Roles of HIF and 2-Oxoglutarate dependent enzymes 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 (2OGDs). 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-OG dioxyenases in the control of gene expression in response to hypoxia and their relevance to human cancers.

. Regulation of HIF levels and activity in normoxia and hypoxia. Under normal oxygen conditions, A, normoxia, HIF-α is constantly hydroxylated by PHDs and FIH. PHD-mediated hydroxylation increases binding affinity with the tumour suppressor VHL, which promotes ubiquitination and degradation by the proteosome. As oxygen levels decrease, in mild hypoxia B, PHDs are inhibited, HIF-α is stabilised, though still hydroxylated by FIH, binds to HIF-1β and is able to induce transcription of certain target genes. With further reduction in oxygen levels, in severe hypoxia C, FIH is also inhibited and HIF is able to become fully active by the recruitment of co-activators such as p300.
In this review, we highlight the importance of oxygen sensing in coordinating an efficient response to hypoxia. We discuss the relevance of HIF transcription factors, and roles of 2-OGDs in controlling almost all aspects of gene expression from chromatin structure, to transcription, translation and post-translational modifications.  Hypoxia-induced changes to transcription are largely mediated by HIF As mentioned earlier HIFs are the master regulators of gene transcriptional changes in hypoxia. HIFs are known to bind to Hypoxia Responsive Elements (HREs) (5-(A/G)CGTG-3) of DNA [8].

Box1. 2-OGDs and their reported affinities for oxygen from in vitro assays
Since the identification of the Erythropoietin gene (EPO) as the first hypoxia responsive HIF target gene, we now know HIF controls a wide range targets, influencing numerous biological processes, antioxidant enzymes [28,29], transcription factors [30], heat shock proteins, S100 family proteins [24],

Chromatin regulation in hypoxia
Central to the hypoxia response is the activation of a dynamic transcriptional programme. HIF transcription factors are the primary mediators of hypoxia induced gene transcriptional changes (reviewed in [8]). Further to HIF stabilisation and activation under low oxygen tensions, the chromatin landscape also plays a complex role in co-ordinating hypoxia inducible changes to gene transcription.
Most aspects of chromatin regulation are altered in response to low oxygen, including histone methylation and acetylation, DNA methylation, actions of chromatin remodeller complexes and non coding RNAs, histone eviction and incorporation of histone variants, and chromatin accessibility (reviewed in [11][12][13]). However, this is still a vastly unexplored aspect of the hypoxia response. As   showed that severe hypoxia (0.5% oxygen) in human and murine cells and tumour hypoxia in multiple human tumours causes DNA hypermethylation at gene promoters correlating with gene silencing at a subset of hypoxia repressed genes and gene silencing linked to hypoxia associated tumour progression. DNA hypermethylation was attributed to oxygen dependent reduction in TET1 and TET2 activity in hypoxia, with a 50% reduction in activity observed at 0.3% oxygen for TET1 and 0.5% for TET2 in vitro. Thus, TET1 and TET2 may be characterised as tumour oxygen sensors, and depending on the context of oxygen deprivation, may remain active in hypoxia environments or display inhibition.
However, more work is needed to establish the oxygen dependence of TET activity in cells and in vivo and the physiological contexts in which TETs can sense changes in oxygen availability as well as the consequences this has for DNA methylation, gene transcription and cellular responses. Indeed, the seemingly contradictory roles for TETs in hypoxia from studies to date may be dependent on the different cell models used and timing/severity of hypoxic stimulus. between oxygen availability and chromatin regulation and future work on oxygen sensing by chromatin will be essential in better hypoxia driven processes.

