MOLECULAR AND CELLULAR BIOLOGY, Mar. 2007, p. 2120–2129
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 6
Structure/Function Analysis of Xenopus Cryptochromes 1 and 2 Reveals
Differential Nuclear Localization Mechanisms and Functional Domains
Important for Interaction with and Repression of CLOCK-BMAL1?
Ellena A. van der Schalie, Francesca E. Conte, Karla E. Marz, and Carla B. Green*
Department of Biology, University of Virginia, Charlottesville, Virginia 22904
Received 1 September 2006/Returned for modification 17 October 2006/Accepted 20 December 2006
Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral
processes within an organism and also synchronize these processes with the external environment. A cell
autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops,
drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a
central part in the negative feedback loop of the molecular clock by translocating to the nucleus and
repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this
cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/
function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails
of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mecha-
nisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the
second half of the photolyase homology region (PHR) of CRY is important for repression through
facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us
to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the
Organisms ranging from cyanobacteria to humans exhibit
circadian rhythms in many processes, from gene expression to
cell physiology and from hormone levels to locomotor activity.
Circadian rhythms are approximately 24 hours in duration and
persist in constant conditions. These oscillations do not accel-
erate or decelerate within a physiological range of tempera-
tures and, importantly, can be reset by cues from the environ-
ment. Having an internal timekeeping mechanism allows an
organism to temporally arrange physiological processes inter-
nally and also to anticipate changes in the external environ-
ment (reviewed in reference 1).
The clocks driving these rhythms are intracellular mecha-
nisms composed of interlocking transcriptional/translational
feedback loops (reviewed in reference 23). At the core of the
vertebrate molecular oscillator is a negative feedback loop that
is necessary for rhythmicity (19). Two positive elements,
CLOCK and BMAL1, which are basic helix-loop-helix PAS
transcription factors, heterodimerize and bind to E-box en-
hancer elements in the promoters of the Period (Per1, Per2, and
Per3) and Cryptochrome (Cry1 and Cry2) genes, activating their
transcription. The Per and Cry mRNAs are then translated,
and the proteins accumulate in the cytoplasm. PER, CRY, and
casein kinase Iε (CKIε) proteins form a complex in the cyto-
plasm, which translocates into the nucleus, where it represses
CLOCK-BMAL1-mediated transcription. The repression com-
plex is eventually degraded or dismantled, CLOCK-BMAL1
transcription is activated, and the cycle begins again (reviewed
in reference 1).
Since the molecular clock is temporally precise and longer in
duration than most intracellular feedback loops, many regula-
tory strategies may be required to maintain this unique oscil-
lation. The detailed mechanisms that contribute to the cycle’s
robust rhythmicity and stable period are largely unknown. We
hypothesize that one of these regulation steps may be to con-
trol the intracellular localization of the CRY proteins, in order
to prevent repression before the appropriate time.
Both CRY proteins, CRY1 and CRY2, are components of
the core molecular oscillator in vertebrates, and it has been
shown that their repression of CLOCK-BMAL1-mediated
transcription is essential for rhythmicity at both the molec-
ular and behavioral levels (19, 21). The inhibition of the
CLOCK-BMAL1 heterodimer is independent of light and is
strong even at low doses of CRY (6, 11, 25). While the
mechanism for how this repression occurs is not known,
some ideas have been proposed. One hypothesis is that
CRYs repress CLOCK-BMAL1 by reducing CLOCK-
BMAL1’s affinity for the E box (17). Another idea is that
CRYs inhibit CLOCK-BMAL1 by interacting with the het-
erodimer and then recruiting histone deacetylases (16) or
inhibiting histone acetylases (5).
In order to gain insight into these aspects of the molecular
clock, we sought to identify and define functional domains in
CRYs that are responsible for their subcellular localization
and ability to repress CLOCK-BMAL1. We chose to examine
CRYs from Xenopus laevis for identification of key regions of
the protein that are involved in the circadian function of
CRYs, because these animals, unlike mammals, possess both
CRYs and also a closely related protein, Xenopus 6-4 photol-
* Corresponding author. Mailing address: 275 Gilmer Hall, Depart-
ment of Biology, University of Virginia, P.O. Box 400328, Charlottes-
ville, VA 22904-4328. Phone: (434) 982-5436. Fax: (434) 982-5626.
