Hypoxic regulation of chromatin and gene transcription

Thesis

The hypoxia-inducible transcription factor (HIF) is a well-described regulator of the cellular response to low oxygen (hypoxia). More recently, there has been published evidence that hypoxia influences gene expression through epigenetic mechanisms. I have further investigated these lesser-understood epigenetic effects of hypoxia, specifically regarding histone methylation. Many histone lysine demethylases (KDMs) are inactivated in hypoxia, leading to global histone hypermethylation. Whether this occurs with greater propensity at certain gene loci is not well understood. To examine hypoxic histone hypermethylation positionally across the genome, I performed ChIP-sequencing for four histone marks in two cancer cell lines (PC3, HCT116), incubated in normoxia (21% O2) or hypoxia (0.5% O2 for 16 hours). I assayed marks typically associated with both active (H3K4me3, H3K36me3) and repressed (H3K9me3, H3K27me3) transcription. For the first time in the hypoxia field, I employed a spike-in normalisation strategy to enable accurate quantification of global hypoxic changes. To relate histone methylation changes to gene expression, I performed mRNA-sequencing, again normalised to a spike-in control. To better elucidate HIF’s role in producing this effect, we performed HIF-1α, HIF-2α, and HIF-1β ChIP-sequencing and repeated experiments in PC3 cells following CRISPR Cas9-mediated HIF-1β deletion.

In hypoxia, genes were upregulated and downregulated in roughly equal numbers. However, I showed for the first time that hypoxia induces histone methylation broadly across all gene loci. This was true for both the ‘active’ and ‘repressive’ modifications and, importantly, revealed that histone methylation changes do not directly instruct hypoxic changes in gene expression. While the induction of ‘active’ marks moderately correlated with hypoxic changes in gene expression, the induction of ‘repressive’ marks bore little relationship to the direction and magnitude of change in expression. Notably, HIF target genes were enriched amongst the genes at which H3K4me3 and H3K36me3 increased the most, demonstrating an element of locus-specificity. After blocking the HIF transcriptional pathway by HIF-1β knock-out, the global induction of histone methylation persisted across the genome. However, the pronounced effect at HIF target genes was lost. Therefore, HIF is not required for hypoxia-induced global histone hypermethylation but is necessary for the augmented effect at its target genes.

Authors: Kindrick, JD

• DOI: 10.5287/ora-14kqbz7dz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: gene transcription and epigenetic in cancer; histone methylation in hypoxia
• Subjects: Biology