CRY1 and CRY2 are integral components of the circadian clock and strong transcriptional repressors of the CLOCK-BMAL1 complex. To define the molecular mechanisms of repression by CRY1 and CRY2, we generated and screened libraries of randomly mutagenized CRY1 and CRY2 clones and identified functionally important residues within each protein crucial for their repression of CLOCK-BMAL1 that have different roles in specific actions of CRY1 and/or CRY2. Utilizing this unbiased approach to identify single point mutations that affected the functionality of both CRYs has led to the identification of novel roles for specific residues. Furthermore, random mutagenesis as opposed to deletion of large portions of the protein has allowed us to explore the structure/function of CRYs without causing gross changes in protein structure and thus affecting many aspects of CRY function.
As a result of our random mutagenesis screen, we generated many novel mutant Cry
alleles. The residue changes corresponding with defects in transcriptional repression were confined to the PHR, and no changes were observed in the C-terminal tail, which has been shown to be important for nuclear localization in mammals (4
) and Xenopus
). This is in line with data from Chaves et al. (4
), who reported that interaction of mCRY1 with BMAL1 via the coiled-coil domain in its PHR could facilitate CRY's nuclear localization, even in the event that its NLSs are mutated. Therefore, since both mCry
are endogenously expressed in the cells used for the screen, it is likely that any mutants with disruptions in the NLS maintained wild-type-like repression in the screen.
When the repressive ability of CRY1 mutants were tested in a dose-response assay, we observed deficiencies in repression that fell into three general categories: no repression, weak repression, and attenuated repression that showed a significant dose response. It is interesting that some of the mutants show partial repression that is independent of dose but that others show partial repression that changes with dose. We speculate that the dose-independent mutants may function in a dominant fashion. While all of the CRY1 mutants showed impaired repression compared to that of wild-type CRY1, many showed partial loss of function. In contrast, we did not observe partial-repression phenotypes in any of our CRY2 mutants. In fact, some of them actually caused transcriptional activation over and above the activation seen in the lysates transfected with Clock, Bmal1, and reporter only. We favor the interpretation that these mutant proteins are gain-of-function mutants with transcriptional-activation activity. The fact that the mutants from the CRY1 and CRY2 screens demonstrated repression profiles distinct from each other was unexpected, considering the high sequence similarly between the two proteins, even in the context of a nonsaturating screen.
In silico modeling of the 3D location of the mutants on the predicted tertiary structure of the CRY2 protein drew our attention to two residues (G351 and G354) that were hits only in the CRY2 screen, shared the same repression profile, and were predicted to be in close proximity on the protein's predicted tertiary structure. Despite the nonsaturating nature of the screen, the fact that these residues were conserved between CRY1 and CRY2 but were identified only in the CRY2 screen suggested to us that perhaps these residues were differentially utilized in the mechanism of repression by CRY2 but not CRY1. In fact, when the analogous mutations were made in the CRY1 protein, one of them (CRY1G336D/CRY2G354D) resulted in complete loss of repression in both CRY1 and CRY2 and is therefore not specific to CRY2 repression, while the other (CRY1G333D/CRY2G351D) rendered CRY2, but not CRY1, unable to repress. This differential effect may represent a biochemical mechanism by which CRY2, but not CRY1, represses the CLOCK-BMAL1 complex. Until now, the only biochemical differences seen between CRY1 and CRY2 were subtle differences in the levels of potency of their repression and binding to some core clock components (9
) and differential nuclear localization mechanisms in Xenopus
and mammals (4
). Our data support the notion that the opposing period length phenotypes in Cry1−/−
mice may be due to innate biochemical differences between the two proteins, as opposed to the idea that CRY proteins are biochemically redundant and the differential behavioral phenotypes can be attributed to differences in timing of their expression.
