The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells.
To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.
Yang
Fang
F
Department of Physiology, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan.
Nakajima
Yoshihiro
Y
Kumagai
Megumi
M
Ohmiya
Yoshihiro
Y
Ikeda
Masaaki
M
D056930:ARNTL Transcription Factors; D000595:Amino Acid Sequence; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D056926:CLOCK Proteins; D002940:Circadian Rhythm / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000378:metabolism*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D004264:DNA Topoisomerases, Type I; D008156:Luciferases; D015534:Trans-Activators; C538961:Bmal1 protein, mouse; C538944:Clock protein, mouse