?Published ahead of print on 8 January 2007.
yase (xPHOTO). Both Xenopus CRYs (xCRYs) and xPHOTO
are members of the CRY/PHOTO family, which share signif-
icant sequence similarity over the main body (known as the
photolyase homology region [PHR]) but have C-terminal tails
that differ greatly in length and amino acid composition (re-
viewed in reference 2)). Despite the high degree of similarity in
the PHR, xCRYs can repress CLOCK-BMAL1-mediated tran-
scription, while xPHOTO, even when expressed at high levels,
cannot (24). We have previously shown that the C-terminal
tails of both CRY proteins are necessary for their nuclear
localization in COS7 cells (24), while the PHRs are sufficient
for repression. The combination of primary amino acid se-
quence similarity and functional diversity between xCRYs and
xPHOTO provides an optimal paradigm for studying structure/
Here, we report that the C-terminal tails of xCRY1 and
xCRY2 (formerly known as xCRY2b) are sufficient for nuclear
localization and appear to have distinct nuclear localization
mechanisms. We also demonstrate, through the generation
and characterization of xCRY/xPHOTO chimeras, that resi-
dues that are conserved in repressive CRYs, but not in
xPHOTO, in the second half of the PHR are required for full
repression of xCLOCK-xBMAL1 via facilitating interaction
with xBMAL1. Interestingly, even with these residues replaced
with residues from xPHOTO, the chimeras still exhibit some
MATERIALS AND METHODS
Plasmids. (i) Cry C terminus-nocturnin constructs. The C termini of the xCry1
and xCry2 genes (residues E550 to R596 for xCRY1 and residues G515 to R556
for xCRY2) were amplified by PCR and then subcloned in frame between
enhanced green fluorescent protein (eGFP) and nocturnin (NOC) in the
peGFP-C2 vector (BD Biosciences, Clontech). To make serial deletions of
the xCRY1 C terminus in this construct, EcoRI sites were inserted using the
QuikChange site-directed mutagenesis kit (Stratagene). The clones were then
digested and religated with T4 ligase, deleting a portion of the region encoding
the C terminus. Four deletion mutants were generated: pEGFP-xCry1Cterm?
1-Noc, which is missing amino acids E550 to G564; pEGFP-xCry1Cterm? 2-Noc,
which is missing amino acids S565 to D574; pEGFP-xCry1Cterm? 1-2-Noc, which
is missing amino acids S550 to D574; and pEGFP-xCry1Cterm? 2-3-Noc, which is
missing amino acids S565 to R596. To introduce amino acid substitutions into the
C terminus of xCRY2, site-directed mutagenesis was also used, to change either
a single residue or a small cluster of residues.
(ii) xCry1/xPhoto chimeras. Both xCry/xPhoto chimeras were cloned in the
pCMV-Tag2b vector (Stratagene) in frame with the FLAG tag in the vector and
verified by sequencing. Both xCry constructs (and the chimeras that were derived
from them) contain a portion of the 5? untranslated region following the N-
terminal FLAG tag that is translated in the resulting fusion proteins. We verified
that the resulting CRY proteins showed repression comparable to that of wild-
type mammalian CRY1 (mCRY1) and mCRY2. To generate the xCry1/xPhoto
chimeras, the region of xCry1 corresponding to amino acids 1 to 257 and the
region of xPhoto corresponding to amino acids 258 to stop were amplified. A
unique AflII site was added to the 3? end of the xCry1 piece and to the 5? end of
the xPhoto piece to facilitate joining of the fragments but was designed to prevent
amino acid substitution. To generate the xCry2/xPhoto chimera, the region of
xPhoto corresponding to amino acids 262 to stop was amplified with a SalI site
added at the 5? end and an ApaI site added at the 3? end. pCMV-xCry2 was
digested with SalI and ApaI, and the xPhoto fragment was inserted. To create the
nuclear localization signal (NLS) chimera constructs, the xCry/xPhoto chimeras
were subcloned into a pCMV vector which had a heterologous NLS (PPKKKR
KVEGEF) cloned in frame with the FLAG tag (24).
(iii) xCry1/Photo chimeras with further xCry1 substitutions. The xCry1/Photo
chimeras with further xCry1 substitutions were made by an overlap extension
cloning method (20). The xCRY1/P1,C2,P3-5 chimera consists of xCRY1 resi-
dues 1 to 257, xPHOTO residues 258 to 284, xCRY1 residues 285 to 315, and
xPHOTO residues 315 to stop. xCRY1/P1-2,C3,P4-5 consists of xCRY1 amino
acids 1 to 257, xPHOTO amino acids 258 to 314, xCRY1 amino acids 316 to 348,
and xPHOTO amino acids 347 to stop. xCRY1/P1-3,C4,P5 consists of xCRY1
residues 1 to 257, xPHOTO residues 258 to 346, xCRY1 residues 349 to 496, and
xPHOTO residues 494 to stop. All constructs were verified by sequencing.
Cell culture, transient transfection, and Western blotting analysis. COS7 cells
were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum and 1 mM sodium pyruvate. COS7 cells were transfected with
expression plasmids, using Fugene (Roche), according to the manufacturer’s
instructions. For Western blotting analysis, lysates were harvested using 1?
passive lysis buffer from the dual luciferase reporter assay system (Promega).