Between CRY1 and CRY2, the N terminus is highly conserved and only the C-terminal tails differ in size and amino acid composition. Since the C-terminal tails of CRY1 and CRY2 are the most obvious structural difference between the two proteins, we hypothesized the differential effects of the same mutation in each protein might be due to functional interplay with the C-terminal tail. Interestingly, when mutation G333D/G351D was introduced into Cry1
constructs consisting of the Cry1
PHR only (without a C-terminal tail) or the Cry1
PHR fused to the Cry2
C-terminal tail, their ability to repress was significantly decreased. The effect was most severe when there was no C-terminal tail. These data strongly suggest that the ability of CRY1 to retain its repressive ability when mutated is dependent on the presence of a C-terminal tail. The ability of the C-terminal tail to affect CRY function has been previously reported for Drosophila melanogaster
), where the C-terminal tail of the CRY protein functionally interacts with the N-terminal portion to regulate its activity (6
). When Drosophila
CRY lacks its C-terminal tail, it is constitutively active (6
). Perhaps CRY1G333D loses its ability to repress normally when the C-terminal tail is altered or removed because the tail interacts with the PHR to facilitate its mechanism of repression. While our studies clearly demonstrate a functional interaction between the N- and C-terminal portions of the mammalian CRY proteins, the presence of a physical interaction has yet to be shown.
In order to explain the altered repression seen when G351D or G354D were introduced into CRY2, both mutants were tested for their ability to bind CLOCK-BMAL1 and both PER proteins. Previous studies have shown that the ability of CRY to bind CLOCK-BMAL1 is required for its repression of CLOCK-BMAL1-mediated transcription (4
). CRY2G354D showed decreased binding to CLOCK-BMAL1, which may partially account for its decreased repression. In addition, this mutant was also highly deficient in binding of PER1 and PER2. Although the CRY-binding domain of PER localizes to its C terminus (16
), the PER-binding domain on CRY has not been well defined. These data identify a new residue in CRY2 important for protein-protein interaction. Whether this residue influences the function of the coiled-coil binding domain in a manner analogous to the one identified in CRY1 (4
) or whether it is an essential residue in a novel binding domain remains to be determined. In addition, it is not clear from our data whether the mutants' decreased PER binding causes their defective repression. Data showing that CRY can repress CLOCK-BMAL1 in transient-transfection luciferase assays without coexpression of PER suggest that PER may not be required for CRY's repression (9
). In contrast to CRY2G354D, CRY2G351D retained its ability to bind CLOCK-BMAL1 and both PERs, which is surprising in light of its complete inability to repress. Collectively, these data show that PER binding by CRY proteins is not necessarily predictive of repression. The fact that CRY2G351D can bind the major clock components but still cannot repress CLOCK-BMAL1 suggests a repression-specific role for this residue in regulating the CLOCK/BMAL1 heterodimer. Therefore, we speculate that this CRY2 loss-of-function mutant functions downstream of its interaction with CLOCK, BMAL1, or the PERs.
We also examined the effect of overexpression of these mutants on circadian clock function in an autonomous cellular model, NIH 3T3 cells. While expression of CRY2 protein (wild type or mutants) did not cause any detectable change in period, the amplitude of the rhythms was sensitive to overexpression of all Cry2 constructs tested, but to various degrees. Expression of CRY2G354D, deficient in its ability to bind CLOCK, BMAL1, and PER, caused a significant increase in rhythm amplitude over that of wild-type CRY2, consistent with its partial loss-of-function phenotype. Interestingly, expression of CRY2G351D, which demonstrates intact interaction with CLOCK, BMAL1, and PERs, had the most severe effect on rhythms, causing complete loss of circadian rhythmicity in kinetic imaging assays. CRY2G351D, which cannot repress but can still bind PER, has a more severe effect on amplitude than wild-type CRY2. In total, our data suggest that CRY2G351D acts as a strong dominant negative in the context of a functional circadian clock. The fact that overexpression of constitutive CRY2G351D leads to the most severe effect on molecular rhythms may be due in part to its ability to bind CLOCK-BMAL1 and the PER proteins and, perhaps in doing so, alter the robustness of the molecular clock in a dominant fashion. Therefore, future work comparing the circadian behavior of a CRY2G351D knock-in mouse to that of the Cry2 null mouse will likely lend valuable insight into the role of CRY2, as opposed to CRY1, in circadian rhythms.
