Transcriptional regulation of theDrosophila rfc1gene by the DRE–DREF pathway

Abstract

The DNA replication-related element (DRE) is a common 8-bp sequence (5'-TATCGATA) found in the promoters of many DNA replication-related genes, to which DRE-binding factor (DREF) specifically binds to activate transcription. Replication factor C (RFC) is an essential five-subunit complex in DNA replication, the largest subunit being RFC140. We first identified the gene (rfc1) encoding the Drosophila RFC140 (dRFC140) protein and then isolated a mutant. The phenotypes suggested that the gene is essential for cell-cycle progression, and immunocytochemical studies also indicated a relation between its expression and the cell cycle. The rfc1 gene contains three DRE-like sequences in its 5'-flanking region, one of them perfectly matching DRE and the other two demonstrating a match in seven of eight nucleotides. These sequences were named DRE1 (-63 to -69), DRE2 (-378 to -385), and DRE3 (-1127 to -1134), respectively. Immunostaining of polytene chromosomes in third-instar larvae using anti-DREF sera detected a specific band in 82E2 of 3R chromosome, containing the rfc1 gene region. Band-mobility shift assays using Drosophila Kc cell nuclear extracts revealed that DREF binds to DRE1, -2, and -3 in vitro, and chromatin immunoprecipitation using anti-DREF IgG confirmed that this occurs in vivo. Luciferase transient expression assays in S2 cells further suggested that DREs in the rfc1 promoter are involved in transcriptional regulation of the gene. Moreover, rfc1 promoter activity was reduced by 38% in DREF double-stranded RNA-treated S2 cells. These results indicate that DREF positively regulates the rfc1 promoter.