Search

Table S1: Phenotypes associated with single R161Q and R200W VHL germline mutations are different from the phenotype associated with the combined mutations. Table S2: VHL mutations associated with polycythemia are different from the VHL mutations involved in severe von Hippel-Lindau disease. Table S3: Members of the family carrying the VHL-R200W+R161Q mutations present normal hematological values. Table S4: Intra-domain hydrogen bonds are severely destabilized by the mutations. Table S5: The double mutant reduced the inter-domain mobility compared to the single one. Table S6: The pVHL/HIF-2α direct target genes are deregulated with a significantly ascending gradient by the pVHL mutants of increased severity. Table S7: The target genes of the pVHL/HIF-2α axis that are deregulated in renal cell carcinoma play a role in oncogenic pathways. Figure S1: Germline mutations in the VHL gene are associated with distinct phenotypes. Figure S2: (A) The crystallographic structure of pVHL contains a seven-stranded β-sandwich connected to four α-helices by a flexible linker. (B) The R200W mutation dramatically weakens hydrogen bonds established in the wild-type form between Arg200 and Glu134, and between Arg197 and Arg120. (C) The R161Q mutation indirectly influences crucial stabilizing interactions involving primarily Arg 167. Figure S3: VHL mRNA expression is equivalent in 786.O cells transduced by the different VHL inducible lentiviral constructs.

Supplementary Material and Methods and Legend of Supplementary Tables and Figures

Hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR) are members of the bHLH-PAS family of transcription factors that underpin cellular responses to oxygen and to endogenous and exogenous ligands, respectively, and have central roles in the pathogenesis of renal cancer. Composed of heterodimers, they share a common HIF-1β/ARNT subunit and similar DNA-binding motifs, raising the possibility of crosstalk between the two transcriptional pathways. Here, we identify both general and locus-specific mechanisms of interaction between HIF and AHR that act both antagonistically and cooperatively. Specifically, we observe competition for the common HIF-1β/ARNT subunit, in cis synergy for chromatin binding, and overlap in their transcriptional targets. Recently, both HIF and AHR inhibitors have been developed for the treatment of solid tumours. However, inhibition of one pathway may promote the oncogenic effects of the other. Therefore, our work raises important questions as to whether combination therapy targeting both of these pro-tumourigenic pathways might show greater efficacy than targeting each system independently.

Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

The Yilgarn Craton, Western Australia is endowed with significant gold, nickel‑copper, Cu-Pb-Zn volcanic-hosted massive sulphide (VHMS) and iron ore deposits. Detailed knowledge of the spatial relationships between mineral systems and the crustal framework of the Yilgarn Craton may be powerful tools in the predictive exploration of undiscovered mineral systems. Both Nd and Hf isotopes have the potential to image crustal residence, mantle tapping structures, and juvenile input over both space and time. This study uses a recent compilation of Hf (1236 zircon analyses) and Nd (462 whole rock analyses) isotopes to image the architecture of the crust and compare this to the distribution of gold, nickel‑copper-platinum group elements (Ni-Cu-PGEs), Cu-Pb-Zn VHMS and iron ore deposits. Spatial correlation plots reveal a statistically significant relationship between Nd and Hf isotopic domains and the occurrence of gold, Ni-Cu-PGE, base metals, and iron confirmed by the coefficient of determination (R2) which measures how differences in metal occurrences can be explained by differences in the isotopic signature. Nickel occurrences show the strongest antithetic spatial correlation (R2 = 0.80) with Hf model ages, followed by gold (R2 = 0.58) and base metals (R2 = 0.35), which implies higher metal endowment in areas of more juvenile crust. In contrast, there is a significant positive spatial correlation (R2 = 0.49) between iron ore occurrence and older model ages. Isotopic data can also be plotted as Hf or Nd time-slices, where the strongest spatial correlation between gold, nickel, and base metal occurrence and juvenile input occurs at ca. 2700–2550 Ma. Generally, a model can be proposed where (1) base metals are more prevalent in juvenile crust because of its thinner nature, greater mantle input, high-heat flow, increased water-magma interaction; (2) iron ore is concentrated in regions of old, mature, and stable crust; (3) komatiite-hosted Ni-Cu-PGE systems are focused in juvenile rift zones at the edge of old crustal blocks; and (4) gold is also localised in juvenile crust at craton edges where several factors are localised, including orogenesis, occurrence of previously fertilised crust (i.e. Fe-rich lithologies, sanukitoids), favourable structures (potential inversion of rift structures), and ‘late’ alkaline magmatism. In gold, Ni-Cu-PGE, and base metal mineral systems, the ability of magmas to access higher crustal levels unimpeded appears vital in regional prospectivity and is itself a function of crustal configuration.

Background & Aims Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. Methods Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle. Results Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family. Conclusions Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection. Lay summary Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets., Graphical abstract, Highlights • Primate hepadnaviridae encode conserved hypoxia response elements. • Hypoxia inducible factors bind HBV DNA and activate the basal core promoter. • Pharmacological stabilization of hypoxia inducible factors and low oxygen increases HBV transcription and particle genesis. • Knockdown studies show a role for both HIF-1α and HIF-2α in regulating HBV transcription.

Clear cell renal cancers (ccRCC) are characterized by inactivation of the VHL (von Hippel–Lindau) tumor suppressor. Work leading to the 2019 Nobel Prize for Physiology or Medicine has shown that this is central to cellular oxygen-sensing, orchestrated by the HIF (hypoxia-inducible factor) transcription factors. These regulate hundreds of genes that underpin many hallmarks of cancer, including angiogenesis, cellular energetics, cell proliferation, resisting cell death, and avoiding immune destruction. However, HIF also promotes processes that are detrimental to cancer cells. Therefore, the overall consequence of HIF pathway activation is a balance of these influences. We explore how variations in the HIF pathway during tumorigenesis alter this balance to promote ccRCC formation.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis. Single-cell RNA sequencing is used to characterize and compare the functional diversity of cells from liver biopsies of human scarred and normal liver, and identifies markers for scar-associated macrophages and endothelial cells.

