Involvement of Sp1 in Transcriptional Regulation of Atp7a During Hypoxia

Abstract

Genes with GC‐rich promoters were preferentially induced in the rodent intestine during iron deficiency. Moreover, promoters of iron homeostasis related genes (e.g. Dmt1, Dcytb) contain evolutionarily conserved Sp1 binding sites (GC‐rich sequences). The Menkes copper ATPase (Atp7a) gene was strongly induced during iron deficiency, and we previously demonstrated that Atp7a was co‐regulated with iron transport related genes by HIF2α. In this study, we sought to test the role of Sp‐like trans‐acting factors in transcriptional regulation of Atp7a. Phylogenetic footprinting revealed several conserved Sp‐like binding sites in the Atp7a promoter. These sites were mutated in a −224 bp promoter construct driving the luciferase reporter gene. Several of these sites participated in the induction of promoter activity in response to hypoxia in transfected rat intestinal epithelial (IEC‐6) cells. Moreover, phosphorylation of Sp1 increased in IEC‐6 cells exposed to hypoxia; Sp1 is known to be regulated by phosphorylation, which increases DNA binding and trans‐activation properties. Further studies will explore Sp factor interaction with the Atp7a promoter by chromatin immunoprecipitation (ChIP) assay in IEC‐6 cells and in intestine of iron deficient rats. Current data suggest that synergistic interactions between Sp1 (or a related factor) and HIF2α mediate induction of Atp7a gene expression during hypoxia.Grant Funding Source : 1R01 DK074867