Effects of hypoxia on protein levels
Protein levels and function are key aspects to achieve the correct hypoxia response. Although transcription is important, mechanisms controlling protein levels and function supersede any change in transcriptional output. In hypoxia, mechanisms exist that control translation but also posttranslation aspects of protein function.
Translation is globally repressed in response to hypoxia In addition to the regulation of gene transcription in hypoxia, gene expression is also controlled through regulation of translation ( Figure 5). The cellular response to hypoxia includes a reduction in the energy demands of the cell due to limited ATP production through oxidative phosphorylation. This adaptation results in a reversible global decrease in energy-expensive protein synthesis (reviewed in [2]). This inhibition of translation is a highly regulated response to low oxygen levels preceding ATP depletion (reviewed in [2]). Elongation is also regulated through mTOR, as well as AMPK through its inhibition of RPS6KB1, for in hypoxia eEF2K is not inhibited, which allows its phosphorylation and inhibition of eEF2. PHD2 can also hydroxylate eEF2K, which in normoxia causes its disassociation from calmodulin, decreasing its autophosphorylation. Termination is regulated by JMJD4-mediated hydroxylation of eRF1 which is required for termination. Selective translation of genes in hypoxia is regulated by UTR sequences such as IRES and uORFs, which allow increased translation specifically in hypoxia. RNA binding proteins can bind to various parts of the mRNA and result in different regulatory outcomes. Hydroxylation of splicing regulatory (SR) proteins results in differential splicing or exon choice, such as skipping the prevents binding with eIF2B for exchange of GDP for GTP, therefore remaining in an inactive state and preventing subsequent rounds of translation from the mRNA [67]. eIF2α normally recruits the initiator aminoacylated tRNA to the 40S ribosome, thus limiting global initiation of translation. This second mechanism is independent of HIF and as of yet has not been linked to any 2-OGD.
Inhibition of translation is also regulated at the stage of polypeptide elongation. Elongation is inhibited by phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) at T56 by eEF2 Kinase (eEF2K) [68]. This process has been shown to be dependent on mTOR and 5'-AMP-activated protein kinase catalytic subunit alpha-1 (AMPK) (gene name PRKAA1) [69,70] Interestingly, eEF2 kinase (eEF2K) is also regulated by hydroxylation by PHD2 at P98 in an oxygen-dependent manner [71]. In hypoxia, when PHD2 inhibited, eEF2K activity is induced.
In and (RPL8), respectively. The hydroxylation occurs at residues close to the peptidyl transfer centre, thereby increasing translation efficiency [74]. RIOX1 and RIOX2 transcription is reduced in hypoxia [72,74]. Furthermore, RPL8 hydroxylation is also reduced in hypoxia [74]. However, it is not yet known whether these enzymes are inhibited by low oxygen levels, or lower hydroxylation is solely due to lower transcription. Additionally, hydroxylation of 40S Ribosomal Protein S23 (RPS23) by the 2-OGD, 2-Oxoglutarate And Iron Dependent Oxygenase Domain Containing 1 (OGFOD1), is required for efficient translation [75]. OGFOD1 transcription is also decreased in hypoxia, but the enzyme remains Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 November 2020 doi:10.20944/preprints202011.0651.v1 mostly active even in acute hypoxia [76], suggesting this mechanism is not through direct 2-OGD oxygen sensing.
Efficient decoding of the mRNA during translation requires the JmjC hydroxylase, AlkB Homolog 8, TRNA Methyltransferase, ALKBH8, which hydroxylates tRNA at the wobble position [77,78]. This 2-OGD has yet to be linked to hypoxia, though it would be interesting to investigate its oxygen sensitivity. Finally, lysyl hydroxylation of eukaryotic release factor 1 (eRF1) by the JmjC hydroxylase Jumonji Domain Containing 4 (JMJD4) is required for proper termination of translation [79], although its activity is not significantly inhibited in hypoxia.
As one of the most widely studied PTMs, the phosphorylation on some transcriptional factors and regulators has been found to be changed under various hypoxic conditions, CAMP Responsive Element Binding Protein 1CREB1, NFKB Inhibitor Alpha (NFKBIA), a regulator of NF-κB, and HIF (reviewed in [89]). More recently, through the analysis of phospho-proteomics in renal clear cell carcinoma cells under VHL-independent hypoxic responses, up-regulation of known biomarkers of RCC and signalling adaptor were found. Meanwhile, such hypoxic responses decreased the phosphorylation on intracellular Carbonic Anhydrase 2 (CA2), which might be an unusual way to control the CA2 expression and enhance the activity of the NFκB pathway, resulting in loss of VHL [82].
In recent years, non-HIF targets have been identified to be hydroxylated on prolines by PHDs (reviewed in [13]), resulting in their degradation and/or changes to downstream activity including Centrosomal Protein 192 (CEP192) [90] and Forkhead Box O3 (FOXO3) [91] by PHD1, Actin Beta (ACTB) by PHD3 [92], and AKT Serine/Threonine Kinase 1 (AKT1) by PHD2 [93]. Interestingly, a new study indicated that prolyl-hydroxylation could be crucial for GMGC kinase activation [94]. This could imply an intricate interplay between these two types of PTMs, suggesting yet another role for oxygendependent signalling in the cell.
Other common PTMs have also been found to responding hypoxia in their own ways.
The deSUMOylation of Transcription Factor AP-2 Alpha (TFAP2A), which is known to interact with HIF-1, could enhance the transcriptional activity of HIF-1 under hypoxic conditions [84]. Hypoxia could increase the NAD+-sensitive Sirtuin 3 (SIRT3) activity, that deacetylates key metabolic enzymes and significantly changes the acetylation pattern within the mitochondria. This results in reduced mitochondrial oxidative capacity to match the lowered oxygen availability [85]. In cancer cells, HIF-1α Thus, unbiased proteomic studies on novel PTMs sites [80,96], system-wide analysis of PHDs substrates other than HIF-α [81] and crosstalk of PTMs on PHDs targets in response to hypoxia are now emerging.
Other potential roles of JmjC 2-OGDs in the hypoxia response Hydroxylase, which has unique activity as both an arginine demethylase and lysine hydroxylase [97,98]. JMJD6 expression is increased in hypoxic conditions in the placenta, and can downregulate HIF-1α [99], though it has been found to operate in diverse pathways. JMJD6 can promote the formation of stress granules through demethylation and de-repression of G3BP Stress Granule Assembly Factor 1 G3BP1, resulting in the cytoplasmic sequestering of stalled mRNA-ribosome complexes to reversibly prevent mRNA degradation [100,101]. This would allow a fast re-start of protein synthesis when oxygen homeostasis is restored. JMJD6 also regulates mRNA splicing through hydroxylating the splicing regulatory (SR) proteins LUC7 Like 2, Pre-MRNA Splicing Factor (LUC7L2, U2 Small Nuclear RNA Auxiliary Factor 2 (U2AF2) [102], and Serine And Arginine Rich Splicing Factor 11 (SRSF11) [98]. The SR proteins are involved in exon definition and alternative splicing, with SRSF11 hydroxylation resulting in skipping of the most 5' exon, and hydroxylation of U2AF65 possibly enacting pre-mRNA looping in order to present to the splicing machinery different cis splice enhancer or silencer sequences [103]. However, this only occurs for selected mRNAs and is not a global effect [103]. Nevertheless, this mechanism would allow selection of alternate splice variants as a response to hypoxia. JMJD6 can also interact with both Bromodomain Containing 4 (BRD4) and the positive Transcription Factor Elongation Factor b (P-TEFb) complex [104], eventually resulting in the release of paused DNA polymerase II and resumption of mRNA synthesis at specifically regulated genes [103].
This implies that hypoxia could use this mechanism to stall transcription of genes that are not required for the stress response to hypoxia and would allow a re-start of gene expression when oxygen levels are restored.
Another JmjC hydroxylase, KMD8, which can hydroxylate arginine residues in both RCC1 Domain Containing 1 (RCCD1) and RPS6 [105]. Although not necessarily dependent on its hydroxylation activity, KDM8 is required for cell proliferation and chromosomal stability [106], and can negatively regulate p53 affecting gene expression and control cell cycle and proliferation [107,108]. Also recently, a biochemical function has been assigned to JMJD7 as a lysyl hydroxylase, which targets Developmentally Regulated GTP Binding Protein 1 (DRG1) and DRG2, which are part of the Translation Factor (TRAFAC) family of GTPases, and could affect their binding with messenger, or ribosomal RNA, though this requires further investigation [109].
The JmjC demethylase, KDM2A represses NF-κB activity via demethylation of RELA, providing a possible link to hypoxia and inflammation crosstalk. [110]. It is more than likely that other JmjC demethylases interact and directly demethylate additional transcription factors which may coordinate transcriptional responses to hypoxia. However, unbiased analysis is required to fully assess this aspect of hypoxia induced gene regulation.   Database [111]). The homozygous VHL mutation R200W, which prevents efficient HIF-α degradation in normoxia, is found in all individuals with Chuvash Polycythemia (CP) [112]. CP is characterized by congenital erythrocytosis, and patients have been associated with pulmonary hypertension, thrombosis, vertebral hemangiomas, cerebral vascular events and other vascular abnormalities [113,114], displaying the role of VHL in HIF-dependent regulation of vasculogenesis and erythropoiesis.