The Ntaka Hill Ni-Cu deposit in Tanzania lies within the Mozambique belt, a complex, long-lived Neoproterozoic suture zone that formed during the amalgamation of Gondwana. The deposit is hosted within the moderately deformed Ntaka Ultramafic Complex, which was emplaced at ~660 Ma, around 20 m.y. before the East African orogeny. Due to strain partitioning into amphibolites and metapyroxenites at the margins of the sills, magmatic cumulate textures are very well preserved. The Ntaka Hill mineralization is predominantly disseminated sulfides within orthopyroxenites and harzburgites. Sulfides show typical magmatic mineralogy and occur as isolated blebs and interstitial networks as well as in soft-walled segregation veins that form localized shoot-like high-grade zones. Deformation had relatively little effect on the deposition of sulfides other than minor localization of strain within some preexisting sulfide-dominant veins. Grade shells define elongate tubular channels that coincide broadly with the more magnesian cumulate rocks. Base and precious metal tenors are widely variable, ranging from 1 to 17% Ni and 0.5 to 10% Cu with no particular correlation with host-rock type, although the tenor range in the more magnesian cumulates is more restricted, with the low-tenor component (

In humans, new-born infants have the ability to regenerate their heart during early life. This is modelled in the mouse, where regenerative capacity is maintained for the first week after birth but lost thereafter. Reactivation of this process holds great therapeutic potential, however, the molecular pathways that might be targeted to extend neonatal regeneration remain elusive. Here, we explore a role for hypoxia and HIF signalling on the regulation of epicardial activity which is essential for heart development and the response to injury. Hypoxic regions were found in the epicardium from mid-gestation, associating with HIF1α and HIF2α, and expression of the epicardial master regulator Wilms’ tumour 1 (WT1). Epicardial deletion of Hif1a reduced WT1 levels, leading to impaired coronary vasculature. Moreover, targeting of the HIF degradation enzyme PHD through pharmacological inhibition with clinically approved drugs or epicardial-specific deletion stabilised HIF and promoted WT1 activity ex vivo. A combination of genetic and pharmacological stabilisation of HIF during neonatal heart injury led to prolonged epicardial activation, increased vascularisation, augmented infarct resolution and preserved function beyond the 7-day regenerative window. Together, these findings suggest pharmacological modulation of HIF signalling may represent a viable therapeutic strategy for treating ischaemic heart disease.

Hypoxia is a key feature of the solid tumour microenvironment, in which it is associated with poor prognosis and resistance to therapy. Hypoxia-inducible factor (HIF) is the central mediator of cellular responses to hypoxia and drives a transcriptional program with central roles in tumorigenesis. Clear cell renal cell cancers (ccRCC) exhibit the most direct and profound upregulation of the HIF pathway - through bi-allelic inactivation of the VHL tumour suppressor, a ubiquitin ligase that targets HIF-alpha subunits for degradation under normal oxygenated conditions. HIF-alpha proteins are members of the bHLH-PAS family and interact with HIF-1beta (also known as ARNT) to form functional heterodimers binding chromatin. However, the aryl hydrocarbon receptor (AHR), another bHLH-PAS protein that dimerises with HIF-1beta, is also up-regulated in ccRCC, raising the possibility of crosstalk between these two transcriptional pathways. Previous studies support an antagonistic relationship between HIF and AHR, with evidence of a context-dependent and gene-specific crosstalk. Here, we investigate the pan-genomic and precise relationship between the HIF and AHR transcription factors in ccRCC. Using immunoprecipitation, we show direct competition for HIF-1beta binding between the HIF-alpha and AHR proteins in renal cancer cell lines. Pan-genomic analysis of HIF-1alpha, HIF-2alpha, HIF-1beta and AHR chromatin binding by ChIP-seq analysis reveals a widespread competitive interaction between HIF and AHR. In RCC4 cells, this interaction is bidirectional, with induction of HIF leading to a reduction in AHR binding, while induction of AHR reduces binding of both HIF-1alpha and HIF-2alpha to chromatin. However, this competition is diminished at sites bound by both HIF and AHR, indicating a cooperative interaction between the two transcription factors in cis. Thus, while the HIF-alpha and AHR proteins compete for the common binding partner HIF-1beta, they appear to form a cooperative interaction when bound to neighbouring sites on chromatin. Linking these findings to changes in gene expression by RNA-seq analysis, we show the global downstream effects of the competition between HIF and AHR, but also reveal a degree of overlap and cooperativity between their transcriptional programs. Furthermore, we provide evidence of this antagonistic relationship in ccRCC tumours (TCGA database), in which we observe a negative correlation between HIF and AHR target gene expression. These findings shed light on the complex crosstalk between the HIF and AHR transcriptional pathways, which encompasses both a widespread inhibitory interaction and specific cooperative events and highlight a role for AHR in modifying the HIF transcriptional output in ccRCC. Citation Format: Véronique N. Lafleur, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Bi-directional crosstalk between the HIF and AHR transcription factors in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 677.

Kidney cancer is the seventh most common malignancy in the Western world and 80% of these are clear cell renal cell cancers (ccRCC). ccRCC arises following inactivation of the VHL gene, which negatively regulates HIF (hypoxia-inducible factor) transcription factors. Therefore, in ccRCC, HIF is constitutively active and drives a gene expression program that is central to tumorigenesis. Whilst this is necessary for ccRCC formation, it is not sufficient, and other mutations must also occur in combination with VHL inactivation to drive ccRCC tumorigenesis. The mechanisms underlying their synergy with VHL inactivation remain poorly understood. These secondary driver mutations are often subclonal in nature, suggesting the presence of phenotypically distinct malignant cells within an individual tumor. This intratumor heterogeneity renders it difficult to study the functional impact of driver mutations in ccRCC by conventional ‘bulk’ analyses such as RNA-seq. Consequently, the molecular mechanisms underlying ccRCC initiation are not known. Furthermore, non-malignant stromal cells (including the vasculature and immune response) can support tumor growth, and little is known as to how they interact with malignant cells in ccRCC. This is particularly important since current therapeutic strategies in kidney cancer target the stromal compartment. Therefore, there is an unmet need to better-understand transcriptional heterogeneity and cellular diversity in ccRCC. To that end, we have sampled multiple regions from ccRCC tumors (and tumor-adjacent kidney tissue) and performed single cell RNA-seq (10x Genomics) on approximately 150,000 individual cells. These encompass malignant cells as well as non-malignant cells (including fibroblasts, endothelial cells, pericytes and immune cells), both of which exhibit pronounced transcriptional heterogeneity within and between tumors. Analysis of these transcriptomes has identified evolutionary gene expression programs underlying cancer cell dedifferentiation. In addition, we observe concurrent heterogeneity in angiogenic signaling between malignant cells and non-malignant blood vessel cells within these tumors. Lastly, we have explored the effect of microenvironmental hypoxia across different ccRCC tumor cell types. In summary, we present a single cell atlas derived from multi-region heterogenous ccRCC samples. This sheds light on the mechanisms underlying tumor evolution and progression, as well as angiogenic heterogeneity (which may contribute to the variable response to receptor tyrosine kinase inhibition) and the effect of hypoxia on ccRCC biology. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Peter J. Ratcliffe, David R. Mole. Multi region single cell sequencing reveals dedifferentiation programs, angiogenic heterogeneity and the effect of microenvironmental hypoxia in ccRCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1587.

