Copper Regulation of HIF-1 Transcription Activity [ChIP-seq]

Copper (Cu) regulates hypoxia-inducible factor-1 (HIF-1) transcription activity by affecting the selectivity of HIF-1a targeting to the promoters of the affected genes. Here, we made an effort to provide a comprehensive understanding of Cu regulation of the selectivity of HIF-1a targeting across genome. We used tetraethylenepentamine (TEPA), a Cu selective chelator, to reduce Cu content in the cells. In hypoxia, we conducted chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) to globally map the binding sites of HIF-1a, Pol ? (RNA polymerase?) and histone H3K27ac. We also performed RNA-sequencing (RNA-seq) in EA.hy926 cells under hypoxia (1% O2) with or without Cu depression to determine the profile of mRNA expression. Our analyses identified 3197 HIF-1a binding sites under hypoxia. Cu depression by TEPA reduced 1820 binding sites from the 3197, but induced additional 274 new binding sites. We analyzed these binding sites in the promoter and putative enhancer regions, coupled with their mRNA expression profiles, and found 281 Cu-dependent and 10 Cu-independent HIF-1a target genes. We found that the core bases “GGAA” and “TTCC” constituted the critical motifs for the binding sites of Cu-dependent genes. This study thus revealed that Cu, by affecting the binding of HIF-1a to the critical motifs in the promoter and putative enhancer regions of HIF-1 regulated genes, leads to the selectivity of HIF-1 regulated expression of Cu-dependent genes. Overall design: Analysis of DNA-binding profiling of HIF-1a, Pol ? and H3K27ac in TEPA treated and -untreated cells. .