A novel E4BP4 element drives circadian expression of mPeriod2.
Period2 (Per2) is an essential component of the mammalian clock mechanism and robust circadian expression of Per2 is essential for the maintenance of circadian rhythms. Although recent studies have shown that the circadian E2 enhancer (a non-canonical E-box) accounts for most of the circadian transcriptional drive of mPer2, little is known about the other cis-elements of mPer2 oscillatory transcription. Here, we examined the contribution of E4BP4 to Per2 mRNA oscillation in the cell-autonomous clock. Knockdown experiments of E4BP4 in both Northern blots and real-time luciferase assays suggested that endogenous E4BP4 negatively regulates Per2 mRNA oscillation. Sequence analysis revealed two putative E4BP4-binding sites (termed A-site and B-site) on mammalian Per2 promoter regions. Luciferase assays with mutant constructs showed that a novel E4BP4-binding site (B-site) is responsible for E4BP4-mediated transcriptional repression of Per2. Furthermore, chromatin immunoprecipitation assays in vivo showed that the peak of E4BP4 binding to the B-site on the Per2 promoter almost matched the trough of Per2 mRNA expression. Importantly, real-time luciferase assays showed that the B-site in addition to the E2 enhancer is required for robust circadian expression of Per2 in the cell-autonomous clock. These findings indicated that E4BP4 is required for the negative regulation of mammalian circadian clocks.
Ohno
Tomoya
T
Clock Cell Biology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.
Onishi
Yoshiaki
Y
Ishida
Norio
N
D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002940:Circadian Rhythm / Q000235:genetics*; D015536:Down-Regulation; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D056950:Period Circadian Proteins; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D018797:Cell Cycle Proteins; C497441:Nfil3 protein, mouse; D009687:Nuclear Proteins; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors