Subcellular localization equilibrium of the mCRY1-mPER2 complex. (A) Western blot of an immunoprecipitation (ip) of COS7 cells transfected with HA-CRY1 (wild type or mutant) and either PER1-EGFP (left) or PER2-EGFP (right). Proteins were precipitated from the lysate with anti-HA antibodies and then analyzed on Western blots using anti-HA (top panels) and anti-GFP (middle panels) antibodies. The bottom panels show Western blots of the total lysate using anti-GFP antibody. (B) Immunofluorescence pictures of COS7 cells transfected with PER2-EGFP alone or with PER2-EGFP and HA-CRY1, HA-CRY1ΔCC, or HA-CRY1mutNLSc. αHA, anti-HA. (C) Quantification of the cellular localization of PER2-EGFP shown in panel B. (D) Immunofluorescence pictures of COS7 cells transfected with HA-CRY1mutNLSc alone (top panels) or with HA-CRY1mutNLSc and PER2-EGFP (bottom panels). Treatment of cells with LMB is indicated (+ LMB). (E) Quantification of the cellular localization of HA-CRY1mutNLSc, as shown in panel D. (F) Immunofluorescence pictures of COS7 cells transfected with HA-CRY1mutNLSc and EGFP-PER2(596-1257) (top) or PER2(1-916)-EGFP (bottom). (G) Fluorescence microscopy pictures of wild-type (WT), Per1^−/−, Per2^Brdm1/Brdm1, and Per1^−/− Per2^Brdm1/Brdm1 MDFs transiently expressing mCRY1-EGFP.

Cross talk between the photolyase-like domain of mCRY1 and its C terminus. (A) Schematic representation of HA-CRY1, (6-4PP)PhL, and the fusion proteins (6-4PP)PhL-CT, (6-4PP)PhL-extCT, and EGFP-extCT. (B) Immunofluorescence pictures of transfected COS7 cells showing the subcellular localization of the aforementioned proteins. αPhL, anti-PhL. (C) Graphic representation of the CLOCK/BMAL1-inhibitory capacity of the proteins in the Dbp E-box promoter-luciferase reporter assay (using 100 ng of plasmid). Error bars represent the standard deviations. The difference between HA-CRY1 and (6-4PP)Phl-extCT is not statistically significant (t test P value is 0.07).

Functional characterization of the C terminus of mCRY1 in nuclear import. (A) Protein sequence alignment of NLSn and NLSc of mCRY1, human CRY1 (hCRY1), and mCRY2 (amino acid numbers are indicated above the sequence; stop codons are represented by a star). For comparison, the bipartite NLS of nucleoplasmin is aligned with NLSc. Conserved basic amino acids in the NLSs are in boldface type and underlined. Amino acid substitutions, as present in HA-CRY1mutNLSc, HA-CRY1mutNLSn, and HA-CRY1mutNLSn+c, are also indicated. (B) Schematic representation of HA-CRY1, HA-CRY1mutNLSc, HA-CRY1mutNLSn, and HA-CRY1mutNLSn+c. Mutations in NLSc and NLSn are indicated by a star. The photolyase-like domain is shown in dark blue, and the C terminus is shown in light blue. (C) Immunofluorescence pictures of COS7 cells transfected with the constructs shown in B. (D) Quantification of the cellular localization of the above-described proteins. Nuclear (N) signal is black, nuclear/cytoplasmic (N/C) signal is gray, and cytoplasmic (C) signal is red. (E) Schematic representation of HA-CRY1ΔCC, HA-CRY1ΔCCmutNLSc, HA-CRY1ΔCCtail, and HA-CRY1Δtail. The tail is indicated in light blue, and the CC domain is shown in yellow. The left and right red bars represent NLSn and NLSc, respectively, and the star is the mutated NLSc. (F) Immunofluorescence pictures of COS7 cells transfected with the constructs shown in E. αHA, anti-HA. (G) Quantification of the cellular localization of the transiently expressed proteins.

Role of the C terminus of mCRY1 in the inhibition of CLOCK/BMAL1-driven transcription. (A) CLOCK/BMAL1 transcription assay using a Dbp E-box promoter-luciferase reporter construct. Luminescence, shown as a severalfold induction from the basal level, is indicated on the y axis. pcDNA3, pRL-CMV, and the pGL3 promoter (Dbp) were added in all conditions. The presence or absence of the other expression plasmids is indicated. We tested HA-CRY1, HA-CRY1Δtail, HA-CRY1mutNLSc, HA-CRY1ΔCC, and HA-CRY1ΔCCtail (100 ng plasmid/assay). Error bars represent the standard deviations. (B) Immunofluorescence pictures of COS7 cells expressing FLAG-BMAL1 and HA-CRY1mutNLSc (top panels), FLAG-BMAL1 and HA-CRY1ΔCCmutNLSc (middle panels), or FLAG-BMAL1, PER2-EGFP, and HA-CRY1mutNLSc (bottom panels). The HA-CRY1 mutants were detected with rabbit anti-HA (αHA) antibodies. (C) Immunofluorescence pictures of COS7 cells expressing HA-CRY1mutNLSc or HA-CRY1ΔCCtail either alone (left panels) or together with CLOCK (right panels). (D) Immunofluorescence pictures of COS7 cells expressing HA-CRY1ΔCCtail, EGFP-BMAL1, and CLOCK (note the higher magnification used).

Model for the mechanism of action of mCRY1 within the mammalian core oscillator. The C terminus of mCRY1 is involved in association with mPER1 and mPER2 proteins and therefore regulates the stability and cellular localization of the latter proteins. The formation of the mCRY/mPER complex occurs possibly through CC interactions, as a predicted CC is also present in the mCRY binding region of mPER proteins (data not shown). To achieve full nuclear localization, this protein complex requires both the mCRY1 NLSc and the mPER2 NLS to counteract the NES-mediated nuclear export of mPER2. This molecular mechanism may explain the synchrony in nuclear localization of these proteins observed in vivo. Moreover, the photolyase-like core domain of mCRY1 interacts with CLOCK, while the CC is involved in association with BMAL1. Our data also suggest that there is competition between mPER2 and BMAL1 for binding with mCRY1, which supports the concept that the periodicity of the oscillator depends on temporal abundance and the strength of the interaction between the partners as well as on the transcription-inhibitory capacity of each complex (23). The inhibitory action of mCRY1 is likely achieved through an intermolecular interaction, which may impose a structural change in the C terminus, thereby allowing this domain to recruit transcriptional corepressor complexes.