Deletion of an intronic HIF-2α binding site suppresses hypoxia-induced WT1 expression

Biochim Biophys Acta Gene Regul Mech. 2019 Jan;1862(1):71-83. doi: 10.1016/j.bbagrm.2018.11.003. Epub 2018 Nov 20.


Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1, marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma.

Keywords: Chromosome conformation capture; Gene expression; Hypoxia; Neuroblastoma; Transcription factor; Tumor suppressor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Basic Helix-Loop-Helix Transcription Factors / physiology
  • Cell Line, Tumor
  • Chromatin Immunoprecipitation
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Hypoxia / metabolism
  • Introns
  • Neuroblastoma / pathology*
  • Promoter Regions, Genetic
  • Response Elements
  • Transcriptional Activation
  • WT1 Proteins / genetics
  • WT1 Proteins / metabolism*


  • Basic Helix-Loop-Helix Transcription Factors
  • WT1 Proteins
  • WT1 protein, human
  • endothelial PAS domain-containing protein 1