Cloning, characterization and analysis of the 5′ regulatory region of zebrafish xpd gene

The gene XPD/ERCC2 encodes an ATP-dependent 5′–3′ helicase of 760 amino acids. There are few transcription factors known to be involved in the regulation of XPD, and these include the hypoxia-inducible factor-1α (HIF-1α), p53, hepatitis B virus x protein (HBx) and specificity protein 1 (Sp1).To identify functional elements of xpd gene in fish we employed a comparative genomic approach. In our in silico attempt to identify cis-regulatory transcription factor binding sites (TFBSs) conserved among the xpd promoter sequences from five available fish species, we verified that in some cases the spatial disposition of the putative TFBSs was not maintained, but they were present in all the sequences, allowing the prediction of conserved regulatory elements within the promoter sequences analysed. We also investigated the transcriptional regulation of the zebrafish xpd gene. Our in vitro transient transfection-reporter analysis identified a region in the zebrafish xpd gene responsible for xpd regulation, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Moreover, we found two p53 binding sites in the xpd promoter and upon co-transfection assays we observed a repression due to p53. This provides the first identification and characterization of promoter regions that regulate the transcription of the zebrafish xpd gene, and offers insights into the regulation of this gene by p53.Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the basic information to explore and better understand the regulation and expression of XPD.