Low oxygen availability (hypoxia) is a common feature of many solid tumors and is associated with poor prognosis and resistance to therapy. The main regulator of cellular responses to hypoxia is the transcription factor HIF (hypoxia-inducible factor). In addition, several histone lysine demethylases (KDMs) are 2-OG dependent dioxygenases and require oxygen for their activity. When oxygen availability is limited, the activity of these KDMs is inhibited leading to global histone hypermethylation, potentially altering gene expression. However, some KDMs are also known to be direct transcriptional targets of HIF, increasing their expression in hypoxia and counteracting this histone hypermethylation. Furthermore, HIF can facilitate recruitment of histone methyltransferase activity to chromatin in cis, leading to local increases in methylation status. Histone methylation can be associated with both gene activation and gene repression, and it remains unclear how the balance of these widespread changes relates to locus-specific regulation of gene expression. Furthermore, previous attempts to analyze regulation of histone methylation by ChIP-seq in hypoxia have not employed normalization methods that take into account global changes in histone modification. We have performed ChIP-seq analysis of histone trimethylation (H3K4me3, H3K9me3, H3K27me3 and H3K36me3) in PC3 prostate cancer cells incubated in normoxia or (16hrs, 1%) hypoxia. We have then examined the effects of the HIF-transcriptional pathway on these marks using the same cell line following deletion of HIF-1b to block the HIF response. We employ an integrated spike-in with drosophila chromatin to provide a normalization control. We then relate our findings to changes in gene expression using RNA-seq analysis, again normalized to a spike-in control. We observe global increases in all 4 histone trimethylations across the genome in hypoxia. In particular, we observe hypoxic increases in histone trimethylation at all gene loci. However, while basal levels of histone trimethylation correlated with basal transcript levels as previously reported, hypoxic increases in promoter-associated histone H3K4me3 trimethylation bore no relation to either the direction or magnitude of gene regulation in hypoxia. Although, more subtle increases were observed across the gene body at gene loci induced by hypoxia. These findings question the currently accepted model that changes in H3K4me3 instruct changes in transcription in hypoxia and suggest that the overall effect of histone trimethylation is a more subtle balance of competing effects. Citation Format: Jessica D. Kindrick, Peter J. Ratcliffe, WIlliam Doug Figg, David R. Mole. Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3766.

Low levels of tissue oxygen, or intra-tumor hypoxia, is a common feature of many solid cancers in which dysregulated cell proliferation often outstrips the ability to develop new functional blood vessels. Furthermore, hypoxia pathways may also be activated in response to mutation in key tumor suppressor genes or oncogenes. Importantly, in many types of cancer, activation of hypoxia pathways is associated with a clinically aggressive phenotype as well as with resistance to therapy. Indeed, hypoxia drives many of the “hallmarks” of cancer, including cell proliferation, apoptosis, metabolism, immune responses, genomic instability, vascularization, invasion and metastasis. However, the full-range of genes activated by hypoxia varies greatly between cell-types and for many this remains uncharacterized. The key mediator of the cellular transcriptional response to hypoxia is the HIF (hypoxia-inducible factor) family of transcription factors. Here, we generate pan-genomic datasets encompassing HIF ChIP-seq and RNA-seq across cancer cell lines from diverse common tumor types. We then employ integrated analysis of our 72 transcriptome and whole-genome-binding datasets to define a core set of HIF-target genes that are conserved across multiple cell types. We show that these genes can be used to identify HIF activation and tumor hypoxia in bulk RNA-seq analysis of solid tumors, and that this can be applied to large tumor databases (TCGA). We then leverage this analysis to identify tumor-type-specific HIF-associated genes in 32 different cancer types, including those lacking cell-culture models. We further show that these conserved HIF-target genes can be applied to single cell RNA-seq datasets from solid tumors and used to examine intra-tumor heterogeneity in HIF-pathway activation within individual cancer samples. Importantly, using a panel of genes reduces the phenomenon of “drop-out” typically observed in gene-level expression in these datasets. Lastly, applying this gene signature to single cell data allows the hypoxia response to be deconvoluted across diverse cell-types in heterogenous tumor samples. This includes not only the identification of HIF-activity in cancer cells, but also in non-cancer cells within the tumor, thereby allowing the effects of intra-tumor hypoxia on stromal responses (such as tumor angiogenesis) to be studied in vivo in solid tumors. Citation Format: Olivia Lombardi, Ran Li, Silvia Halim, Peter J. Ratcliffe, David R. Mole. Pan-cancer analysis of HIF pathways defines a robust molecular signature that reflects tissue and cellular hypoxia in bulk and single-cell RNA-seq analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 119.