Total protein levels were measured using a Bradford-based protein assay kit
(Bio-Rad), and 20 ?g of total protein from each sample was run on a 10%
sodium dodecyl sulfate-polyacrylamide gel and then transferred onto a poly-
vinylidene difluoride membrane (Bio-Rad). The membrane was then blocked
with BLOTTO solution (0.1% Tween 20 and 5% dry nonfat milk powder in
Tris-buffered saline [TBS], pH 7.4) overnight at 4°C. The membrane was treated
for 1 h at room temperature (RT) with the primary antibody, monoclonal mouse
anti-FLAG M2 antibody (Sigma), which was diluted 1:1,000 in BLOTTO. The
membrane was then washed twice for 10 min at RT with BLOTTO and then
twice more for 10 min at RT with TBS-Tween (0.1% Tween 20 in 1? TBS, pH
7.4), followed by incubation for 30 min at RT with the secondary antibody,
anti-mouse immunoglobulin G-peroxidase (Chemicon), which was diluted
1:1,500 in BLOTTO. The membrane was washed four times with TBS-Tween for
15 min at RT. The bound antibody was visualized using a chemiluminescence
Western blotting kit (Roche).
Immunocytochemistry. COS7 cells were seeded on coverslips in six-well cell
culture dishes, transfected 24 h later with a total of 1 ?g DNA as described
above, and then allowed to incubate for 24 h before fixation. Cells expressing
eGFP constructs were rinsed with phosphate-buffered saline (PBS), fixed with
4% paraformaldehyde for 15 min, washed two more times with PBS, and then
mounted onto slides, using Fluoromount G (Electron Microscopy Sciences).
Cells expressing FLAG-tagged constructs were fixed with ice-cold methanol for
15 min and were put in blocking solution (0.3% Triton, 1% protease-free bovine
serum albumin, 0.25% L-carageenan, 0.1% sodium azide in 1? TBS, pH 7.6)
overnight at 4°C. These slides were then treated with monoclonal mouse anti-
FLAG (Sigma), diluted 1:1,000 in blocking solution for 1.5 h at RT, washed with
PBS three times, and then treated with digoxigenin (DIG)-conjugated sheep
anti-mouse secondary antibody (Chemicon), diluted 1:1,000 in blocking solution,
for 1 h at RT. The coverslips were then washed with PBS, treated with 1:1,000
rhodamine-conjugated rabbit anti-DIG tertiary antibody (Boehringer) for 1 h at
RT, washed, stained for 5 min with Hoechst’s stain (Sigma), and mounted as
described above. The cells were viewed using an Olympus inverted epifluores-
cence microscope (IX-70). A blind count of 200 cells for each sample was done,
and the percentage of cells in each cell compartment (nuclear, cytoplasmic, or
both) was calculated.
Immunoprecipitation. COS7 cells, which were 60% confluent in 10-cm dishes,
were transfected in duplicate with 150 ng of null-Renilla luc, 2 ?g of per-luciferase
reporter gene, 1.5 ?g of xClock, 1.5 ?g of xBmal1, and either 1.5 ?g of wild-type
Cry or 3.0 ?g of xCry/xPhoto in 600 ?l Opti-MEM (Invitrogen) and 22.5 ?l
Fugene (Roche). After 24 h, the cells were washed with sterile PBS, harvested by
trypsinization, and solublized in 0.7 ml TGED buffer (50 mM Tris [pH 7.4], 100
mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol) with protease
inhibitors (Sigma) and 0.5% Triton. The duplicate lysates were pooled, nutated
at 4°C for 10 min, and centrifuged for 20 min at 13,000 rpm. Total protein levels
were measured using a Bradford-based protein assay kit (Bio-Rad). Lysates were
analyzed by Western blotting to confirm protein expression levels. Equal
amounts of total protein, 1.2 mg, were loaded on anti-FLAG-conjugated resin
(Sigma), which was prepared according to the manufacturer’s instructions. The
lysate-resin mix was nutated overnight at 4°C. Bound proteins were washed three
times with wash buffer (50 mM Tris HCl [pH 7.4], 150 mM NaCl) and then were
eluted with 0.25 ?g/?l 3XFLAG peptide (Sigma) in wash buffer for 1 h at 4°C.
The eluted protein was resolved on a sodium dodecyl sulfate-polyacrylamide gel,
transferred, and then blotted with monoclonal mouse anti-V5 (Invitrogen) or
monoclonal mouse anti-FLAG M2 (Sigma) antibody to detect xBMAL1-V5 or
FLAG-xCRY protein, respectively.
