Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type-Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney.

Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type-specific responses to Vhl inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma. SIGNIFICANCE: Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.

Multi-level interaction between HIF and AHR transcriptional pathways in kidney carcinoma.

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-1ß/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-1ß/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.

Pan-cancer analysis of tissue and single-cell HIF-pathway activation using a conserved gene signature.

Activation of cellular hypoxia pathways, orchestrated by HIF (hypoxia-inducible factor) transcription factors, is a common feature of multiple tumor types, resulting from microenvironment factors and oncogenic mutation. Although they help drive many of the "hallmarks" of cancer and are associated with poor outcome and resistance to therapy, the transcriptional targets of HIF vary considerably depending on the cell type. By integrating 72 genome-wide assays of HIF binding and transcriptional regulation from multiple cancer types, we define a consensus set of 48 HIF target genes that is highly conserved across cancer types and cell lineages. These genes provide an effective marker of HIF activation in bulk and single-cell transcriptomic analyses across a wide range of cancer types and in malignant and stromal cell types. This allows the tissue-orchestrated responses to the hypoxic tumor microenvironment and to oncogenic HIF activation to be deconvoluted at the tumor and single-cell level.

Altered regulation of DPF3, a member of the SWI/SNF complexes, underlies the 14q24 renal cancer susceptibility locus.

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 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.

Oxygen isotopes trace the origins of Earth's earliest continental crust.

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.

Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.

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.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

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.

Hypoxic Regulation of Gene Transcription and Chromatin: Cause and Effect.

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.

Hypoxic microenvironment shapes HIV-1 replication and latency.

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.

Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.

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.

Co-incidence of RCC-susceptibility polymorphisms with HIF cis-acting sequences supports a pathway tuning model of cancer.

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.

No evidence for high-pressure melting of Earth's crust in the Archean.

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.

Case report of oxalate nephropathy in a patient with pancreatic metastases from renal carcinoma.

BACKGROUND: Patients with metastatic renal carcinoma frequently have pre-existing renal impairment and not infrequently develop worsening renal function as a complication of their treatment. The presence of pancreatic metastases in patients with metastatic renal carcinoma, often confers a more favourable prognosis and as a consequence this patient group may be exposed to such treatments for more prolonged periods of time. However, the development of renal failure may also be a consequence of the cancer itself rather than its treatment. CASE PRESENTATION: We present an 84-year-old patient receiving the tyrosine kinase inhibitor (TKI) pazopanib for metastatic renal carcinoma who developed oxalate nephropathy as a consequence of pancreatic exocrine insufficiency resulting from pancreatic metastases. CONCLUSIONS: This case demonstrates the importance of investigating unexpected toxicities and highlights the potential consequences of pancreatic insufficiency and its sequelae in patients with pancreatic metastases.

MITF controls the TCA cycle to modulate the melanoma hypoxia response.

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.

Inherent DNA-binding specificities of the HIF-1α and HIF-2α transcription factors in chromatin.

Hypoxia-inducible factor (HIF) is the major transcriptional regulator of cellular responses to hypoxia. The two principal HIF-α isoforms, HIF-1α and HIF-2α, are progressively stabilized in response to hypoxia and form heterodimers with HIF-1ß to activate a broad range of transcriptional responses. Here, we report on the pan-genomic distribution of isoform-specific HIF binding in response to hypoxia of varying severity and duration, and in response to genetic ablation of each HIF-α isoform. Our findings reveal that, despite an identical consensus recognition sequence in DNA, each HIF heterodimer loads progressively at a distinct repertoire of cell-type-specific sites across the genome, with little evidence of redistribution under any of the conditions examined. Marked biases towards promoter-proximal binding of HIF-1 and promoter-distant binding of HIF-2 were observed under all conditions and were consistent in multiple cell type. The findings imply that each HIF isoform has an inherent property that determines its binding distribution across the genome, which might be exploited to therapeutically target the specific transcriptional output of each isoform independently.

The SIN3A histone deacetylase complex is required for a complete transcriptional response to hypoxia.

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.

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway.

Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.

Genetic variation at the 8q24.21 renal cancer susceptibility locus affects HIF binding to a MYC enhancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel-Lindau tumour suppressor (VHL) and unrestrained activation of hypoxia-inducible transcription factors (HIFs). Genetic and epigenetic determinants have an impact on HIF pathways. A recent genome-wide association study on renal cancer susceptibility identified single-nucleotide polymorphisms (SNPs) in an intergenic region located between the oncogenes MYC and PVT1. Here using assays of chromatin conformation, allele-specific chromatin immunoprecipitation and genome editing, we show that HIF binding to this regulatory element is necessary to trans-activate MYC and PVT1 expression specifically in cells of renal tubular origins. Moreover, we demonstrate that the risk-associated polymorphisms increase chromatin accessibility and activity as well as HIF binding to the enhancer. These findings provide further evidence that genetic variation at HIF-binding sites modulates the oncogenic transcriptional output of the VHL-HIF axis and provide a functional explanation for the disease-associated effects of SNPs in ccRCC.