Hypoxia-inducible transcription factors (HIFs) are fundamental to the cellular adaptation to low oxygen levels but how they interact with chromatin and efficiently activate their target genes is unclear. Using genome-wide mutagenesis in human cancer cells, we define genes required for HIF transcriptional activation, and identify a requirement for the Histone 3 lysine 4 (H3K4) methyltransferase SET1B. Loss of SET1B leads to a selective reduction in HIF transcriptional activity in hypoxia, with SET1B driving expression of genes involved in angiogenesis rather than glycolysis, resulting in impaired tumour establishment in SET1B deficient xenografts. Mechanistically, we show that SET1B is itself oxygen regulated, accumulates on chromatin in hypoxia, and is recruited to HIF target genes through HIF-1α. Accordingly, we show that the hypoxic induction of H3K4me3 at specific HIF targets is both HIF and SET1B dependent, and when impaired, decreases promoter acetylation and gene expression. Together, these findings reveal SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Cellular responses to low oxygen (hypoxia) are fundamental to normal physiology and to the pathology of many common diseases. Hypoxia-inducible factor (HIF) is central to this by enhancing the transcriptional activity of many hundreds of genes. The cellular response to HIF is cell-type-specific and is largely governed by the pre-existing epigenetic landscape. Prior to activation, HIF-binding sites and the promoters of HIF-target genes are already accessible, in contact with each other through chromatin looping and display markers of activity. However, hypoxia also modulates the epigenetic environment, both in parallel to and as a consequence of HIF activation. This occurs through a combination of oxygen-sensitive changes in enzyme activity, transcriptional activation of epigenetic modifiers, and localized recruitment to chromatin by HIF and activated RNApol2. These hypoxic changes in the chromatin environment may both contribute to and occur as a consequence of transcriptional regulation. Nevertheless, they have the capacity to both modulate and extend the transcriptional response to hypoxia.

Hypoxia-inducible transcription factors (HIFs) are fundamental to the 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 reveal SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

Viral replication is defined by the cellular microenvironment and one key factor is local oxygen tension, where hypoxia inducible factors (HIFs) regulate the cellular response to oxygen. Human immunodeficiency virus (HIV) infected cells within secondary lymphoid tissues exist in a low-oxygen or hypoxic environment in vivo. However, the majority of studies on HIV replication and latency are performed under laboratory conditions where HIFs are inactive. We show a role for HIF-2α in restricting HIV transcription via direct binding to the viral promoter. Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin, mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. Our data support a model where the low-oxygen environment of the lymph node may suppress HIV replication and promote latency. We identify a mechanism that may contribute to the limited efficacy of latency reversing agents in reactivating HIV and suggest new strategies to control latent HIV-1., Zhuang et al. investigate how low oxygen levels affect HIV-1 replication in cell culture models. They find that the hypoxia-induced transcription factor HIF-2α represses HIV transcription and that low oxygen reduces the reactivation of latent HIV, suggesting that the hypoxic environment in the lymph node may influence HIV replication and latency.

Much of the current volume of Earth’s continental crust had formed by the end of the Archaean eon1 (2.5 billion years ago), through melting of hydrated basaltic rocks at depths of approximately 25–50 kilometres, forming sodic granites of the tonalite–trondhjemite–granodiorite (TTG) suite2–6. However, the geodynamic setting and processes involved are debated, with fundamental questions arising, such as how and from where the required water was added to deep-crustal TTG source regions7,8. In addition, there have been no reports of voluminous, homogeneous, basaltic sequences in preserved Archaean crust that are enriched enough in incompatible trace elements to be viable TTG sources5,9. Here we use variations in the oxygen isotope composition of zircon, coupled with whole-rock geochemistry, to identify two distinct groups of TTG. Strongly sodic TTGs represent the most-primitive magmas and contain zircon with oxygen isotope compositions that reflect source rocks that had been hydrated by primordial mantle-derived water. These primitive TTGs do not require a source highly enriched in incompatible trace elements, as ‘average’ TTG does. By contrast, less sodic ‘evolved’ TTGs require a source that is enriched in both water derived from the hydrosphere and also incompatible trace elements, which are linked to the introduction of hydrated magmas (sanukitoids) formed by melting of metasomatized mantle lithosphere. By concentrating on data from the Palaeoarchaean crust of the Pilbara Craton, we can discount a subduction setting6,10–13, and instead propose that hydrated and enriched near-surface basaltic rocks were introduced into the mantle through density-driven convective overturn of the crust. These results remove many of the paradoxical impediments to understanding early continental crust formation. Our work suggests that sufficient primordial water was already present in Earth’s early mafic crust to produce the primitive nuclei of the continents, with additional hydrated sources created through dynamic processes that are unique to the early Earth. Oxygen isotopes and whole-rock geochemistry show that the water required to make Earth’s first continental crust was primordial and derived from the mantle, not surface water introduced by subduction.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Intraplate continental magmatism represents a fundamental mechanism in Earth's magmatic, thermal, chemical and environmental evolution. It is a process intimately linked with crustal development, large-igneous provinces, metallogeny and major global environmental catastrophes. As a result, understanding the interactions of continental magmas through time is vital in understanding their effect on the planet. The interaction of mantle plumes with the lithosphere has been shown to significantly affect the location and form of continental magmatism, but only at modern mantle conditions. In this study, we perform numerical modelling for Late Archean (1600 °C), Paleoproterozoic (1550 °C), Meso-Neoproteroic (1500 °C) and Phanerozoic (1450 °C) mantle potential temperatures ( Tp ) to assess the time-space magmatic effects of ambient-mantle- and plume- lithosphere interaction over Earth's thermal history. Within these experiments, we impinge a mantle plume, with a time-appropriate Tp , onto a ‘step-like’ lithosphere, to evaluate the effect of craton margins on continental magmatism through time. The results of this modelling demonstrate that lithospheric architecture controls the volume and location of continental magmatism throughout Earth history, irrespective of ambient mantle or plume Tp . In all plume models, mantle starting plumes (diameter 300 km) impinge on the base of the lithosphere, and spread laterally over > 1600 km, flowing into the shallowest mantle, and producing the highest volume magmas. In ambient-mantle only models, Archean and Paleoproterozoic Tp values yield significant sub-lithospheric melt volumes, resulting in ‘passive’ geodynamic emplacement of basaltic magmatic provinces, whereas no melts are extracted at > 100 km for Meso-Neoproterozoic and Phanerozoic Tp . This indicates a major transition in non-subduction related continental magmatism from plume and ambient mantle to a plume-dominated source around the Mesoproterozoic. While the experiments presented here show the variation in plume-lithosphere interaction through time, the consistency in melt localisation indicates the lithosphere has been a first-order control on continental magmatism since its establishment in the Mesoarchean.

