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.
Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, California 92121, USA.
Trey K
TK
Sato
Rikuhiro G
RG
Yamada
Hideki
H
Ukai
Julie E
JE
Baggs
Loren J
LJ
Miraglia
Tetsuya J
TJ
Kobayashi
David K
DK
Welsh
Steve A
SA
Kay
Hiroki R
HR
Ueda
John B
JB
Hogenesch
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