Luciferase repression assay. The luciferase reporter assay was carried out as
previously described (24). COS7 cells were transfected with 200 ng of per-
luciferase reporter gene and 15 ng of null-Renilla luc (14), which was used as an
internal control, and 150 ng of Clock, Bmal1, wild-type xCry, and/or chimera
plasmid was added as indicated below each graph in the figures. The total
amount of DNA transfected was kept constant at 1 ?g by supplementation with
empty pCMV-Tag2b vector (Stratagene). Transcriptional activity was assessed
VOL. 27, 2007IDENTIFICATION OF XENOPUS CRY FUNCTIONAL DOMAINS2121
VAN DER SCHALIE ET AL.MOL. CELL. BIOL.
with the dual-luciferase reporter assay system (Promega) by measuring the ratio
of firefly luciferase activity to Renilla luciferase activity in each cellular lysate.
xCRY1 PHR structure model. A homology model of the xCRY1 PHR was
generated and evaluated using tools available at the SWISS-MODEL Workspace
(http://swissmodel.expasy.org/workspace/). The model was constructed by the
SWISS-MODEL protein homology modeling server (7) using the sequence of
the xCRY1 PHR and the coordinates of an Anacystis nidulans cyclobutane
pyrimidine dimer photolyase structure (9) (Protein Data Bank [PDB] code
1OWL). The stereochemical characteristics of the model, analyzed using
PROCHECK (12), were similar to those of the template structure, with 88.5%
and 9.3% of the residues in the core and allowed regions, respectively, of the
Ramachandran plot (compared to 91.9 and 7.8% in the template) and an overall
G-factor of 0.05 (compared to 0.42). This model was then visualized using
PyMOL (4) (http://www.pymol.org).
In order to gain insight into the role of CRYs in the molec-
ular circadian clock, we sought to identify and further define
functional domains in the CRY proteins of Xenopus laevis. We
have previously shown that the C-terminal tails of both xCRY
proteins are necessary for each protein’s nuclear localization in
vitro (24). To determine whether these regions are sufficient
for nuclear localization, we fused these portions of the C-
terminal tail of each xCRY protein in frame with eGFP and
NOC, a cytoplasmic protein, and assayed these constructs for
subcellular localization (Fig. 1). While eGFP-NOC was local-
ized in the cytoplasm as expected, both eGFP-xCRY1Cterm-
NOC and eGFP-xCRY2Cterm-NOC were found predomi-
nantly in the nucleus (Fig. 1A). Therefore, residues E550 to
R596 of xCRY1 and residues G515 to R556 of xCRY2 are not
only necessary but also sufficient for nuclear localization in
Next, we set out to define which residues within these do-
mains are important for nuclear localization. In the C-terminal
tail of xCRY1, residues 595 to 612 comprise a sequence that
weakly resembles a bipartite NLS. This sequence is highly
conserved in mCRY1 and has been shown to be functional in
that protein. To determine if this sequence is sufficient for
localization of xCRY1, we cloned it in frame with eGFP and
NOC and assayed for subcellular localization. We found that
there was only a small increase of this NOC fusion protein in
the nucleus or in both the nucleus and the cytoplasm compared
to eGFP-NOC without the putative NLS (37% versus 22%,
respectively). It is for this reason that we did not include this
putative NLS in our eGFP-xCry1Cterm-NOC constructs.
Since we found that the putative NLS in the C-terminal
portion of xCRY1 was not sufficient for nuclear localization,
we then investigated the area comprised of residues 550 to 596
into three regions, i.e., residues 550 to 564, residues 565 to 574,
and residues 575 to 596, and then made various deletions in the
xCRY1 C-terminal portion of eGFP-xCRY1Cterm-NOC. Any
region or combination of regions that we deleted within the
xCRY1 C terminus in the eGFP-xCRY1Cterm-NOC fusion
protein led to partial or complete loss of nuclear localization
(Fig. 1B). Deletion of region 1 from the xCRY1 C-terminal
region had the most severe effect on NOC localization, but
when it alone was fused to NOC, it was not sufficient to bring
NOC into the nucleus. While we have shown that the xCRY1
C-terminal tail, residues 550 to 596, is sufficient for nuclear
localization in these cells, it seems that this function is not
confined to a single small domain.
In contrast, the C-terminal tail of xCRY2 contains a putative
canonical nuclear NLS, which consists of six positively charged
residues in a K(X9)KRK(X10)K(X)R pattern (15). In order
to test the functionality of this sequence, we replaced each
cluster of basic residues with noncharged residues in the
xCRY2Cterm-NOC fusion protein. When tested for subcellu-
lar localization, all of the mutated fusion proteins exhibited
decreased nuclear localization; in fact, mutation of KRK541-
543 or KVR554-556 resulted in complete loss of nuclear local-
ization (Fig. 1C). Mutation of these same residues in full-
length xCRY2 also resulted in complete loss of nuclear
localization (data not shown). Therefore, xCRY2 has a func-
tional bipartite NLS in its C-terminal tail.
