Dataset Information

Oxygen-dependent changes in HIF binding partners and post-translational modifications regulate stability and transcriptional activity C4PR_LIV

ABSTRACT: Adaption of cells to low oxygen environments is an essential process mediated in part by the Hypoxia Inducible Factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs, and consequently the hypoxic response, are regulated by post-translational modification (PTM) and changes in biomolecular interactions. However, our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies, with validation typically having been conducted by in cellulo fragment expression and hypoxia mimicking drugs. Consequently, we still lack an understanding of true oxygen deprivation signaling via HIFα. Using an immunoprecipitation-based, mass spectrometry approach, we characterize the regulation of in cellulo expressed full-length HIF-1α and HIF-2α, in terms of both PTM and binding partners, in response to normoxia (21% oxygen) and hypoxia (1% oxygen). These studies revealed that a change in oxygen tension significantly alters the complexity and composition of HIF-α protein interaction networks, with HIF-2α in particular having an extended hypoxia-induced interactome, most notably with mitochondrial-associated proteins. Both HIFα isoforms are heavily covalently modified: we define ~40 different sites of PTM on each of HIF-1α and HIF-2α, comprising 13 different PTM types, including multiple cysteine modifications and a highly unusual phosphocysteine. Over 80% of the PTMs identified are novel, and approximately half exhibit oxygen-dependency under these conditions. Combined with domain and evolutionary analysis of >225 vertebrate species, we validate Ser31 phosphorylation on HIF-1α as a regulator of transcription, and propose functional roles for Thr406, Thr528 and Ser581 on HIF-2α.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: leonard daly

LAB HEAD: Professor Claire

PROVIDER: PXD022479 | Pride | 2026-03-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	LD_181013_HIF2-21_-Digest-rep1-C-MAX.mgf	Mgf
	LD_181013_HIF2-21_-Digest-rep1-C-MAX.raw	Raw
	LD_181013_HIF2-21_-Digest-rep1-E-MAX.mgf	Mgf
	LD_181013_HIF2-21_-Digest-rep1-E-MAX.raw	Raw
	LD_181013_HIF2-21_-Digest-rep1-T-MAX.mgf	Mgf

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Publications

Oxygen-dependent changes in binding partners and post-translational modifications regulate the abundance and activity of HIF-1α/2α.

Daly Leonard A LA Brownridge Philip J PJ Batie Michael M Rocha Sonia S Sée Violaine V Eyers Claire E CE

Science signaling 20210720 692

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs-and consequently, the hypoxic response-are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies typically validated in fragment-expressing ...[more]

PMID: 34285132

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Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response. Global gene expression profiling in colon RNAs isolated from 7-week-old Hif-2αF/F (n=6, Shah 007) and Hif-2αΔIE (n=5, Shah 008).