The Lamboo Province of the East Kimberley region of Western Australia contains a record of crustal and tectonic evolution from ca. 1912 to 1808 Ma. In this interval, current models indicate an ocean basin was systematically closed from ca. 1880 Ma until collision of the Kimberley (Western Zone) and North Australia Cratons (Eastern Zone) at the ca. 1830 Ma Halls Creek Orogeny. Within this orogen, the Central Zone has two proposed origins: (1) an intra-oceanic island-arc; and (2) an exotic continental fragment. In this study, we investigated the origin of Ni-Cu mineralized mafic-ultramafic intrusions found in the Central Zone. We performed U-Pb SHRIMP geochronology on 21 samples, together with O-isotopes in zircon and whole-rock geochemistry, to understand the formation of the intrusions and their host mineralization within the tectonic evolution of the Lamboo Province. Geochronology demonstrates that the majority of intrusions are synchronous at ca. 1848–1838 Ma (ca. 1844 ± 2 Ma), and were intruded into the

The ca. 2770–2690 Ma Fortescue Group of the Pilbara Craton in Western Australia represents one of the oldest known large igneous provinces (LIP) on Earth. Existing and new data show that the Fortescue LIP lavas have relatively evolved Nd isotopic compositions and are enriched in highly-incompatible elements. This is exemplified by mafic lavas of the 2775 Ma Mt Roe, 2740 Ma Kylena, 2715 Ma Bunjinah-Maddina and 2690 Ma Jeerinah Formations, with Th/Ti ca. 25, Th 13–5 ppm and eNdi around −4 (High-Th suite). Basalts and rare Al-depleted komatiites of the 2724–2715 Ma Pyradie Formation show more primitive compositions, with Th/Ti ca. 4, Th ca. 3–0.5 ppm and eNdi of 0 to −1 (Intermediate-Th suite). In this paper, we show that: (1) Parent magmas to all Fortescue LIP lavas were probably komatiitic; (2) Fortescue lavas contain 3.5–3.3 Ga xenocrystic zircons that correlate with the known age of Pilbara crust; and (3) the widespread enriched compositions in the Fortescue lavas require contributions to the ultramafic parent magmas from highly-enriched 3.7–3.5 Ga continental crust of the type available in the underlying Pilbara Craton. These results indicate that enriched chemical/isotopic compositions of the Fortescue mafic lavas result from large-scale crustal assimilation rather than enriched mantle/lithospheric sources. This implies that komatiite magmas were emplaced through Eo- to Paleoarchean enriched crust for ca. 90 Ma, via either (1) a long-lived source of high-temperature mantle melts such as a mantle plume; or (2) multiple mantle plumes, possibly represented by the 2775–2763 Ma Mt Roe and 2724–2715 Ma Tumbiana-Pyradie-Maddina-Bunjinah basalts. This study demonstrates that mafic magmas, with arc-like geochemical character and intermediate to felsic continental crustal compositions, can form via contamination of plume magmas by existing (local) crustal reservoirs, and the employment of modified lithospheric sources or enriched mantle is not required.

Mafic and ultramafic magmas that intrude into the lower crust can preserve evidence for metal and sulfur transfer from the lithospheric mantle into the lower continental crust. Here we focus on a series of ultramafic, alkaline pipes in the Ivrea Zone (NW Italy), which exposes deeply buried (6–11 kbar), migmatitic metasedimentary rocks intruded by voluminous basaltic magmas of the Mafic Complex, a major crustal underplating event precisely dated via U/Pb CA-IDTIMS on zircon at 286.8 ± 0.4 Ma. The ultramafic pipes postdate the Mafic Complex and from 100 to 300 m wide cumulate-rich conduits. They are hydrated and carbonated, have unusually high incompatible element concentrations and contain blebby and semi-massive Ni-Cu-PGE sulfide mineralisation. The sulfides occur as coarse intergranular nodules (>10 mm) and as small intragranular blebs (

The Neoproterozoic geology of East Africa records the events leading up to the formation of Gondwana and subsequently Pangea, and hence understanding the geodynamic history of this area offers a unique insight into supercontinent development. Eighteen new U-Pb zircon and two U-Pb monazite ages were collected in conjunction with 110 whole-rock lithogeochemistry analyses from a region of south-east Tanzania we designate the ‘Ntaka Terrane’. These new data suggest this area of the East African Orogen constitutes an exotic nappe terrane that was part of the Mozambique Ocean in the Neoproterozoic. The lithological sequence in the region consists of a 770–720 Ma ‘basement’ of intermediate-felsic paragneiss possibly derived from a volcano-sedimentary sequence and recording arc-like geochemical signatures. These are intruded by 700–650 Ma amphibolites (±garnet) with mafic-intermediate magmatic protoliths that also have arc-like geochemistry. This sequence is then intruded by a set of ca. 670–650 Ma ultramafic, dominantly pyroxenitic sills collectively termed the Ntaka Ultramafic Complex, which have a much more mantle-like, non-arc origin, possibly formed as the island-arc intersected a mantle plume ‘hot spot’. The mafic-intermediate intrusives occur synchronous with, and outlast, the high-Mg basalt magmatism, suggesting that this latter event was transient in the context of the overall tectonic regime. The final magmatic event in the terrane is the intrusion of 640–620 Ma syn-orogenic granites that are coeval with 640–600 Ma upper amphibolite-granulite facies metamorphism. The broader geodynamic setting consisted of the Congo-Tanzania-Bangweulu Block of east Africa to the west and the Azania Block to the east of the Neomozambique Ocean, both of which contain Archean-Paleoproterozoic detritus. Based on the island arc-like geochemical characteristics of the 700–650 Ma mafic intrusive rocks and the lack of detrital zircons >1000 Ma, we suggest that the Ntaka Terrane represents a Neoproterozoic intra-oceanic island arc which was accreted to either East Africa or Azania shortly before full continental collision (the East African Orogeny) at ca. 640–620 Ma. Data from the Ntaka Terrane correlate with the Vohibory Terrane (Madagascar), Eastern Granulites (northern Tanzania) and Cabo Delgado Nappe Complex (Mozambique), suggesting these areas represent a set of broadly correlative oceanic terranes.

