Regulation of chromatin accessibility by hypoxia and HIF.

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) co-ordinate a transcriptional programme required for survival and adaptation to hypoxia. However, the effects of HIF on chromatin accessibility are currently unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility are enriched at a subset hypoxia transcriptionally responsive genes, agreeing with previous data using other models. We show for the first time that hypoxia inducible changes in chromatin accessibility across the genome are predominantly HIF dependent, rapidly reversible upon reoxygenation and partially mimicked by HIF-α stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that HIF is central to chromatin accessibility alterations in hypoxia, and has implications for our understanding of gene expression regulation by hypoxia and HIF.

Von Hippel-Lindau (VHL) small-molecule inhibitor binding increases stability and intracellular levels of VHL protein.

Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.

Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia.

Hypoxia-reduction in oxygen availability-plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

Background: Hypoxia-inducible factor (HIF) transcription factors are well known to control the transcriptional response to hypoxia. Given the importance of cellular response to hypoxia, a number of pharmacological agents to interfere with this pathway have been developed and entered pre-clinical or clinical trial phases. However, how similar or divergent the transcriptional response elicited by different points of interference in cells is currently unknown. Methods: We performed RNA-sequencing to analyse the similarities and differences of transcriptional response in HeLa cells treated with hypoxia or chemical agents that stabilise HIF by inhibiting components of the hypoxia signalling pathway - prolyl hydroxylase (PHD) inhibitor or von Hippel-Lindau (VHL) inhibitor. Results: This analysis revealed that hypoxia produces the highest changes in gene transcription, with activation and repression of genes being in large numbers. Treatment with the PHD inhibitor IOX2 or the VHL inhibitor VH032 led mostly to gene activation, majorly via a HIF-dependent manner. These results were also confirmed by qRT-PCR using more specific and/or efficient inhibitors, FG-4592 (PHDs) and VH298 (VHL). Conclusion: PHD inhibition and VHL inhibition mimic gene activation promoted by hypoxia via a HIF-dependent manner. However, gene repression is mostly associated with the hypoxia response and not common to the response elicited by inhibitors of the pathway.

Hypoxia induces rapid changes to histone methylation and reprograms chromatin.

Oxygen is essential for the life of most multicellular organisms. Cells possess enzymes called molecular dioxygenases that depend on oxygen for activity. A subclass of molecular dioxygenases is the histone demethylase enzymes, which are characterized by the presence of a Jumanji-C (JmjC) domain. Hypoxia can alter chromatin, but whether this is a direct effect on JmjC-histone demethylases or due to other mechanisms is unknown. Here, we report that hypoxia induces a rapid and hypoxia-inducible factor-independent induction of histone methylation in a range of human cultured cells. Genomic locations of histone-3 lysine-4 trimethylation (H3K4me3) and H3K36me3 after a brief exposure of cultured cells to hypoxia predict the cell's transcriptional response several hours later. We show that inactivation of one of the JmjC-containing enzymes, lysine demethylase 5A (KDM5A), mimics hypoxia-induced cellular responses. These results demonstrate that oxygen sensing by chromatin occurs via JmjC-histone demethylase inhibition.

Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298).

The von Hippel-Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein-protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure-activity relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways and new VHL ligands for next-generation PROTACs.

Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition.

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.