Despite the fact that the C-terminal tails of xCRYs seem to
be necessary and sufficient for the proteins’ nuclear localiza-
tion, the PHR of xCRY1 and xCRY2 is sufficient for repres-
sion of CLOCK-BMAL1-mediated activation when it is local-
ized to the nucleus by a heterologous NLS (24). Although the
primary amino acid sequences of xCRY1, xCRY2, and
xPHOTO are 85% similar in this region (Fig. 2A), the PHRs of
xCRYs can repress CLOCK-BMAL1-mediated activation, but
xPHOTO cannot (24). The fact that these proteins share a high
degree of sequence similarity yet have distinct functions al-
lowed us to use a chimeric protein approach to characterize the
function of the PHR of xCRY in the repression mechanism of
CRYs and also to define functional domains within this region,
while preserving the protein’s structural integrity.
xCRY/xPHOTO chimeras which had the first half of either
xCRY1 or xCRY2 and the second half of PHOTO were gen-
erated (Fig. 2B). Since xCRYs and xPHOTO are so similar,
this was equivalent to making 46 amino substitutions in the
second half of the PHR of xCRY. These chimeras were then
tested for their ability to repress CLOCK-BMAL1-mediated
transcription in a luciferase repression assay in COS7 cells.
Both chimeras were still able to repress CLOCK-BMAL1, but
they exhibited a significant decrease in repression compared to
wild-type xCRY (P ? 0.01) (Fig. 2C).
FIG. 1. The C termini of xCRY1 and xCRY2 are sufficient for nuclear localization of the CRY protein when expressed in COS7 cells.
(A) Visualization of NOC, when cloned in frame with the C terminus of either xCRY1 (residues 550 to 596) or xCRY2 (residues 515 to 556). All
constructs were cloned in the pEGFP-C2 vector and therefore were tagged with eGFP on their N terminus, as indicated in each schematic. One
microgram of each plasmid was transfected into COS7 cells. N, N/C, and C, nuclear, nuclear and cytoplasmic, and cytoplasmic localization,
respectively. (B) Effect of serial deletions of the C terminus of xCRY1 on the localization of GFP-CRYCterm-NOC. The C terminus was divided
into three regions: 1, residues 550 to 564; 2, residues 565 to 574; and 3, residues 575 to 596. Deletions were made as indicated in each schematic,
and then each construct was tested for localization, as described for panel A. (C) Effect of amino acid substitutions in the putative canonical
bipartite NLS in the C terminus of xCRY2 on the localization of a cytoplasmic protein. Substitutions were made as indicated in each schematic,
and then the localization of each mutant was tested using immunocytochemistry as described for panel A.
VOL. 27, 2007 IDENTIFICATION OF XENOPUS CRY FUNCTIONAL DOMAINS2123
The decreased repression shown by the chimeras in the lucif-
erase assay could be for many reasons; some examples include
decreased protein stability, improper subcellular localization, or
decreased interaction with CLOCK-BMAL1. To see if the chi-
meras’ decreased repression is due to decreased protein expres-
chimeric proteins were present at somewhat reduced levels com-
pared to their wild-type xCRY counterparts: xCRY1/xPHOTO
expression was reduced approximately twofold, and xCRY2/
xPHOTO expression was reduced approximately fourfold (Fig.
3A). When the luciferase repression assay was repeated using
doses of plasmid adjusted to yield comparable protein expression
levels, the chimeras’ abilities to repress CLOCK-BMAL1 im-
proved slightly but still were significantly lower than that of wild-
type xCRY (P ? 0.01) (Fig. 3B). Therefore, the decrease in
repression shown by the chimeras cannot be fully explained by
decreased protein expression or stability.
FIG. 2. xCRY/PHOTO chimeras show reduced repression of CLOCK-BMAL1-mediated activation. (A) Protein sequence alignment of the PHRs
of xCRY1, xCRY2, and xPHOTO. The xCRY sequences are very similar to those of xPHOTO in the PHR, which includes all of the protein except the
highly variable C terminus. Conserved amino acid residues are indicated by gray shading, while the nonconserved amino acids are not shaded.
(B) Generation of xCry/xPhoto chimeras. Both chimeras were tagged with a FLAG tag on their N terminus. xCRY1/xPHOTO consists of xCRY1 amino
acids 1 to 257 and xPHOTO amino acids 258 to stop. xCRY2/xPHOTO consists of xCRY2 amino acids 1 to 261 and xPHOTO amino acids 262 to stop.
Both constructs were fused in a conserved region to avoid amino acid substitution at the fusion site. (C) Luciferase repression assay. In this experiment,
COS7 cells were transfected with the Per-luciferase reporter gene, and 150 ng of Clock, Bmal1, wild-type xCry, and/or chimera plasmids were added as
indicated. Each data point is averaged from six replicates, with the error bars representing the standard error of the mean. Both chimeras, xCRY1/
xPHOTO and xCRY2/xPHOTO, exhibit decreased repression significantly different from each wild-type CRY (P ? 0. 01).
VAN DER SCHALIE ET AL.MOL. CELL. BIOL.
Next, the subcellular localization of each chimera was inves-
tigated to see if the decrease in repression is due to mislocal-
ized protein. While wild-type xCRY1, xCRY2, and xCRY2/
xPHOTO were found predominantly in the nucleus, xCRY1/
xPHOTO was localized mostly in the cytoplasm (Fig. 3C).
Coexpression of CLOCK and BMAL1 with the xCRY1 chi-
mera did not alter its localization (data not shown). To further
elucidate whether the decrease in repression exhibited by
xCRY1/PHOTO was due to its cytoplasmic localization, both
Xenopus chimeras were cloned in frame with a heterologous
NLS. Although both NLS-xCRY/xPHOTO proteins were lo-
calized to the nucleus (Fig. 3D), neither protein’s repressive
ability improved significantly (Fig. 3E), suggesting that im-
proper localization of the chimera cannot explain its decreased
Previous studies have shown that CRY binds to the
CLOCK-BMAL1 heterodimer (6, 11, 13) and that this binding
is required for repression (19). In light of these data, we hy-
pothesized that that the disruption of repression seen with the
xCRY chimeras may be due to an inability to bind the
CLOCK-BMAL1 heterodimer. To test this hypothesis, we
transfected COS7 cells with xCLOCK, xBMAL1-V5, and ei-
ther FLAG-xCRY1, FLAG-xCRY2, FLAG-xCRY1/PHOTO,
or FLAG-xCRY2/PHOTO. We then examined whether
xBMAL1 immunoprecipitated with each CRY protein. While
wild-type xCRY1 and xCRY2 exhibit a strong interaction with
xBMAL1, neither chimera showed detectable interaction
with xBMAL1 (Fig. 4). This suggests that reduced interac-
tion with theBMAL1 explains the decrease in repression by
We could not determine whether CLOCK can bind to the
chimeras since CLOCK was present at very low levels in our
immunoprecipitation lysates and did not coimmunoprecipitate
even with wild-type xCRY1 (data not shown). This is not sur-
prising, since it has been previously shown that when CLOCK
and BMAL are coexpressed in cell culture, CLOCK is rapidly
The characterization of the xCRY/xPHOTO chimeras in-
dicates that the second half of the xCRY PHR is required
for full repression of CLOCK-BMAL1. To further define
functionally important regions of this second half, we re-
placed portions of the xPHOTO moiety of the xCRY1/
xPHOTO chimera with the equivalent portions of xCRY1 to
see if restoration of xCRY1 residues would rescue repres-
sion of CLOCK-BMAL1. These substitutions corresponded
to clusters of residues that are conserved in repressive-type
vertebrate CRYs but not in xPHOTO (Fig. 2A and 5A and
B). When tested in the luciferase repression assay at a sub-
saturating dose (150 ng), these chimeras were expressed at
levels comparable to those of wild-type xCRY1 and higher
than those of the original xCRY1/xPHOTO chimera (Fig.
5C). However, the substitutions in these chimeras did not
improve their repression of CLOCK-BMAL transcriptional
activity, and in fact, the chimeras with further substitutions
were less effective (Fig. 5C). From these data, we can con-
clude that restoration of any single cluster of xPHOTO
residues to xCRY1 residues in the second half of the PHR
was insufficient for full repression.
Although both CRY1 and CRY2 are potent repressors of
CLOCK-BMAL1-mediated transcription, mice lacking either
protein individually have opposing circadian locomotor phe-
notypes (21). Knocking out CRY1 makes the clock run fast,
while knocking out CRY2 makes the clock run slow. In this
paper, we have shown that the C-terminal tails of both xCRY1
and xCRY2 are sufficient for nuclear localization of the protein
in cell culture, but by different mechanisms. Differential regu-
lation of nuclear translocation is one potential mechanism by
which CRYs could share a common function but affect the
molecular circadian clock in opposite ways.
We report that in xCRY1, the entire region of amino acids
550 to 596 is both necessary and sufficient for localization when
expressed in COS7 cells. Although in the C-terminal tail of
xCRY1, residues 595 to 612 comprise a sequence that weakly
resembles a bipartite NLS that has shown to be functional in
mCRY1 (3), we show that this sequence is not sufficient for
NLS on its own. In addition, previous studies from our lab
showed that an xCRY1 truncation mutant missing a large por-
tion of this sequence did show partial loss of nuclear localiza-
tion. These data suggest that this sequence may have a role in
determining the localization of xCRY1 in the context of the
entire protein (24).
Interestingly, Chaves et al. also have reported a second
mechanism by which mCRY1 can gain entry into the nucleus
via interaction with BMAL1 though mCRY1’s coiled-coil re-
gion, located at amino acids 471 to 493 (3). At first glance, it
appears that this mechanism may not be conserved in Xenopus,
since we have previously reported that xCRY1 with an intact
coiled-coil region but lacking a C-terminal tail exhibits cyto-
plasmic localization (24). In that localization experiment, how-
ever, the xCRY1 mutant was not coexpressed with xBMAL1,
leaving the possibility open that xBMAL1 can affect the sub-
cellular localization of xCRY1. In addition, we have previously
shown that xCRY1 with a coiled-coil domain but without a
C-terminal tail exhibited partial repression of CLOCK-
BMAL1 (24), providing further evidence that this second
mechanism for nuclear localization may be conserved in
Xenopus. The C-terminal tail of xCRY2 contains a canonical
bipartite NLS, which is necessary and sufficient for localization
in COS7 cells, while the region most important for localization
in xCRY1 appears to be noncanonical. This bipartite NLS is
conserved in mouse CRY2 and has been shown to mediate
nuclear localization through active nuclear import via importin
alpha/beta (18). Similar sequences in other proteins are tightly
regulated through posttranslational modifications, such as
In this paper, we have characterized two functional domains
that are important for nuclear localization of the CRY proteins
when expressed in COS7 cells. Many groups have reported
that in cell culture CRY proteins are only observed in the
nucleus, but curiously, in vivo there is a constant pool of
CRY1 and CRY2 in the cytoplasm and only a portion of the
CRY protein population rhythmically translocates to the
nucleus (13). This suggests that nuclear translocation of
CRY protein is tightly regulated in cells with a functional
circadian clock. We hypothesize that nuclear translocation
of CRY in the molecular clock may be important for estab-
VOL. 27, 2007IDENTIFICATION OF XENOPUS CRY FUNCTIONAL DOMAINS 2125
FIG. 3. Decreased repression exhibited by chimeras is not due to decreased protein expression or improper subcellular localization. (A) Protein
expression levels of xCRY and chimera proteins. COS7 cells were transfected with the indicated plasmids. A DNA dose of 150 ng was used for
each clock plasmid, except for the chimeras, of which 300 ng was added to compensate for decreased expression levels. Twenty micrograms of total
protein lysate was run, and both membranes were blotted with anti-FLAG (1:1,000; Sigma) and anti-mouse-POD (1:1,500; Chemicon). The gray
arrowheads denote the wild-type CRY band, while the black arrowhead denotes the chimera band. Nonspecific bands are indicated with asterisks.
(B) Luciferase repression assay with protein normalization. Instead of using the same DNA dose for each construct, these data were normalized
VAN DER SCHALIE ET AL.MOL. CELL. BIOL.
lishing period length. Furthermore, the fact that nuclear
localization of CRY1 and CRY2 is achieved by different
mechanisms offers a potential way for the two CRY proteins
to have different effects on period length.
One potential mechanism for the regulation of the subcel-
lular localization of CRYs is through changing their binding
partners. It is known that CRY translocates to the nucleus in a
complex with PERs and CKIε (13). Perhaps there is some
other mechanism by which CRYs are retained in the cyto-
plasm until PER protein levels trigger nuclear localization.
It is important to mention that other researchers have
shown that CRY affects the localization of PER and that
PER can also influence the localization of CRY (11, 13, 22).
Although it is clear that in vivo CRYs, PERs, and CKIε
translocate to the nucleus as a complex (13), it is not clear
whether CRY, being the only nuclear protein when ex-
pressed alone in cell culture, is the driving force for the
movement of the entire complex. The contribution of each
protein, and each localization domain within each protein,
to the localization of the complex as a whole has been and
continues to be debated in the literature.
Our laboratory has previously shown that the PHR of xCRY,
when attached to an NLS, is sufficient for repression of
CLOCK-BMAL1. In this report, we showed that when the
second half of the PHR of xCRY is replaced with the homol-
ogous region of xPHOTO, repression is significantly de-
creased. This cannot be explained by decreased protein expres-
sion or mislocalization. While the xCRY1 chimera is localized
predominantly to the cytoplasm, this does not explain its de-
creased repression, since when it is pulled into the nucleus by
a heterologous NLS, its repressive ability does not improve.
This suggests that even though xCRY1/PHOTO appears to be
cytoplasmic in our immunocytochemistry assay, enough pro-
tein enters the nucleus to repress CLOCK-BMAL1.
