327–1334
37. Hughes, M. E., DiTacchio, L., Hayes, K. R., Vollmers, C., Pulivarthy, S.,
Baggs, J. E., Panda, S., and Hogenesch, J. B. (2009) Harmonics of circadiangene transcription in mammals. PLoS Genet. 5, e1000442
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T. J., Welsh, D. K., Kay, S. A., Ueda, H. R., and Hogenesch, J. B. (2006)
Feedback repression is required for mammalian circadian clock function.
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39. Ueda, H. R., Hayashi, S., Chen, W., Sano, M., Machida, M., Shigeyoshi, Y.,
Iino, M., and Hashimoto, S. (2005) System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nat. Genet.
37, 187–192
40. Kumaki, Y., Ukai-Tadenuma, M., Uno, K. D., Nishio, J., Masumoto, K. H.,
Nagano, M., Komori, T., Shigeyoshi, Y., Hogenesch, J. B., and Ueda, H. R.
(2008) Analysis and synthesis of high-amplitude cis-elements in the mammalian circadian clock. Proc. Natl. Acad. Sci. U.S.A. 105, 14946 –14951
41. Roy, A., Kucukural, A., and Zhang, Y. (2010) I-Tasser. a unified platform
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43. Zhang, Y. (2008) I-TASSER server for protein three-dimension structure
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Supplemental Data
Sanjoy K. Khan1, Haiyan Xu1, Maki Ukai-Tadenuma2, Brittany Burton5, Yongmei Wang5,
Hiroki R. Ueda2,3,4, and Andrew C. Liu1*

Identification of a Novel Cryptochrome Differentiating Domain Required for Feedback Repression in
Circadian Clock Function

Table of contents♦Supplemental Table.
Table S1. Primer list for generation of domain swapped chimeric constructs.
♦Supplemental Figures.
Figure S1. Western blot analysis of CRY proteins.
Figure S2. Sequence alignment and key structural features of mCRY1 and mCRY2.
Figure S3. Roles of coiled-coil 2 (CC2)-containing C region and the CTD of CRY in clock function.
Figure S4. CRY1 and CRY2 display differential repression activity.
Figure S5. Mutation of each of the six critical residues within the CRY1-PHR(313-426) of CRY*
abolished transcriptional repression activity.

S-1

Table S1. Primer list for generation of domain swapped chimeric constructs.
Name of theconstruct
pMU2-P(Cry1)Cry1 intronA1B2C2D2

Roundof PCRFirst

Primer sequence
Reaction 1
Reaction 2

SecondpMU2-P(Cry1)Cry1 intronA1B1C2D2First

Reaction 1
Reaction 2

SecondpMU2-P(Cry1)Cry1 intronA1B1C1D2

FirstReaction 1
Reaction 2

SecondpMU2-P(Cry1)Cry1 intronA2B1C1D1FirstReaction 1
Reaction 2

SecondpMU2-P(Cry1)Cry1 intronA2B2C1D1

First

Reaction 1
Reaction 2

SecondpMU2-P(Cry1)Cry1 intronA2B1C2D2First

Reaction 1
Reaction 2
Reaction 3

SecondpMU2-P(Cry1)Cry1 intronA1B2C1D1

First

Reaction 1
Reaction 2
Reaction 3

Secondf-primer: ggtagagctgtcaagattac
r-primer: ctggatgcagatggggttcccttccattttgtcaaa
f-primer: tttgacaaaatggaagggaaccccatctgcatccag
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-primer: gcctttcagtttgggcagataacgcctaatatagtc
f-primer: gactatattaggcgttatctgcccaaactgaaaggc
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f- primer: ggtagagctgtcaagattac
r- Primer: caatagacagagtccccggtaccgggaaagctgctgatagat
f-primer: atctatcagcagctttcccggtaccggggactctgtctattg
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: ctgaacacagatggggttcccctccattcggtcaaa
f-primer: tttgaccgaatggaggggaaccccatctgtgttcag
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gcctcttaggacaggtaagtaccgccggatgtagtc
f-primer: gactacatccggcggtacttacctgtcctaagaggc
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: ctgaacacagatggggttcccctccattcggtcaaa
f-primer: tttgaccgaatggaggggaaccccatctgtgttcag
r-Primer: gcctttcagtttgggcagataacgcctaatatagtc
f-primer: gactatattaggcgttatctgcccaaactgaaaggc
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: ctggatgcagatggggttcccttccattttgtcaaa
f-primer: tttgacaaaatggaagggaaccccatctgcatccag
r-Primer: gcctcttaggacaggtaagtaccgccggatgtagtc
f-primer: gactacatccggcggtacttacctgtcctaagaggc
r-Primer: gtatcttatcatgtctgctcg
f-primer: ggtagagctgtcaagattac
r-Primer: gtatcttatcatgtctgctcg

