Nucleic Acids Research Advance Access originally published online on December 19, 2006
Nucleic Acids Research 2007 35(2):648-655; doi:10.1093/nar/gkl868
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Nucleic Acids Research, 2007, Vol. 35, No. 2 648-655
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
A novel E4BP4 element drives circadian expression of mPeriod2
1 Clock Cell Biology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST) Tsukuba 305-8566, Japan 2 Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba 305-8576, Japan
*To whom correspondence should be addressed at Clock Cell Biology, National Institute of Advanced Industrial Science and Technology, Central 6-5, 1-1-1 Higashi, Tsukuba 305-8566, Japan. Tel: +81 298 61 6053; Fax: +81 298 61 9499; Email: n.ishida{at}aist.go.jp
Received August 23, 2006. Revised October 3, 2006. Accepted October 4, 2006.
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.
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