Hypoxia research, where to now?

Investigating how cells and organisms sense and respond to O2 levels is essential to our understanding of physiology and pathology. This field has advanced considerably since the discovery of the major transcription factor family, hypoxia-inducible factor (HIF), and the enzymes that control its levels: prolyl hydroxylases (PHDs). However, with its expansion, new complexities have emerged. Herein we highlight three main areas where, in our opinion, the research community could direct some of their attention. These include non-transcriptional roles of HIFs, specificity and O2 sensitivity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), and new tools and methods to detect O2 concentrations in cells and organs. A greater understanding of these areas would answer big questions and help drive our knowledge of cellular responses to hypoxia forward.

LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein.

The interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to Coronavirus Disease 2019 (COVID-19). The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary angiotensin-converting enzyme 2 (ACE2) receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was functionally restricted to SARS-CoV-2 by accessibility. We localized the interaction to the C-terminus of the S1 domain and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Levels of LRRC15 were greatly elevated by inflammatory signals in the lungs of COVID-19 patients. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence that it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.

CDK-dependent phosphorylation of PHD1 on serine 130 alters its substrate preference in cells.

PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.

Cell cycle progression in response to oxygen levels.

Hypoxia' or decreases in oxygen availability' results in the activation of a number of different responses at both the whole organism and the cellular level. These responses include drastic changes in gene expression, which allow the organism (or cell) to cope efficiently with the stresses associated with the hypoxic insult. A major breakthrough in the understanding of the cellular response to hypoxia was the discovery of a hypoxia sensitive family of transcription factors known as the hypoxia inducible factors (HIFs). The hypoxia response mounted by the HIFs promotes cell survival and energy conservation. As such, this response has to deal with important cellular process such as cell division. In this review, the integration of oxygen sensing with the cell cycle will be discussed. HIFs, as well as other components of the hypoxia pathway, can influence cell cycle progression. The role of HIF and the cell molecular oxygen sensors in the control of the cell cycle will be reviewed.

Hypoxia drives HIF2-dependent reversible macrophage cell cycle entry.

Low-oxygen conditions (hypoxia) have been associated primarily with cell-cycle arrest in dividing cells. Macrophages are typically quiescent in G0 but can proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in macrophages. Cell cycle progression is largely limited to G0-G1/S phase transition with little progression to G2/M. This cell cycle transitioning is triggered by an HIF2α-directed transcriptional program. The response is accompanied by increased expression of cell-cycle-associated proteins, including CDK1, which is known to phosphorylate SAMHD1 at T592 and thereby regulate antiviral activity. Prolyl hydroxylase (PHD) inhibitors are able to recapitulate HIF2α-dependent cell cycle entry in macrophages. Finally, tumor-associated macrophages (TAMs) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at the single-cell level. These findings have implications for inflammation and tumor progression/metastasis where low-oxygen environments are common.

VHL synthetic lethality screens uncover CBF-ß as a negative regulator of STING.

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor ß (CBF-ß) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-ß. Mechanistically, CBF-ß loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-ß at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-ß in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

A synthetic system for expression of components of a bacterial microcompartment.

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Cigarette smoke preferentially induces full length ACE2 expression in differentiated primary human airway cultures but does not alter the efficiency of cellular SARS-CoV-2 infection.

Cigarette smoking has many serious negative health consequences. The relationship between smoking and SARS-CoV-2 infection is controversial, specifically whether smokers are at increased risk of infection. We investigated the impact of cigarette smoke on ACE2 isoform expression and SARS-CoV-2 infection in differentiated primary human bronchial epithelial cells at the air-liquid-interface (ALI). We assessed the expression of ACE2 in response to CSE and therapeutics reported to modulate ACE2. We exposed ALI cultures to cigarette smoke extract (CSE) and then infected them with SARS-CoV-2. We measured cellular infection using flow cytometry and whole-transwell immunofluorescence. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated isoform (dACE2) that lacks the capacity to bind SARS-CoV-2. CSE did not have a significant impact on key mediators of the innate immune response. Importantly, we show that, despite the increase in flACE2, CSE did not alter airway cell infection after CSE exposure. We found that nicotine does not significantly alter flACE2 expression but that NRF2 agonists do lead to an increase in flACE2 expression. This increase was not associated with an increase in SARS-CoV-2 infection. Our results are consistent with the epidemiological data suggesting that current smokers do not have an excess of SARS-CoV-2 infection. but that those with chronic respiratory or cardiovascular disease are more vulnerable to severe COVID-19. They suggest that, in differentiated conducting airway cells, flACE2 expression levels may not limit airway SARS-CoV-2 infection.

A HIF independent oxygen-sensitive pathway for controlling cholesterol synthesis.

Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.

Genetic approaches to understand cellular responses to oxygen availability.

Oxygen-sensing mechanisms have evolved to allow organisms to respond and adapt to oxygen availability. In metazoans, oxygen-sensing is predominantly mediated by the hypoxia inducible factors (HIFs). These transcription factors are stabilised when oxygen is limiting, activating genes involved in angiogenesis, cell growth, pH regulation and metabolism to reset cell function and adapt to the cellular environment. However, the recognition that other cellular pathways and enzymes can also respond to changes in oxygen abundance provides further complexity. Dissecting this interplay of oxygen-sensing mechanisms has been a key research goal. Here, we review how genetic approaches have contributed to our knowledge of oxygen-sensing pathways which to date have been predominantly focused on the HIF pathway. We discuss how genetic studies have advanced the field and outline the implications and limitations of such approaches for the development of therapies targeting oxygen-sensing mechanisms in human disease.

The impact of hypoxia on B cells in COVID-19.

BACKGROUND: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. METHODS: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice. FINDINGS: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions. INTERPRETATION: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes. FUNDING: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.

The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex.

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.

Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP-HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.

PITX1, a specificity determinant in the HIF-1α-mediated transcriptional response to hypoxia.

Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1ß and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers.

PHD1 links cell-cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192.

PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.