S-2

Figure S1

anti CRY1

anti CRY2

NS

anti β-actin

Vector control

P(Cry1)-Intron-Cry*(I372V)

P(Cry1)-Intron-Cry*(V316I)

P(Cry1)-Intron-Cry*(N393L)

P(Cry1)-Intron-A1B1C2D2P(Cry1)-Intron-Cry*

P(Cry1)-Intron-A1B2C2D2P(Cry1)-Intron-Cry2

P(Cry1)-Intron-Cry2

Antibody

P(CMV)-3xFlag-Cry2

Vector control

P(Cry1)-Intron-Cry*(N425S)

Bmal1/Clock
P(Cry1)-Intron-Cry*(S404A)

P(Cry1)-Intron-Cry*(I392V)

P(Cry1)-Intron-Cry1(D387N)

P(Cry1)-Intron-A1B2C1D1P(Cry1)-Intron-A2B2C1D1P(Cry1)-Intron-A2B1C1D1P(Cry1)-Intron-Cry1

P(Cry1)-Intron-Cry1

P(CMV)-3xFlag-Cry1

NS

3xFlag-Cry1
3xFlag-Cry2

5000
4000

Antibody

3000
2000

anti Flag

1000
0

P(Cry1)-Intron-3xFlag-Cry2
with Bmal1/Clock

Bioluminescence

6000

P(Cry1)-Intron-3xFlag-Cry27000

Vector control

B

P(Cry1)-Intron-3xFlag-Cry1

anti β-actin

P(Cry1)-Intron-3xFlag-Cry1
with Bmal1/Clock

Antibody

Bmal1/Clock

P(Cry1)-Intron-A1B1C1D2

A

1

2

3

Days

4

5

anti β-actin

Figure S1. Western blot analysis of CRY proteins.
(A) HEK-293T cells were transfected with different Cry constructs in the absence or presence of Bmal1/Clock as indicated. Total cell extracts wereprobed with either anti-CRY1 (left panel) or anti-CRY2 (right panel) antibody. Compared to CMV promoter, P(Cry1)-Intron drives low levels of protein
expression, which can be further induced by cotransfected Bmal1/Clock to a higher level. Detection of CRY proteins indicates that all Cry constructs
are able to express the proteins. Because the regulation of Cry expression in functional clock cells (e.g., those that are rescued by P(Cry1)-Intron-Cry1)
is different from that in arrhythmic cells (e.g., those expressing Cry2), to directly compare Cry expression, transfected 293T cells provide a better means
to test these constructs for their ability to express proteins. 3xFlag-CRY1 and CRY2, positive controls. Arrow, non-specific (NS) band.
(B) Cry1 and Cry2 are expressed to similar levels in Cry1–/–:Cry2–/– fibroblasts in the rescue assay. Cry1–/–:Cry2–/– fibroblasts were transfected with
3xFlag-Cry1 or 3xFlag-Cry2 constructs in the absence or presence of Bmal1/Clock as indicated. 3xFlag-Cry1 and 3xFlag-Cry2 are functionallycomparable to Cry1 and Cry2, respectively, in the rescue assay (left panel). Western blot analysis with anti-Flag antibody shows that CRY1 and CRY2
are expressed to similar levels in these cells (right panel).

Khan et al. Western blot analysis of CRY proteinsS-3

Figure S2

PHR

α/β
domainCry1 1
Cry2 1

loop
A1
A2

CTD

helicaldomainloop

PHR(313-426)

CC2

B1
B2

C1
C2

D1
D2

606
592

A (α/β domain, interdomain loop)
mCRY1
mCRY2
Consensus(1)(1) - - - - - - - - - - - - - - - - - - M G V N A V H W F R K G L R L H D N P A L K E C I Q G A D T I R C V Y I L D P W F A G S S N V G I N R W R F L L Q C L E D L D A N L R K L N S R L F V I R G Q P A D V(1) M A A A A V V A A T V P A Q S M G A D G A S S V H W F R K G L R L H D N P A L L A A V R G A R C V R C V Y I L D P W F A A S S S V G I N R W R F L L Q S L E D L D T S L R K L N S R L F V V R G Q P A D V
G
AVHWFRKGLRLHDNPALI GA
IRCVYILDPWFAASS VGINRWRFLLQ LEDLDLRKLNSRLFVIRGQPADV
(1)

