Feedback repression is required for mammalian circadian clock function

Abstract

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

Figure 1

Mutations in CLOCK and BMAL1 confer insensitivity to CRY-mediated transcriptional repression without affecting CLOCK/BMAL1 transcriptional activity. (a,c,e) Results of cell-based transcriptional PER1-luciferase reporter assays with mutant CLOCK and BMAL1 clones in HEK293T cells. Plasmids expressing Flag-tagged wild-type or mutant CLOCK (a) or BMAL1 (c,e) cDNAs were transiently cotransfected with wild-type (WT) BMAL1 or CLOCK, respectively, PER1-luciferase reporter and 0–5 ng of CRY1 plasmid. Activity is expressed as the percentage of normalized PER1-luciferase activity in cells transfected with wild-type CLOCK/BMAL1 alone. Data are mean ± s.e.m. from independent experimental triplicates. (b,d,f) Domain locations of causative mutations. Schematic locations of amino acid changes within the CLOCK PAS-B domain (b) and BMAL1 C terminus (d) that confer CRY1 desensitization are indicated. Locations of protein domains are indicated for CLOCK (bHLH, blue, amino acids 35–85; PAS domain, green, amino acids 113–377; PAS-A repeat, orange, amino acids 128–170; PAS-B repeat, yellow, amino acids 283–329) and BMAL1 (bHLH, blue, amino acids 73–126; PAS domain, green, amino acids 148–439; PAS-A repeat, orange, amino acids 163–206; PAS-B repeat, yellow, amino acids 344–391). (f) Schematic location of amino acid changes within the N terminus of BMAL1 that confer CLOCK/BMAL1 hyperactivity: Bmal1-5, S10L and V160I; Bmal1-6, S9F.

Figure 2

Coexpression of CLOCK and BMAL1 desensitized mutants confers synergistic insensitivity to CRY1 in HEK293T cells. (a–c) Various combinations of single and double CRY-desensitized CLOCK and BMAL1 mutant cDNAs were cotransfected with the PER1 reporter and 0–50 ng of CRY1 plasmid. PER1 reporter activity alone (blue triangle) or with wild-type CLOCK/BMAL1 (orange triangle) are also displayed. Activity is expressed as the percentage of normalized PER1-luciferase activity in cells transfected with wild-type CLOCK/BMAL1 alone. Solid lines: CLOCK and BMAL1 single mutants. Dashed lines: double mutants. (d) Luciferase activities were analyzed from combinations of single and double Bmal1-3 and Clock-3 mutant cDNAs that were cotransfected with the PER1 reporter and 0–25 ng of CRY2 plasmid. PER1 reporter activity alone (blue triangle) or with wild-type CLOCK/BMAL1 (green triangle) are also shown. Activity is expressed as the percentage of normalized PER1-luciferase activity in transfection with wild-type CLOCK/BMAL1 and no CRY1 plasmid. Data are mean ± s.e.m. determined from independent experimental triplicates.

Figure 3

Mutations in the CLOCK PAS domain and BMAL1 C terminus abrogate interactions between the CLOCK/BMAL1 complex and transcriptional repressors CRY1 and PER2. (a–c) Native coimmunoprecipitations (co-IPs) were performed on cell extracts from HEK293T cells transiently transfected with plasmids expressing epitope-tagged circadian components. (a) Flag-tagged wild-type or mutant BMAL1 protein was coexpressed with or without CRY1 and various untagged CLOCK alleles and precipitated with anti-Flag. Copurified proteins were visualized by protein blotting with antibodies to Flag or CLOCK. (b) Co-IPs were performed with anti-MYC on extracts from cells coexpressing MYC-tagged CRY1, combinations of various Flag-tagged CLOCK and BMAL1 CRY-desensitized alleles and Flag-tagged PER2. Anti-Flag and CRY1 antibodies were used in protein blotting. (c) Flag-PER2 was coprecipitated (using anti-Flag) from cells coexpressing additional combinations of untagged CLOCK and BMAL1 alleles. Protein blotting with anti-Flag, anti-CLOCK and anti-BMAL1 was used to identify copurified proteins.

Figure 4

Coexpression of CLOCK/BMAL1 mutant heterodimers that are insensitive to CRY repression ablates circadian E-box and RORE activities in NIH3T3 cells. Plasmids expressing Flag-tagged CLOCK and BMAL1 alleles were transiently cotransfected with the PER2-destabilized luciferase (dLuc) reporter plasmid into NIH3T3 cells. (a,b) PER2 promoter activities in NIH3T3 cells transfected with single (a) or double (b) CRY1-insensitive CLOCK and BMAL1 mutants were monitored over 5 d. (c,d) BMAL1 promoter activities in NIH3T3 cells transfected with single (c) or double (d) CRY-insensitive mutants of CLOCK and BMAL1 were monitored over 6 d. All reporter activities were normalized such that the median wild-type luciferase activity over the time-course was 100%.

Figure 5

Coexpression of CLOCK/BMAL1 mutant heterodimers impairs circadian rhythmicity in individual cells. (a–d) PER2-luciferase reporter activities from individual NIH3T3 cells (n = 133) transfected with Flag-tagged wild-type CLOCK/BMAL1 (a,c) or double-mutant Clock-1/Bmal1-3 (b,d) were monitored over 3 d. Reporter activities from each wild-type (a) or double-mutant (b) cell were normalized such that the maximum bioluminescence value was set to 100% for each panel. The mean reporter activity for all analyzed single cells at each time point is indicated by a black line. Normalized bioluminescence activities from each wild-type (c) or double-mutant (d) cell were detrended and ranked according to corresponding significance by autocorrelation (upper panels) or COSOPT (lower panels). Autocorrelation and MMC-β values for each cell are depicted to the right of each heat map from 0 (bottom, dark blue) to 1 (top, white). Red and green denote high and low normalized reporter activities, respectively. Results shown are representative of duplicate experiments.