Abstract
ABSTRACT Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.
Highlights
In response to hypoxia in animals, adaptive pro cesses take place in cells and tissues that work to maintain oxygen supply and/or increase the effi ciency of its use
Changes in the inten sities of other H4 peaks, relative to the 11306 Da peak, were observed between the hypoxic and Hypoxia mimetics: iron chelators We investigated the changes in histone post-translational modifica tions (PTMs) profiles observed on treatment of cells with the iron chelating drugs, DFO and CP20 (Supplementary Figure S1)
All changes were much less prominent than for cells grown under hypoxia or treated with the iron chelating drugs or DMOG (Figure 2,3, Supplementary Figure S10). These results suggest that the majority of changes in H3.1 PTMs observed under hypoxia or with the other mimics tested are not directly caused by prolyl hydroxylases (PHDs)-mediated hypoxia-inducible factor (HIF) upregulation
Summary
In response to hypoxia in animals, adaptive pro cesses take place in cells and tissues that work to maintain oxygen supply and/or increase the effi ciency of its use. Such processes include erythro poiesis, angiogenesis, and the partial shutdown of high-energy consuming processes[1]. HIF-α and by hindering its interaction with the histone acetyltransferases CBP/p300 Another layer of hypoxic gene regulation is provided by histone post-translational modifica tions (PTMs) [7,8,9]. Many of the enzymes responsible for modifying these PTMs are them selves HIF target genes (e.g. G9a, KDM3A), and their levels, and thereby activity, are regulated by oxygen availability [9,11]. The transcriptional response to hypoxia and its regulation is both com prehensive and highly complex
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