(102)
mCRY1 (84) F P R L F K E W N I T K L S I E Y D S E P F G K E R D A A I K K L A T E A G V E V I V R I S H T L Y D L D K I I E L N G G Q P P L T Y K R F Q T L V S K M E P L E M P A D T I T S D V I G K C M T P L S D
mCRY2 (102) F P R L F K E W G V T R L T F E Y D S E P F G K E R D A A I M K M A K E A G V E V V T E N S H T L Y D L D R I I E L N G Q K P P L T Y K R F Q A L I S R M E L P K K P A V A V S S Q Q M E S C R A E I Q E
SHTLYDLDKIIELNG
PPLTYKRFQ LISKME
PA
ISS
I
C
I D
Consensus (102) F P R L F K E W I T K L S E Y D S E P F G K E R D A A I K L A E A G V E V I(203)
mCRY1 (185) D H D E K Y G V P S L E E L G F D T D G L S S A V W P G G E T E A L T R L E R H 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 C L S C R L F Y F K L T D L Y K K V K K N S S P P L S
mCRY2 (203) N H D D T Y G V P S L E E L G F P T E G L G P A V W Q G G E T E A L A R L D K H 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 C L S C R L F Y Y R L W D L Y K K V K R N S T P P L S
Consensus (203) H D D Y G V P S L E E L G F T D G L A V W G G E T E A L R L D K H 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 C L S C R L F Y F K L D L Y K K V K K N S S P P L S
p-binding loop
protrusion motif

B (PHR(313-426))

V316

K322

I372
α15 R358(304)
mCRY1 (286) L Y G Q L L W R E F F Y T A A T N N P R F D K M E G N P I C V Q I P W D K N P E A L A K W A E G R T G F P W I D A I M T Q L R Q E G W I H H L A R H A V A C F L T R G D L W I S W E E G M K V F E E L L L
mCRY2 (304) L F G Q L L W R E F F Y T A A T N N P R F D R M E G N P I C I Q I P W D R N P E A L A K W A E G K T G F P W I D A I M T Q L R Q E G W I H H L A R H A V A C F L T R G D L W V S W E S G V R V F D E L L L
Consensus (304) L F G Q L L W R E F F Y T A A T N N P R F D K M E G N P I C I Q I P W D K N P E A L A K W A E G K T G F P W I D A I M T Q L R Q E G W I H H L A R H A V A C F L T R G D L W I S W E G M K V F D E L L L
sulfur loop

C

D387 I392 N393

S404

α18

N425

(405)
mCRY1 (387) D A D W S I N A G S W M W L S C S S F F Q Q F F H C Y C P V G F G R R T D P N G D Y I R R Y L P V L R G F P A K Y I Y D P W N A P E G I Q K V A K C L I G V N Y P K P M V N H A E A S R L N I E R M K Q I
mCRY2 (405) D A D F S V N A G S W M W L S C S A F F Q Q F F H C Y C P V G F G R R T D P S G D Y I R R Y L P K L K G F P S R Y I Y E P W N A P E S V Q K A A K C I I G V D Y P R P I V N H A E T S R L N I E R M K Q I
Consensus (405) D A D F S I N A G S W M W L S C S A F F Q Q F F H C Y C P V G F G R R T D P G D Y I R R Y L P L K G F P A K Y I Y D P W N A P E I Q K A K C I I G V Y P K P I V N H A E S R L N I E R M K Q I
recognition loop
CC2

D (CTD)(506)
mCRY1 (488) Y Q Q L S R Y R G L G L L A S V P S N S N G N G G L M G Y A P G E N V P S C S S S G N G G L M G Y A P G E N V P S C S G G N C S Q G S G I L H Y A H G D S Q Q T H S L K Q G R S S A G T G L S S G K R P S
mCRY2 (506) Y Q Q L S R Y R G L C L L A S V P S C V E D L S H P V A E P G S S Q A G S I S N T G P R A L - - - - - - - - - - - - - - - - - - - - - - - - - - S S G P A S P K R K L E A A E E P P G E E L T K R A R V T
MANS S SG
AL
A G A
L
A
G
LS
R S
Consensus (506) Y Q Q L S R Y R G L L L A S V P S(607)
mCRY1 (589) Q E E D A Q S V G P K V Q R Q S S N
mCRY2 (581) E M P T Q E P A S K D S - - - - - Consensus (607)

