Poster Topical Area: Vitamins and Minerals
Location: Hall D
Poster Board Number: 501
Objective: Under hypoxic condition copper is required for the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), but not for that of insulin-like growth factor 2 (IGF2), although both genes are regulated by hypoxia-inducible factor-1α (HIF-1α). The study was undertaken to understand how copper regulates the target gene selectivity of HIF-1α by determining the effect of copper on the binding of HIF-1α to target genes.
Methods: Human umbilical vein endothelial cells were treated with hypoxia or CoCl2 to increase HIF-1α accumulation. The hypoxia-responsive element (HRE) bound by HIF-1α in the promoters of BNIP3 and IGF2 were predicted by ChIP-sequencing data and computer analysis, then confirmed by chromatin immunoprecipitation (ChIP) and luciferase reporter assay. A copper chelator tetraethelenepentamine (TEPA) was used to reduce the availability of copper in the nuclei. The endonuclear copper concentration and protein level of HIF-1α were detected by atomic absorption spectrometry and western blot respectively. The binding of HIF-1α to its binding sites in the BNIP3 and IGF2 were detected by ChIP assay, and confirmed by luciferase reporter assay and qRT-PCR detection of mRNA expression.
Results: ChIP-sequencing data and computational analysis revealed three HRE sites in the promoter of BNIP3 and two HRE sites in the IGF2. The functional HRE sites that increased HIF-1α binding under CoCl2 treatment were confirmed as follows: -412/-404 (5'-CCTGCACGT-3') at BNIP3 and -354/-347 (5'-GACGTGAC-3') at IGF2. The reduction of copper concentration by TEPA did not affect the accumulation of HIF-1α induced by hypoxia or CoCl2. Both under hypoxia and CoCl2 treatment, the binding of HIF-1α to the HRE site of BNIP3 was suppressed, but to the HRE site of IGF2 was not affected by copper deficiency.
Conclusion: Copper regulates the target gene selectivity of HIF-1α via at least partially its influence on the binding of HIF-1α to the HRE sites of target genes. This relationship between copper and HIF-1α provides a clinic application potential to target HIF-1 as a therapy of diseases related to hypoxia and abnormal copper metabolism, such as ischemic heart diseases and cancer.
This work was supported by National Science Foundation of China (grants: 81230004 to Y. James Kang and 81600214 to Wenjing Zhang).
Sichuan University West China Hospital
Chengdu, Sichuan, China (People's Republic)