The use of Shortwave Infrared (SWIR) and Thermal Infrared (TIR) hyperspectral data in mineral exploration has been well documented in many mineralisation types, but is limited in komatiite-hosted nickel sulphide deposits. This project combines hyperspectral, Portable X-ray Fluorescence (pXRF) and whole-rock geochemical data to assess different analytical techniques in the exploration of these deposits. We use the Fisher East nickel sulphide prospects, Western Australia for our case study. The Fisher East prospects lie in an area of the eastern goldfields that has historically been underexplored and understudied. The rocks have undergone intense deformation with primary igneous textures being destroyed, along with strong alteration to talc carbonate assemblages. Combining different analytical tools allowed for differentiation of A and B-zones of original komatiite flows, and the reconstruction of original volcanological facies in a setting where whole rock chemistry as well as igneous textures have been substantially modified by metamorphism. By using different lithogeochemical techniques including pXRF, this study shows the Fisher East prospects are hosted within channelised komatiite flows, and have similar characteristics to Kambalda style deposits.

Economic volcanic-hosted massive sulfide (VHMS) deposits of the Archaean Yilgarn Craton of Western Australia are restricted to zones of juvenile crust as revealed through regional Nd, Pb and Hf isotopic variations and the geochemistry of felsic volcanic rocks. Interpreted as Archaean paleo-rift zones, one of these runs N-S through the Eastern Goldfields Superterrane (broadly coincident with the Kurnalpi Terrane) and is associated with the high grade ca. 2690 Ma Teutonic Bore, Jaguar and Bentley deposits, plus sub-economic VHMS mineralization further south. To date, only small historic Cu occurrences (e.g. Anaconda) and barren pyritic lenses have been recognized in the older >2.7 Ga plume-dominated lower stratigraphy of the Eastern Goldfields Superterrane. The Nimbus Ag-Zn-(Au) deposit (12.1 Mt at 52 g/t Ag, 0.9% Zn and 0.2 g/t Au) is located approximately 10 km east of Kalgoorlie, near the margin of the Kurnalpi Terrane. Its origin has been contentious for a number of years, with previous models favouring seafloor/sub-seafloor VHMS mineralization or a high sulfidation fault-hosted system. We present a detailed account of the deposit, its host stratigraphy and associated hydrothermal alteration, plus two new SHRIMP U-Pb zircon ages, Pb isotope (galena), and O isotope (zircon) constraints. Compared to other VHMS occurrences in the Yilgarn Craton, the Nimbus deposit is unusual in terms of its tectono-stratigraphic position, the geochemistry of its host sequence (i.e. FI-affinity felsic volcanic rocks, ocean-plateau-like low-Th basalts), mineralogy (e.g. abundance of Ag-Sb-Pb-As bearing sulfosalts, high Hg, low Cu) and quartz-carbonate-sericite dominated alteration assemblages. Classification of Nimbus as a shallow water and low temperature VHMS deposit with epithermal characteristics (i.e. a hybrid bimodal-felsic deposit) is consistent with its position near the margin of the Kurnalpi paleo-rift zone and radiogenic μ (238U/204Pb) values. The recognition that the Nimbus deposit is associated with c. 2705 Ma plume magmatism opens new areas for VHMS exploration in the Eastern Goldfields Superterrane over a strike length exceeding 500 km.

Emerging evidence suggests that dysregulation of oncogenic pathways requires precise tuning in order for cancer to develop. To test this, we examined the overlap between cis-acting elements of the hypoxia-inducible factor (HIF) pathway and cancer-susceptibility polymorphisms as defined in genome-wide association studies (GWAS). In renal cancer, where HIF is constitutively and un-physiologically activated by mutation of the von Hippel-Lindau tumour suppressor, we observed marked excess overlap, which extended to potential susceptibility polymorphisms that are below the conventional threshold applied in GWAS. In contrast, in other cancers where HIF is upregulated by different mechanisms, including micro-environmental hypoxia, we observed no excess in overlap. Our findings support a ‘pathway tuning’ model of cancer, whereby precise modulation of multiple outputs of specific, activated pathways is important in oncogenesis. This implies that selective pressures to modulate such pathways operate during cancer development and should focus attempts to identify their nature and consequences.

Much of the present-day volume of Earth’s continental crust had formed by the end of the Archean Eon, 2.5 billion years ago, through the conversion of basaltic (mafic) crust into sodic granite of tonalite, trondhjemite and granodiorite (TTG) composition. Distinctive chemical signatures in a small proportion of these rocks, the so-called high-pressure TTG, are interpreted to indicate partial melting of hydrated crust at pressures above 1.5 GPa (>50 km depth), pressures typically not reached in post-Archean continental crust. These interpretations significantly influence views on early crustal evolution and the onset of plate tectonics. Here we show that high-pressure TTG did not form through melting of crust, but through fractionation of melts derived from metasomatically enriched lithospheric mantle. Although the remaining, and dominant, group of Archean TTG did form through melting of hydrated mafic crust, there is no evidence that this occurred at depths significantly greater than the ~40 km average thickness of modern continental crust., Some of Earth’s earliest continental crust has been previously inferred to have formed from partial melting of hydrated mafic crust at pressures above 1.5 GPa (more than 50 km deep), pressures typically not reached in post-Archean continental crust. Here, the authors show that such high pressure signatures can result from melting of mantle sources rather than melting of crust, and they suggest there is a lack of evidence that Earth’s earliest crust melted at depths significantly below 40 km.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

In response to the dynamic intra‐tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia‐associated transcription factor (MITF). The response to hypoxia is driven by hypoxia‐inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA‐binding specificity, it is unclear whether they co‐regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up‐regulated by HIF1α and co‐regulates a subset of HIF targets including VEGFA. Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo‐hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.

The formation of the continental crust in the early Earth, and the geodynamics that drove it, are fundamental to understanding the evolution of our planet, but remain intensely debated. Here, we analysed 148 archived zircon separates of magmatic rocks for in-situ zircon U-Pb-Hf-O-trace element data, and compiled published geochronological, whole-rock geochemical and Sm-Nd isotopic data from across the south-east Superior Craton, Canada. We combine these data spatially and temporally to investigate the crustal evolution of this part of the craton in the Neoarchean, with a view to understanding its tectonic setting. In terms of zircon data, at >2704–2695 Ma, the central and north-west Abitibi demonstrate more juvenile eHf, light to mantle-like δ18O, lower (Eu/Eu*)/Y (drier/shallower crust), reduced ΔFMQ, less continental initial-U (Ui)/Yb, and more mantle-like Ui/Nb, relative to surrounding crust, which contains older, ca. 2800–2750 Ma inherited and magmatic zircon ages. Furthermore, whole-rock Sr/Y and La/Sm demonstrate the presence of a high Sr/Y TTG component (mainly intrusive) surrounding zones of low Sr/Y (mainly volcanic) component, the latter of which shows contamination trends with Mesoarchean crust. We interpret this to represent a continental-rift setting, driven by plume magmatism as represented by multiple komatiite suites. At ca. 2704–2695 Ma, there is a marked transition in multiple datasets, including; increases in δ18O, (Eu/Eu*)/Y, ΔFMQ, Ui/Yb and Ui/Nb data, together with more distinct arc-like trace element trends, suggesting a transition to north-dipping subduction. This process closed the rift system and initiated orogenesis. Subsequently, this study constrains the geodynamic setting which formed the majority of Neoarchean continental crust in the south-east Superior Craton, and the timing at which it transitioned to subduction. If these findings are replicated in other cratons, it suggests that plate tectonics was active by, or started at, ca. 2.7 Ga.

