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Copper manipulation of target gene selectivity of hypoxia‐inducible factor‐1
Author(s) -
Zhang Wenjing,
Liu Xiaojuan,
Wu Zhijuan,
Kang Y. James
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.301.1
Subject(s) - chromatin immunoprecipitation , copper , chemistry , reporter gene , hypoxia inducible factors , microbiology and biotechnology , luciferase , umbilical vein , gene , gene expression , messenger rna , promoter , biology , biochemistry , transfection , in vitro , organic chemistry
Previous studies demonstrated that under hypoxic conditions 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 (IGF‐2), although both genes are regulated by hypoxia‐inducible factor‐1 (HIF‐1). The present study was undertaken to understand the mechanism by which copper regulates the target gene selectivity of HIF‐1. Human umbilical vein endothelial cells (HUVECs) were treated with CoCl 2 to increase HIF‐1α accumulation in the cells. The binding of HIF‐1α to hypoxia‐responsive elements (HREs) of BNIP3 or IGF‐2 under copper deprivation was examined. Computational analysis revealed three HRE sites in the promoter regions for both BNIP3 and IGF‐2 genes. Chromatin Immunoprecipitation (ChIP) assay and luciferase reporter assay identified that the binding of HIF‐1α to HRE (−412/−403) of BNIP3 and HRE (−355/−347) of IGF‐2 was increased after CoCl 2 treatment. A copper chelator tetraethelenepentamine (TEPA) was used to reduce the availability of copper in the nucleus and CuSO 4 was used to replenish copper in the presence of TEPA. Reduction in nuclear copper concentrations by TEPA did not affect the HIF‐1α accumulation induced by CoCl 2 . ChIP assay revealed that the binding of HIF‐1α to the HRE (−412/−403) site of BNIP3 was suppressed, but to the HRE (−355/−347) site of IGF‐2 was not affected under copper deficiency. Luciferase reporter assay and detection of mRNA expression by real‐time RT‐PCR both confirmed the result obtained from ChIP assay. Thus, our data demonstrate that copper regulates the target gene selectivity of HIF‐1α via at least partially its effect on the binding of HIF‐1α to the HRE site of target genes. Support or Funding Information This work was supported by National Science Foundation of China (grant number: 81230004 to Y. J. Kang) 1HRE sites in the promoter of BNIP3 and IGF‐2.(A) The potential HRE sites of BNIP3 and IGF‐2 was predicted by computer analysis according to the core consensuses sequence of HRE. (B and C) ChIP assay showing binding of HIF‐1α to the HREs of BNIP3 (B) and HREs of IGF‐2 (C). (D and E) Luciferase reporter assay confirmed the transcriptional activity of HREs found in (B and C). HUVECs were treated with CoCl 2 for 16 h at a final concentration of 100 μM. *, p < 0.05.2Copper depletion does not affect the protein level of HIF‐1α in nucleus under CoCl2 treatment.(A) Nuclear extract. (B) Copper concentrations in the nucleus of different groups were detected by AAS. (C) HIF‐1α protein level in nucleus after different treatment. (D) Quantitation analysis of Western bolt of HIF‐1α protein level. HUVECs were treated with 50 μM TEPA for 6 h, and/or 100 μM CoCl 2 for 16 h, 50 μM CuSO 4 for 4 h. *, p < 0.05.3Copper deprivation suppressed HIF‐1α binding to the HRE of BNIP3, but not to that of IGF‐2.(A and D) Binding of HIF‐1α to HRE of BNIP3 (A) or HRE of IGF‐2 (D) after different treatment of HUVECs were detected by ChIP assay. (B and E) Luciferase reporter assay showing the transcriptional activity of HRE of BNIP3 (B) or HRE of IGF‐2 (E). (C and F) Real‐Time RT‐PCR detected the mRNA expression of BNIP3 (C) and IGF‐2 (F) after different treatment. *, p < 0.05.