Role of DREF in transcriptional regulation of the Drosophila p53 gene

Abstract

The tumor suppressor protein p53 has a critical role in safeguarding the integrity of the genome. Its functions are well understood but factors responsible for the transcriptional regulation of the p53 gene are almost entirely unknown. The DNA replication-related element (DRE)/ DNA replication-related element-binding factor (DREF) transcriptional regulatory system is established as a master key to cell proliferation in Drosophila. DREF binds specifically to DRE sequences in the Drosophila p53 (dmp53) gene promoter as shown using anti-DREF antibodies in chromatin immunoprecipitation assays. Furthermore, a rough eye phenotype because of overexpression of DREF in Drosophila eye imaginal disks could be suppressed by half dose reduction of the dmp53 gene. In addition, the level of mRNA of dmp53 was decreased in DREF-knockdown cells and transient expression of the luciferase gene under control of the wild-type dmp53 gene promoter showed strong promoter activity in S2 cells, but this was almost completely abrogated with a DRE-mutated promoter. Requirement of DREs for dmp53 promoter activity was further confirmed by anti-b-galactosidase antibody-staining of various tissues from transgenic flies carrying dmp53 promoter-lacZ fusion genes. These results indicate that DREF is necessary for dmp53 gene promoter activity.

Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle

“…4,[6][7][8][9][10][11][12] ChIP-seq data confirms enrichment of DREF at these loci, including DNA polymerase I, p53 and warts (Fig. 2B).…”

“…12 Overexpression of DREF in the eye imaginal disc causes excessive cell proliferation, resulting in a rough eye phenotype. Expression of a dominant-negative form of DREF protein in larval salivary glands shows defective endo-reduplication.…”

NF-Y transcriptionally regulates the Drosophila p53 gene

“…1A). Interestingly, the DRE1 (5'-TATCGATT) element of the DREF regulated pathway (Matsukage et al, 2008;Trong-Tue et al, 2010), partially overlaps with site 1b. In addition, the site 1a CCAAT box shows high conservation among Drosophila species, but not with the other sites (Fig.…”

“…Then the protein-DNA crosslinks were reversed by heating at 65°C for 4 h. After deproteinization with proteinase K, DNA was recovered by phenol-chloroform extraction and ethanol precipitation. Immunoprecipitated DNA fragments were detected by quantitative real-time PCR using SYBR Green I (Takara, Kyoto, Japan) and the Applied Biosystems 7500 real-time PCR system (Phuong Thao et al, 2006;Ida et al, 2007;Trong-Tue et al, 2010). Primer pairs for PCR were as described above.…”

“…Since controlling p53 gene expression is necessary to improve efficiency of establishment of iPS cells (Kawamura et al, 2009), understanding expression control mechanisms is increasingly important. We have reported that the DNA replication-related element (DRE) and DRE-binding factor (DREF) play important roles in dmp53 gene promoter activity (Trong-Tue et al, 2010), but involvement of other transcription factor(s) is suggested by the only partial decrease of dmp53 mRNA levels on knockdown of DREF. In the present study, we have identified four potential nuclear factor Y (NF-Y)-binding sites located in the 5'-flanking region of the dmp53 gene.…”