We did, however, observe that the chimeras do not interact
with BMAL1, which we believe to play a central role in their
decreased repressive ability. These data are consistent with
data reported by Chaves et al., showing that residues 471 to 493
comprise the BMAL interaction domain in mCRY1 (3). Inter-
estingly, replacement of these residues from xPHOTO back to
xCRY1 was not sufficient to restore the repressive ability of our
xCRY1 chimera. These data imply that CRY’s interaction with
BMAL1 may depend on more than just the coiled-coil domain
or that the decreased interaction with BMAL1 may not fully
explain their decreased abilities. Although it is clear that in-
teraction with BMAL1 is required for full repression by CRY,
the significance of this binding is for repression is not clear. It
may be that CRY binding decreases the affinity of the CLOCK-
BMAL1 heterodimer for the E box, allows recruitment of
corepressors, or inhibits coactivators. Our experiments do not
distinguish between these possibilities.
While the chimeras exhibit significantly decreased repres-
sion of CLOCK-BMAL1, it is important to emphasize that the
chimeras still repress significantly. One interpretation of these
data is that the first half of the PHR contains a region that is
sufficient for some repression. An alternative interpretation
could be that it is the amino acids that are conserved between
xPHOTO and CRYs in the second half of the PHR that are
important for repression. In this scenario, residues in the PHR
that are not conserved between CRYs and xPHOTO and res-
idues in the C-terminal tail confer specificity, facilitating inter-
action with BMAL1. This interaction allows CRYs to repress
CLOCK-BMAL1, while xPHOTO cannot. This idea is consis-
tent with the publication by Chaves et al., which reported that
when PHOTO is fused to the last 100 amino acids of the PHR,
coil-coil domain, and C-terminal tail of mCRY1, it can repress
CLOCK-BMAL1 (3). The fact that the chimeras still repress
well is especially interesting since we have shown that the
chimeras do not bind xBMAL1.
In summary, our data suggest that the C-terminal tails of
to protein level. The experiment was done as described for Fig. 2, except for the following changes. The black bars indicate activation seen when
chimera plasmids or wild-type Cry DNA (300 ng and 150 ng, respectively) was added as indicated. These DNA doses lead to equal protein levels
of wild-type CRYs and the chimeras, as verified by Western blotting with anti-FLAG antibody. Both chimeras, xCRY1/xPHOTO and xCRY2/
xPHOTO, still exhibit decreased repression significantly different from both wild-type CRYs (P ? 0.01). (C) Immunocytochemistry of wild-type
xCRYs and chimeras. One microgram of each plasmid was transfected separately into COS7 cells. These cells were then fixed and incubated with
monoclonal mouse anti-FLAG M2 primary antibody, DIG-conjugated sheep anti-mouse secondary antibody, rhodamine-conjugated goat anti-DIG
tertiary antibody, and Hoechst’s stain. The florescence shown here is the rhodamine signal. The graphs below each immunocytochemistry picture
denote the percentage of cells showing nuclear (N), nuclear and cytoplasmic (N/C), or cytoplasmic (C) localization of the expressed protein. (D)
Schematic and immunocytochemistry of NLS-chimera proteins. The heterologous NLS was cloned between the FLAG-tag and the beginning of
the Xenopus chimera coding sequence. Immunocytochemistry experiments were done as described for panel C, revealing that both chimeras reside
only in the nucleus. (E) Luciferase repression assay, comparing chimeric CRYs to NLS-chimeric CRYs. There was no significant difference in the
repression ability of either chimera versus its NLS-fused counterpart.
FIG. 4. Chimeras do not interact with xBMAL1, unlike wild-type
CRY proteins. IP, immunoprecipitation. COS7 cells were transfected
with xCLOCK, xBMAL1-V5, and FLAG-xCRY constructs as indi-
cated. CRY and chimera proteins were pulled down, using anti-FLAG-
conjugated resin (Sigma). Copurified proteins were detected by West-
ern blotting, using mouse anti-V5 (Invitrogen) or monoclonal mouse
anti-FLAG M2 (Sigma) antibodies.
VOL. 27, 2007 IDENTIFICATION OF XENOPUS CRY FUNCTIONAL DOMAINS2127
both xCRY proteins are sufficient for nuclear localization of
the proteins but act through different mechanisms. We also
have identified regions in the PHR of CRYs that are im-
portant for interaction with BMAL1 and also repression of
the CLOCK-BMAL1 heterodimer. While these data give
insight into the structure/function relationships of CRYs,
the true value of these insights will emerge as the resulting
hypotheses are tested in the context of a functioning molec-
This work was supported by grants from the National Institute of
Mental Health (MH-61461) and the Jack Kent Cooke Foundation.
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