The late Proterozoic Ntaka Ultramafic Complex is a body of dominantly pyroxenitic cumulate rocks containing cyclic alternations of olivine-orthopyroxene cumulates. Chemical zoning in the pyroxenes has been imaged at 25-40 μm resolution using desktop microbeam X-ray fluorescence mapping followed up with laser ablation-inductively coupled plasma mass spectrometry analysis for minor and trace elements on selected samples. Poikilitic and granular harzburgites are finely intermingled, in some cases on a centimetre scale in the same thin section. Poikilitic varieties display spectacular textures, ranging from isolated equant orthopyroxene oikocrysts within olivine-rich heteradcumulate harzburgites to rocks composed entirely of interlocking centimetre-sized anhedral orthopyroxene oikocrysts containing sharply bounded idiomorphic Cr-enriched cores. The poikilitic harzburgites are interlayered with cumulate pyroxenites in which orthopyroxene grains show a variety of zoning patterns: Cr-rich cores similar to those in the oikocrysts; sharply bounded oscillatory zoned cores; and reverse zoning with Cr-poor cores and Cr-enriched rims. A further variation is the presence of a mingled harzburgite lithology in which dunite or poikilitic harzburgite is invaded on a centimetre scale by diffuse vein networks or patches of coarse orthopyroxenite. This range of textures and lithologies attests to a more complex set of processes than implied by the standard cumulus theory model in which oikocrysts are considered to have crystallized from intercumulus liquid within a permeable crystal mush. A range of hypotheses is proposed, including infiltration metasomatism of original olivine cumulates by migrating orthopyroxene-saturated pore fluid; however, the textural relationships, whole-rock chemistry and Cr zoning within the grains can best be explained by a model in which the orthopyroxene oikocrysts form in part or whole as mechanically accumulated cumulus grains. The complexity of zoning patterns is attributed to stirring of entrained olivine and orthopyroxene crystals within a heterogeneous flowing crystal mush, where the transporting magma has a wide range of silica contents owing to poorly stirred incorporation of siliceous country-rock material. The Cr-rich orthopyroxenite component grew from Sienriched chromite-saturated magma. Mingled lithologies developed after accumulation as a result of percolation and infiltration metasomatism by Si-enriched liquid derived by melting of xenoliths within the crystal pile. The model may be more generally applicable: dunite-harzburgite cycles, common in many layered intrusions, may reflect variable degrees of contamination rather than cycles of fractional crystallization and replenishment.

The purpose of this study was to determine whether specific components of the HIF-transcriptional pathway are under selective pressure during the development of cancer. Induction of HIF is a common feature of most cancers that frequently results from intra-tumour hypoxia but may also be activated as a result of oncogenic switching. Activation of such large transcriptional pathways commonly leads to heterogeneous effects on tumour biology that entrain multiple responses, which both promote and inhibit tumour formation. This generates a Darwinian pressure that selects for variants (either germline or somatic) that re-balance the numerous (often small) opposing effects of pathway activation to promote tumourigenesis. We have undertaken pangenomic analyses of the direct and indirect transcriptional targets of the HIF pathway in cell lines from six common cancer types (breast, prostate, lung, colon, kidney and liver) using combined ChIP-seq and RNA-seq assays. This identifies a small set of common HIF-target genes affecting discrete pathways as well as much larger sets of target genes that are cancer-type specific. Correlating these findings with RNA-seq analyses across 20 tumour types in the TCGA database confirms distinct patterns of expression and the importance of these genes to tumour biology. Furthermore, by relating our datasets to GWAS signals of cancer-susceptibility in each tumour type, we have identified a significant excess overlap between cancer-susceptibility loci and HIF-target genes. Notably, despite the importance of HIF in other tumour types, this overlap is specific to kidney cancer in which, by contrast, HIF activation, resulting from oncogenic switching, is inappropriate to the level of intra-tumour oxygenation. This analysis provides a comprehensive analysis of the HIF pathway across multiple cancer types and provides evidence that specific aspects of the HIF-pathway are under Darwinian selection in kidney cancer. Since these alter kidney cancer susceptibility, this analysis helps distinguish those components of the HIF transcriptional response that drive tumour formation from those that are simply co-activated as a consequence of large-pathway activation. Finally, this work supports a ‘pathway tuning' model of cancer and suggests that subtle re-balancing of inappropriately activated, cancer-specific pathways is important in oncogenesis. Citation Format: David R. Mole, Virginia Schmid, Olivia Lombardi, Silvia Halim, Veronique N. Lafleur, Ran Li, Rafik Salama, Leandro Colli, Stephen Chanock, Peter J. Ratcliffe. Pan-cancer analysis of the HIF-transcriptional pathway and its association with genetic susceptibility to cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3436.

