Dissecting the genome-wide dynamic responses of the HIF transcriptional system

Hypoxia is an important stimulus for physiological processes such as development and adaptation to high altitude. It is also important in the pathology of many human diseases such as cancer, cardiovascular and diabetic disorders. The sequence-specific, heterodimeric transcription factor HIF (Hypoxia Inducible Factor) has been shown to play a key role in cellular adaptation to hypoxia. This work aimed to better understand the contribution of HIF to hypoxia-inducible gene expression through determining the range of HIF-target genes (both direct and indirect) by sequencing the whole transcriptome (RNA-seq) in HKC-8 wild type and a series of mutant cells (in which one or more components of the HIF-VHL pathway are inactivated). The work went on to examine the pan-genomic binding distribution of HIF polypeptides in response to different severity and duration of hypoxia and in cells bearing introduced mutations in the von Hippel- Lindau tumour suppressor (pVHL), the chromatin remodelling component BRG1-associated factor 180 (BAF180, encoded by PBRM1 gene), and the HIF asparaginyl hydroxylase, FIH (Factor Inhibiting HIF). This was performed using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), and was subjected to a range of quantitative and qualitative analyses. Analyses of hypoxic gene expression profiles demonstrated that the vast majority of hypoxia-dependent genes in HKC-8 cells were regulated by the canonical HIF pathway i.e. via dimerisation of either HIF-1α or HIF-2α with HIF-1β. There was little evidence for any HIFindependent contributions to gene expression in hypoxia under the conditions analysed. Analyses of hypoxia-dependent genes in HIF mutant cells revealed some persistent regulation by hypoxia that was less than in the wild type cells, and also a set of genes with a lipid metabolism signature that were more regulated by hypoxia in HIF-deficient cells. This observation might represent an adaptive response to enable cell survival in the absence of a functional HIF response. Having confirmed the critical role of the canonical HIF pathway in regulating hypoxic gene expression in the wild type cells, I then went on to study the canonical HIF DNA-binding profiles across the genome in response to various physiological and pathophysiological conditions as outlined above. It was hypothesised that these conditions might alter the profile of HIF binding as a result of changes in HIF protein abundance, or transcriptional function, as well as by the potential rearrangement of chromatin accessibility. Highly stringent canonical HIF binding sites were defined in each condition, and quantitative analyses of ChIP-seq signals were performed. Overall, the HIF binding signal at any one site was broadly proportional to the total binding for that HIF protein, with HIF-1α and HIF-2α behaving largely independently of each other and showing little evidence for redistribution of HIF DNA-binding profiles in response to different severity and duration of hypoxia, or in the mutant cells. In summary, this study demonstrates that the canonical HIF pathway is central to hypoxia-dependent gene regulation. HIF DNA-binding occurs over a quantitative continuum, which appears to be qualitatively unaltered under the conditions examined, with the applied interventions only altering the magnitude of HIF binding at the existing sites, rather than qualitatively generating new sites.