Identification of the negative regulatory elements for the mPer2 transcription

Abstract

A mouse gene, mPer2, having all the properties expected of a circadian clock gene, was reported recently. This gene is expressed in a circadian pattern in the suprachiasmatic nucleus(SCN), the mammalian circadian center. The mPer2 maintains this pattern of circadian expression in the constant darkness and can be entrained to a new light/dark cycle. The mPer2 is assumed to be a key molecule in the regulation and functioning of the mammalian circadian clock, which is based on the oscillation generated by a transcription-(post) translation feedback loop of a set of clock genes. To investigate the mechanism underlying the complex regulation of mPer2 expression, we isolated and characterized the promoter region of the mPer2. To gain insight into the regulation of the mPer2, we functionally analyzed 7,748-bp BamH I fragment (containing 1,158-bp 5`` upstream region and 6,590-bp intron 1) and 3,280-bp Xba I fragment (containing 1,158-bp 5`` upstream region and 2,122-bp intron 1) of the mPer2. These regions lack a TATA box, but has one E-box in the first intron. Transient transfection assays using a mPer2 promoter-luciferase reporter gene in human liver-derived HepG2 cell line revealed that potential negative regulatory element exists in the 4,468-bp intron 1. Transient transfection assays of progressively deleted fragments of the 4,468-bp region were used to identify two regions from +3082 to +3239 (NRI: negative regulation 1) and from +5743 to +5908 (NR2 : negative regulation 2) that contain putative negative regulatory elements. Binding affinities of DNA-protein interactions were defined by electrophoretic mobility shift assays and the protected nucleotide positions were determined by DNase I footprinting. Site-directed deletion mutagenesis of those protected regions revealed nucleotides at positions from +3165 to +3185 (NR1D2) and from +5795 to +5825 (NR2D1) as being necessary for negative control. EMSA and SDS-PAGE analysis using DNA sequences containing +3165 to +...