2-OGDs -hydroxylases
Similar to PHDs, prolyl-4-hydroxylases P4HA1 and P4HA2 are hypoxia-inducible, but P4HA1/2 prolyl hydroxylation is required for different processes, that is collagen fiber formation. Consistent to their roles in collagen synthesis, P4HA1 and P4HA2 mutations are found in patients with collagenrelated extracellular matrix disorders (Table 1; Supplementary Table 1). Furthermore, homozygous deletion of P4HA1 is embryonic lethal with base membrane rupture due to defective collagen IV assembly (Table 1). PAHX is another hydroxylase, but of phytanoyl-CoA; essential for breaking down phytanic fatty acid. Mutations in PAHX is well associated with Refsum disease, a rare inherited neurological disorder caused by neurotoxic phytanic acid as these mutations result in an enzymatically inactive protein, thus leading to phytanic acid accumulation.
The roles of TET1-3 in development are demonstrated in knockout mouse models (reviewed in [283]). TET1-null mice present several defects but these depend on the mode of genetic deletion.
TET3 deletion results in neonatal lethality, highlighting TET3 role in development. TET3 mutations have been found in patients with intellectual disability and/or delayed global development (Table 1; Supplementary acute myeloid leukemia, or chronic myelomonocytic leukemia [284]. Most of these mutations are predicted to lead to partial or total loss of function due to protein truncation.

2-OGDs -JmjC demethylases
Many of the JmjC-demethylase genes have been associated with human diseases. In particular, several of them are mutated in patients with neurodevelopmental disorders, midline defects and cancers (Table 1; Supplementary Table 1). Although KDM3A is found to be mutated in infertile males [206], its role in infertility is not clear. Mutations in KDM3B are frequently implicated with intellectual disability, but also found in cancers including myeloid leukemias (Table 1). Similarly, JMJD1C mutations have been identified in individuals with autism spectrum disorder and intellectual disability JMJD1C is also associated with congenital heart disease manifestation in 22q11.  [256,258,[285][286][287]. However, whether the phenotypes observed are due to loss of demethylase activity solely is currently unknown.
Overall, the presence and connections of HIFs or dioxygenase mutations in human disorders and the knockout studies demonstrate the essential roles of these genes.