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.