The Savannah (formerly Sally Malay) Ni-Cu-Co Camp is composed of a group of mineralised intrusions (Savannah and Savannah North) and petrologically similar mafic-ultramafic intrusions without known mineralisation (Sub-chamber D, Dave Hill, and Wilson’s Creek). The results of a wide range of analytical techniques (geochemical, geochronological and geophysical) are integrated to gain a better understanding of the geometry of the Savannah mineralised magmatic intrusions and the timing of events. Magnetic anisotropy results suggest that Savannah and Savannah North have both been affected by similar deformation events, implying near-synchronous intrusion. However, Savannah and Savannah North show evidence of fundamentally different styles of deformation (strong folding at Savannah, as opposed to gentle tilting at Savannah North). This discrepancy can be reconciled with a resolvable variation in age, with Savannah North being the younger by ca. 0.8–2.5 Ma, as well as difference in the geometry and associate rheological properties of the intrusions: the larger more equant Savannah North intrusion may have been more resistant to deformation than the thinner elongated Savannah body. Isotopic and geochemical data on the mafic-ultramafic intrusions of the Savannah Ni-Cu-Co Camp indicate that the entire intrusive suite, with the exception of the early mafic granulite dykes, was derived from a similar parent magma of basaltic composition with likely continental arc affinity. Results of sulfide tenor calculations combined with mineral chemistry and in-situ S isotope data provide insight into ore genetic processes: in both the Savannah and Savannah North intrusions tenors are low, with median values in semi-massive ores of 3.8% Ni and 0.9% Cu in 100% sulfide for Savannah and 2.7% Ni and 1.0% Cu for Savannah North. PGE tenors are similar and extremely low in both deposits, ranging from below 5 ppb to 200 ppb for Pt (median 6 ppb) and from 40 ppb to 480 ppb (median 110) for Pd, with Pd/Ir of 25–160 and Pd/Pt of 3–50 (10th and 90th percentiles). For both Savannah and Savannah North, S isotopic analyses indicate a limited amount of incorporation of crustal S, and the contaminant does not appear to have been the immediately adjacent Tickalara Metamorphics, implying either a deeper crustal contaminant, or a component of subduction-derived crustal S in the mantle source . Finally, olivine records a history of postcumulus re-equilibration with trapped silicate melt and cumulus sulfide liquid, giving rise to distinctive compositional trends. The Savannah Ni-Cu-Co Camp provides further evidence that arc and collisional environments are conducive to the formation of intrusion-hosted orthomagmatic Ni-Cu-Co deposits. The multidisciplinary approach that we describe here allowed us to unravel the petrological, structural, and mineralisation history of a cryptic but prospective region, and provided insights to help evaluate the prospectivity of neighbouring intrusions. This approach can be used in a wide range of settings to resolve critical factors such as S source, timing of intrusion and mineralisation, and structural controls.

Hypoxia is associated with aggressive and poor prognosis of breast cancer. Generally, pan-genome analyses of hypoxia have focussed on protein-coding genes, however, the role of non-coding RNAs, in particular long non-coding RNAs (lncRNA) in hypoxia is not well characterised. We undertook an integrated genomic analysis of the hypoxic transcriptome in MCF7 breast cancer cells, employing total RNA-seq together with ChIP-seq for the hypoxia-inducible transcription factor (HIF) and for epigenetic marks of transcriptional activation (RNApol2 and histone H3K4me3). Analyses revealed that all classes of RNA are significantly regulated by hypoxia including piwiRNA, miRNA, tRNA, and sn/snoRNA. Significant numbers of lncRNAs were upregulated in hypoxia and were associated with increased RNApol2 and H3K4me3 markers and with HIF binding, indicating direct transcriptional activation of lncRNAs by HIF. The most hypoxiclly upregulated lncRNA was NEAT1, which is a direct transcriptional target of HIF-2a but not HIF-1a. The role of NEAT1 in cancer has not been previously studied. We demonstrated that hypoxic NEAT1 induction is common in breast cancer cell lines and xenografts models treated with bevacizumab. NEAT1 directly induces the formation of nuclear paraspeckle bodies in hypoxia. Moreover, it contributes to tumourigenicity by increasing cell proliferation, colony formation, and reducing apoptosis. In addition, we report that NEAT1 is required to retain hypoxia induced hyper edited Junctional Adhesion Molecule A (JAM-A) mRNA in the nucleus, thus preventing export into the cytoplasm for translation. Finally, in a large cohort of 2000 breast cancers, high levels of NEAT1 were associated with poor clinical outcomes and clinicopathological features. Our results extend knowledge of the hypoxic transcriptional response into the spectrum of non-coding transcripts. These findings provide novel mechanisms of transcriptional regulation in hypoxia and open new avenues to find novel pathways and targets to develop therapies for breast cancer. Citation Format: Hani Choudhry, Johannes Schodel, Ashwag Albukhari, Syed Haider, Francesca Buffa, Peter J Ratcliffe, David R Mole, Ioannis Ragousis, Adrian L Harris. The non-coding transcriptome of hypoxic breast cancer: Novel insights of clinical relevant long non-coding RNA in hypoxia signalling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-01.

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. OA gold

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output., Ministerio de Ciencia e Innovacion (Spanish Ministry of Science and Innovation, MICINN) [SAF2011 24225 to L.d.P., SAF2014–53819-R to L.d.P., B.J.]; Canadian Institutes of Health Research (CIHR) [MOP-82875 to W.W.).]; Natural Sciences and Engineering Research Council of Canada (NSERC) [RGPIN355532–10 to W.W.W.]; National Institutes of Health [1R01GM084875 to W.W.W.]; CIHR Fellowship (to R.W.H.); Michael Smith Foundation for Health Research Fellowship (to R.W.H.); Caja Madrid Foundation for Visiting Professor Fellowship (to L.d.P). Funding for open access charge: Spanish Ministry of Science and Innovation, MICINN, [SAF2014-53819-R].

The formation of structurally controlled lode-gold deposits is a controversial subject with both magmatic and metamorphic fluids considered a feasible source for gold. Here we present a study from the Southern Cross district (Yilgarn Craton, Western Australia), where structurally controlled Neoarchean gold deposits are hosted in amphibolite facies greenstones. We combine geochronological data with structural analysis to constrain the timing and structural control of ore deposition. We show that gold mineralization took place between c. 2638 and 2630 Ma, contemporaneous with the onset of voluminous intrusions of Low-Ca, I-type granites, which are ubiquitous in Southern Cross and elsewhere in the Yilgarn Craton. We argue that the timing of the intrusions, their spatial association with gold deposits, their chemical composition, and isotopic signature are consistent with Low-Ca granites being a major fluid source for mineralization. We propose that the fluid release by cooling plutons was facilitated by transport along active amphibolite facies shear zones. As gold mineralization was synchronous with Low-Ca magmatism across large areas of the Yilgarn Craton, our model may be applicable to orogenic gold deposits in other parts of the Yilgarn Craton as well as other Archean granite-greenstone terrains with a ‘late granite bloom’.