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.

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.

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.

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.

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%.