Nucleic Acids Research Advance Access originally published online on February 5, 2008
Nucleic Acids Research 2008 36(4):e23; doi:10.1093/nar/gkn018
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Nucleic Acids Research, 2008, Vol. 36, No. 4 e23
© 2008 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.
Methods Online |
Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector-based strategy
1Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, 2COE Unit of Circadian System, Nagoya University Graduate School of Science, Furo-cho, Chikusa-ku, Nagoya 464-8602 and 3Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minaminachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
*To whom correspondence should be addressed. Tel: +81 6 6879 3124; Fax: +81 6 6879 3129; Email: kyagita{at}anat1.med.osaka-u.ac.jp
Received October 18, 2007. Revised December 27, 2007. Accepted January 12, 2008.
In mammals, the expression of 5–10% of genes occurs with circadian fluctuation in various organs and tissues. This cyclic transcription is thought to be directly or indirectly regulated through circadian transcriptional/translational feedback loops consisting of a set of clock genes. Among the clock genes in mammals, expression of the Dbp mRNA robustly oscillates both in vivo and in culture cells. Here, we present circadian enhancer detection strategy using prokaryotic transposon system. The mDbp promoter drives reporter gene expression in robust circadian cycles in rat-1 fibroblasts. To identify the circadian enhancer generating this robust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitro transposition. Using this system, we identified a strong circadian enhancer region containing the CATGTG sequence in the 5' flanking region of the mDbp gene; this enhancer region is critical for the ability of the mDbp promoter to drive robust oscillation in living cells. This enhancer is classified as a CANNTG type non-canonical E-box. These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression. Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.
Present address: Keigo Nishii, Kowa Company, Tokyo Laboratory, Noguchi-cho 2-17-43, Higashimurayama, Tokyo 189-0022, Japan.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.