Figure S2. Sequence alignment and key structural features of mCRY1 and mCRY2.
mCRY1 and mCRY2 share ~70/80% sequence identity/similarity. Amino acid sequence alignment was generated using Vector NTI (Invitrogen).
Above the sequence alignment, a schematic diagram of domain structure of CRY proteins, as in Figure 2A. Amino acid sequence alignment: blue,
identical; green, similar; red, divergent. Domain assignment: region A (blue), B (green), C (cyan), and D (yellow). Secondary structures: arrow,alpha helix (numbers are assigned based on Arabidopsis UVR3); barrel, beta strand; CC2: coiled coil 2. Key amino acid residues: black star, FAD-binding residues; gray star, Trp triad; red star, arrhythmic when mutated in Cry*; green star, rhythmic when mutated.

Khan et al. Amino acid sequence alignment of mouse CRY proteinsS-4

Figure S3
A

B
A1
A2
A1

Cry1
Cry2
Cry1(ΔCC2-CTD)

B1
B2
B1

C1
C2
C1

D1
D2
D1

R
AR
AR

A2
A1
A1
A2

A2B1C1D1
A1B1C1D2
A1B1C2D2
Cry*

6000

80000

60000

40000

Cry1
Cry2
Cry1(ΔCC2-CTD)

20000

C1
C1
C2
C2

D1
D2
D2
D2

Cry1
A2B1C1D1
A1B1C1D2
A1B1C2D2
Cry*

5000

Bioluminescence

Bioluminescence

B1
B1
B1
B1

4000
3000
2000
1000

0
1

2

3

4

Days

0
1

2

3

Days

4

5

Figure S3. Roles of coiled-coil 2 (CC2)-containing C region and the CTD of CRY in clock function.
(A) In the truncation construct, Cry1(∆CC-CTD) (top panel), both the CTD and the C region containing CC2 are deleted from the full-length Cry1.
This Cry1 truncation mutant failed to restore cellular rhythms in Cry1–/–:Cry2–/– fibroblasts (bottom panel; representative bioluminescence records).
(B) Representative bioluminescence records from Cry1–/–:Cry2–/– fibroblasts expressing various Cry chimeras. The Cry rescue assay was performed
as in Figure 1B. While the chimeras that contain the CTD from CRY1 (Cry1 and A2B1C1D1) restored rhythms with a period length of ~27 hr (Figure 3A),
those that contain the CTD from CRY2 (A1B1C1D2, A1B1C2D2 and Cry*) restored rhythms with a period length of ~24 hr (Figure 3A), implicating the
CTD in regulating period length.

Khan et al. The roles of CC2 and CTD of CRY in clock function

S-5

0.8

0.8

0.8

2.5

1.0

2.0

0.8

1.5
1.0

M
V)
P(
S
P(
V4
C
0)
ry
1)
-In
tro
n

P(
C

1)

-In

tro

n

P(
C
ry

M
V)
V4
0)
P(
S

P(
C

1)

-In

tro

n

ry
P(
C

M
V)
V4
0)
P(
S

P(
C

Cry1
Cry2
Cry1
Cry2
Cry1
Cry2

0.0

VectorVector

0.0

Cry1
Cry2
Cry1
Cry2
Cry1
Cry2

0.0

VectorVector

0.0

Cry1
Cry2
Cry1
Cry2
Cry1
Cry2

0.2

VectorVector

0.5

Bmal1/Clock

Cry1

Cry2

0.4

0.2

Bmal1/Clock

Cry1

0.6

0.2

Bmal1/Clock

Cry2

Vector

P(
C

M
V)
P(
SV
P(
40
C
)
ry
1)
-In
tro
n

Cry1

Cry2

Cry1

Cry2

1.2

n

0.4

3.0

P(SV40)-dLuc
w/ Bmal1/Clock

tro

0.4

0.6

1.4

-In

0.6

0.8

Luc/Rluc

Luc/RLuc

0.8

3xE-P(SV40)-dLuc
w/ Bmal1/Clock

1)

1.0

1.0

Cry1

P(
C
3.5

ry

1.2

3xE’-P(SV40)-dLuc
w/ Bmal1/Clock

P(
C

1.2

Luc/RLuc

P(Per2)-dLuc
w/ Bmal1/Clock

Cry2

Vector

M
V)
P(
SV
P(
40
C
)
ry
1)
-In
tro
n

Cry1

Cry2

Cry1

Vector

M
V)
P(
SV
P(
40
C
)
ry
1)
-In
tro
n

P(
C

P(
C

M
V)
P(
SV
P(
40
C
)
ry
1)
-In
tro
n

Cry2

0.0

Cry1

0.0

Cry2

0.0

Cry1

0.0

Cry2

0.2

Cry1

0.2

Cry2

0.2

Cry1

0.2

Cry1

0.4

Cry1
Cry2
Cry1
Cry2
Cry1
Cry2

0.4

0.6

M
V)
V4
0)

0.4

0.6

P(
S

0.4

0.6

Luc/RLuc

0.8

Luc/RLuc

1.0

Luc/RLuc

1.0

0.6

P(SV40)-dLuc

1.2

Cry2

3xE-P(SV40)-dLuc

VectorVector

1.2

1.0

1.4

Luc/RLuc

3xE’-P(SV40)-dLuc1.0

Vector

Luc/RLuc

1.2

P(
C

P(Per2)-dLuc

1.2

Cry2

Figure S4

Bmal1/Clock

Figure S4. Cry1 and Cry2 display differential repression activity.
Dual Luciferase reporter assay in Cry1–/–:Cry2–/– fibroblasts was performed to test repression activity of Cry1 and Cry2 in the absence (top panel)or presence (bottom panel) of exogenous Bmal1 and Clock. For Cry expression, three different promoters were tested: P(CMV), P(SV40) orP(Cry1)-Intron. Each Cry construct was cotransfected with P(SV40)-dLuc (control) or the P(Per2)-dLuc reporter. A Renilla Luciferase (RLuc) was
added in each transfection to normalize transfection efficiency. Under all three promoters, particularly the Cry1-phase promoter, CRY1 acted asa much more potent repressor than CRY2. Similar results were obtained using 3x E'-box-P(SV40) or 3xE-box-P(SV40) promoter, and when Bmal1
and Clock are ectopically over-expressed. Mean and SD (error bar) of two independent experiments are shown (n = 3 for each experiment).

Khan et al. Differential repression by CRY1 and CRY2.
S-6

Figure S5

1.0

0.8

0.8

P(Cry1)-Intron

Cry*(V316I)

Cry*(S404A)

Cry*(N425S)

Cry*(K322R)

Cry*(I392V)

Cry*(V316I)

Cry*(S404A)

Cry*(N425S)

Cry*(I392V)

Cry*(K322R)

Cry*

0.0

Cry*(I372V)

0.0

Cry1

0.2

Cry2

0.2

Cry*(I372V)

0 .4

Cry2

0.4

0.6

Cry1

0.6

Vector

Luc/RLuc

1.0

Vector

Luc/RLuc

P(SV40)-dLuc

1.2

Cry*

P(Per2)-dLuc

1.2

P(Cry1)-Intron

Figure S5. Mutation of each of the six critical residues within the CRY1-PHR(313-426) of CRY*
abolishes transcriptional repression activity.
Dual Luciferase reporter assay in Cry1–/–:Cry2–/– fibroblasts was performed to test repression activityof the CRY* single amino acid mutants. Each mutant contains a mutation in one of the 6 critical residues
within the CRY1-PHR(313-426) of CRY* (as in Figure 2D). All mutants displayed weak repression(similar to Cry2), corroborating circadian phenotypes as shown in Figure 2D.

Khan et al. Mutation of the critical residues within PHR(313-426) of Cry* impaires repression

S-7

Identification of a Novel Cryptochrome Differentiating Domain Required for
Feedback Repression in Circadian Clock Function
Sanjoy K. Khan, Haiyan Xu, Maki Ukai-Tadenuma, Brittany Burton, Yongmei Wang,
Hiroki R. Ueda and Andrew C. Liu
J. Biol. Chem. 2012, 287:25917-25926.
doi: 10.1074/jbc.M112.368001 originally published online June 12, 2012

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