Feedback repression is required for mammalian circadian clock function.
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.
Sato
Trey K
TK
Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, California 92121, USA.
Yamada
Rikuhiro G
RG
Ukai
Hideki
H
Baggs
Julie E
JE
Miraglia
Loren J
LJ
Kobayashi
Tetsuya J
TJ
Welsh
David K
DK
Kay
Steve A
SA
Ueda
Hiroki R
HR
Hogenesch
John B
JB
D016475:3T3 Cells; D056930:ARNTL Transcription Factors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D056926:CLOCK Proteins; D002460:Cell Line; D002940:Circadian Rhythm / Q000502:physiology*; D005246:Feedback; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D049449:Luminescence; D051379:Mice; D010957:Plasmids; D013995:Time; D015534:Trans-Activators / Q000235:genetics*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D008156:Luciferases; D015534:Trans-Activators; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; C538943:CLOCK protein, human; C538944:Clock protein, mouse