Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

Nucleic Acids Res. 2012 Mar;40(5):1928-43. doi: 10.1093/nar/gkr978. Epub 2011 Nov 10.

Abstract

The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenovirus E1A Proteins / metabolism*
  • Binding Sites
  • Breast Neoplasms / metabolism
  • Cell Line, Tumor
  • Female
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / chemistry
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • Hypoxia-Inducible Factor-Proline Dioxygenases
  • Procollagen-Proline Dioxygenase / genetics*
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-ets / metabolism
  • Proto-Oncogene Proteins c-fos / metabolism
  • Signal Transduction
  • Transcriptional Activation*
  • Transferrin / genetics
  • p300-CBP Transcription Factors / metabolism

Substances

  • Adenovirus E1A Proteins
  • ETV4 protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-ets
  • Proto-Oncogene Proteins c-fos
  • Transferrin
  • EGLN1 protein, human
  • Procollagen-Proline Dioxygenase
  • Hypoxia-Inducible Factor-Proline Dioxygenases
  • p300-CBP Transcription Factors

Associated data

  • GEO/GSE32385