The question remains of how identical amino acid substitutions in two clustered residues in CRY2 can lead to such distinct effects on CRY function. Both residues in CRY2 are mutated from glycine, an amino acid with a noncharged side group, to aspartic acid, which has an acidic side chain. Our in silico modeling predicts that this difference may be due to the locations of these residues. G351 is closer to the surface of the protein, while G354 is less superficial and localized closer to the coiled-coil domain, which has previously been shown to be involved in binding to CLOCK-BMAL1 and the PER proteins (4
). Our immunoprecipitation data demonstrated that the G354D mutation disrupts CRY2's binding to CLOCK-BMAL1, PER1, and PER2 (Fig. ), which is similar in phenotype to when the coiled-coil domain is deleted from CRY1 (4
). On the other hand, G351D, which is located on a face of the protein different from this previously described interaction domain, does not show disrupted binding to these core clock components.
Another interesting issue that our data raise is how mutation of this conserved residue, G333/G351 in the CRY proteins, results in much stronger loss of repression in CRY2 than in CRY1. G333/G351 is immediately upstream of a small patch of 13 residues that are highly conserved between repressive CRYs, but not in Photolyase, a close family member that cannot repress CLOCK-BMAL1 (Fig. ). Many of the residues that are neutral or basic in repressive CRYs are acidic or neutral in Photolyase. G351D introduces an acidic residue that confers a negative charge and may change CRY1 and CRY2 to be more like Photolyase. These data suggest that this small region may be vital for the ability of CRYs to repress through interactions of polar amino acids.
FIG. 8. CRY2G354 is immediately upstream of a region of amino acids conserved in repressive CRYs but not Photolyase. CRY2 3D homology model. This model was generated as described for Fig. . The N-terminal residue is shown in black, while the coiled-coil (more ...)
One hypothesis to explain the differential impact of this region on CRY2, but not CRY1, function is that this residue is involved in recruitment of a phosphatase to the promoter by CRY2. Dardente et al. (5
) reported that only CRY2 causes stabilization of the unphosphorylated, transcriptionally inactive form of BMAL1. One possibility is that CRY2 may recruit to the promoter a phosphatase that is able to dephosphorylate BMAL1, leading to its repression. The mutation G351D may change CRY2's ability to bind the phosphatase. Another possible explanation is that this amino acid impacts chromatin remodeling. Several groups have shown that chromatin remodeling is integral to molecular clock function. Naruse et al. (17
) showed that CRY1 binds to mSin3B, a histone deacetylase, and that this leads to downregulation of the Per1
gene. Perhaps the residue we have isolated is involved in recruitment of histone deacetylases to the promoter. It is possible that CRY2, compared to CRY1, may bind different corepressors via different domains, explaining why mutation G351 has different effects on CRY1 and CRY2.
In conclusion, our unbiased mutagenesis screen of the CRY proteins has generated an invaluable allelic series of mutant Crys that can be used to elucidate the multifaceted roles of CRY1 and CRY2 in the molecular clock. In addition, through careful analysis of two particular mutants, we identified a novel residue that is important for CRY2's protein-protein interaction with other clock proteins and another residue that has functionally distinct roles in CRY1 and CRY2. This work has made progress in elucidating the molecular mechanisms underlying the differential roles of the CRY proteins in the molecular circadian clock and will form the basis for future studies that will integrate our new biochemical knowledge about the role of the CRY proteins with other clock components to allow a clearer picture of the molecular timekeeping mechanism to emerge.