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Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs. We thank W. Liu and L. Xu from the Huazhen Laboratory Animal Breeding Centre for helping in the collection of monkey tissues, D. Zhu and H. Li from the Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory) for technical help, G. Guo and H. Sun from Zhejiang University for providing HCL and MCA gene expression data matrices, G. Dong and C. Liu from BGI Research, and X. Zhang, P. Li and C. Qi from the Guangzhou Institutes of Biomedicine and Health for experimental advice or providing reagents. This work was supported by the Shenzhen Basic Research Project for Excellent Young Scholars (RCYX20200714114644191), Shenzhen Key Laboratory of Single-Cell Omics (ZDSYS20190902093613831), Shenzhen Bay Laboratory (SZBL2019062801012) and Guangdong Provincial Key Laboratory of Genome Read and Write (2017B030301011). In addition, L.L. was supported by the National Natural Science Foundation of China (31900466), Y. Hou was supported by the Natural Science Foundation of Guangdong Province (2018A030313379) and M.A.E. was supported by a Changbai Mountain Scholar award (419020201252), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502), a Chinese Academy of Sciences–Japan Society for the Promotion of Science joint research project (GJHZ2093), the National Natural Science Foundation of China (92068106, U20A2015) and the Guangdong Basic and Applied Basic Research Foundation (2021B1515120075). M.L. was supported by the National Key Research and Development Program of China (2021YFC2600200). Sí

Supplementary Figure 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Figure 2 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Table 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development. This work is part of the ‘‘SpatioTemporal Omics Consortium’’ (STOC) paper package. A list of STOC members is available at: http://sto-consortium.org. We would like to thank the MOTIC China Group, Rongqin Ke (Huaqiao University, Xiamen, China), Jiazuan Ni (Shenzhen University, Shenzhen, China), Wei Huang (Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China), and Jonathan S. Weissman (Whitehead Institute, Boston, USA) for their help. This work was supported by the grant of Top Ten Foundamental Research Institutes of Shenzhen, the Shenzhen Key Laboratory of Single-Cell Omics (ZDSYS20190902093613831), and the Guangdong Provincial Key Laboratory of Genome Read and Write (2017B030301011); Longqi Liu was supported by the National Natural Science Foundation of China (31900466) and Miguel A. Esteban’s laboratory at the Guangzhou Institutes of Biomedicine and Health by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502), National Natural Science Foundation of China (92068106), and the Guangdong Basic and Applied Basic Research Foundation (2021B1515120075). Sí

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

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation

People can clearly incubate and transmit SARS-CoV-2 in the absence of symptoms. Nonetheless, the value of mass testing (large scale asymptomatic screening to identify cases) has been contested. In principle, isolation of people with pre-symptomatic or asymptomatic SARS-CoV-2 infection will prevent further spread. In practice, it is less clear whether enough infectious people can be identified through screening to have a quantitatively important effect on transmission, and whether the direct benefits of enhanced case ascertainment are outweighed by direct or indirect costs. The debate is complicated by an absence of data from randomised controlled trials, and controversy about the suitability of lateral flow tests for this purpose.

Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This brought to mind the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. We demonstrated the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes we also observed in B cell VHL-deficient mice. This was corroborated by hypoxia-related transcriptional changes in COVID-19 patients, and by similar B cell abnormalities in mice kept in hypoxic conditions, including reduced marginal zone and germinal center B cells. Thus hypoxia might contribute to B cell pathology in COVID-19, and in other hypoxic states. Through this mechanism it may impact on COVID-19 outcome, and be remediable through early oxygen therapy.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

A new phase I clinical trial has demonstrated that belzutifan provides a route to sustained inhibition of hypoxia-inducible factor 2α in humans that is well tolerated and could be useful in the treatment of advanced clear cell kidney cancer.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice

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.

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.

Genomics and other large-scale analyses have drawn increasing attention to the potential impacts of transposable elements (TEs) on their host genomes. However, it remains challenging to transition from identifying potential roles to clearly demonstrating the level of impact TEs have on genome evolution and possible functions that they contribute to their host organisms. I summarize TE content and distribution in four well-characterized yeast model systems in this review: the pathogensCandida albicansandCryptococcus neoformans, and the nonpathogenic speciesSaccharomyces cerevisiaeandSchizosaccharomyces pombe. I compare and contrast their TE landscapes to their lifecycles, genomic features, as well as the presence and nature of RNA interference pathways in each species to highlight the valuable diversity represented by these models for functional studies of TEs. I then review the regulation and impacts of the Ty1 and Ty3 retrotransposons fromSaccharomyces cerevisiaeand Tf1 and Tf2 retrotransposons fromSchizosaccharomyces pombeto emphasize parallels and distinctions between these well-studied elements. I propose that further characterization of TEs in the pathogenic yeasts would enable this set of four yeast species to become an excellent set of models for comparative functional studies to address outstanding questions about TE-host relationships.

Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans.Bullet pointsWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19.ACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2.ACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities.We unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry.

Background Mannose-binding lectin (MBL) is an important component of innate immune defense. MBL undergoes oligomerization to generate high mol weight (HMW) forms which act as pattern recognition molecules to detect and opsonize various microorganisms. Several post-translational modifications including prolyl hydroxylation are known to affect the oligomerization of MBL. Yet, the enzyme(s) which hydroxylate proline in the collagen-like domain residues have not been identified and the significance of prolyl hydroxylation is incompletely understood. Methods To investigate post-translational modifications of MBL, we stably expressed Myc-DDK tagged MBL in HEK293S cells. We used pharmacologic and genetic inhibition of 2-oxoglutarate–dependent dioxygenases (2OGDD) to identify the enzyme required for prolyl hydroxylation of MBL. We performed mass spectrometry to determine the effects of various inhibitors on MBL modifications. Results Secretion of HMW MBL was impaired by inhibitors of the superfamily of 2OGDD, and was dependent on prolyl-4-hydroxylase subunit α1. Roxadustat and vadadustat, but not molidustat, led to significant suppression of hydroxylation and secretion of HMW forms of MBL. Conclusions These data suggest that prolyl hydroxylation in the collagen-like domain of MBL is mediated by collagen prolyl-4-hydroxylase. Reduced MBL activity is likely to be an off-target effect of some, but not all, prolyl hydroxylase domain (PHD) inhibitors. There may be advantages in selective PHD inhibitors that would not interfere with MBL production.

Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1, and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons.

Von Hippel–Lindau (VHL) disease is a rare autosomal dominant condition with a high risk of renal carcinoma. The underlying tumour suppressor gene is also mutated in most sporadic clear cell renal carcinomas. The VHL gene product functions as an E3 ubiquitin ligase that mediates the degradation of hypoxia-inducible factor (HIF). Loss of VHL leads to constitutive activation of HIF target genes that normally mediate responses to hypoxia, including those that regulate diverse processes such as angiogenesis and cellular metabolism. Novel therapies for renal cancer target angiogenesis mediated by vascular endothelial growth factor (VEGF). A number of other kidney cancer genes have been found from studying other familial cancer syndromes. These genes mediate responses to cellular stress and nutrient deprivation. Key Concepts: Von Hippel–Lindau (VHL) disease is a dominantly inherited familial cancer syndrome caused by mutations in the VHL tumour suppressor gene. VHL disease exhibits striking genotype–phenotype correlation. The great majority of sporadic clear cell renal cancers also show loss of VHL function. The VHL gene product functions as an E3 ubiquitin ligase that mediates the degradation of hypoxia-inducible factor (HIF) alpha. Loss of VHL leads to constitutive activation of HIF target genes that play key roles in angiogenesis and metabolism. Novel therapies for renal cancer target specific effects of VHL loss, including inhibitors of angiogenesis. A number of other kidney cancer genes have been found to be involved in cellular pathways involved in cellular stress and nutrient deprivation. Keywords: tumour suppressor gene; ubiquitin; hypoxia-inducible factor; proteolysis; angiogenesis

The ability of cells to sense and adapt to changes in oxygen is mediated by hypoxia-inducible factor (HIF). Immune cells function in physiologically complex and varying environments whereby oxygen, pH, nutrients, metabolites and cytokines are continuously fluctuating. HIF is well known to play an important role in coordinating the adaptation and function of both innate immune cells and T cells in these complex environments. This review summarises recent discoveries concerning how hypoxia and HIF control B cell behaviour, and regulate antibody quality and decisions concerning tolerance. Hypoxia and HIF activation may provide an important context; coordinating metabolism with variable demands for quiescence, rapid proliferation, and differentiation. Understanding when and how HIF is activated during B cell development and response is important as drugs targeting HIF could influence antibody responses, providing novel therapeutic opportunities for vaccine adjuvants and in treating autoimmunity.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

Funder: Cancer Research UK; doi: http://dx.doi.org/10.13039/501100000289, Background: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. Methods: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. Results: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. Conclusions: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Brexit and the troubled state of the NHS call for re-thinking the UK's approach to health. The EU referendum vote reveals deep social divisions as well as presenting the country with important decisions and negotiations about the future. At the same time, health problems are growing; the NHS faces severe financial constraints and appears to lurch from crisis to crisis, with leaving the European Union likely to exacerbate many problems including staffing issues across the whole sector. However, new scientific developments and digital technology offer societies everywhere massive and unprecedented opportunities for improving health. It is vital for the country that the NHS is able to adopt these discoveries and see them translated into improved patient care and population health, but also that the UK benefits from its capabilities and strengths in these areas.

Background: Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.Methodology/Principal Findings: Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1 alpha or HIF-2 alpha knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.Conclusions/Significance: Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis. ispartof: NATURE vol:565 issue:7740 pages:511-+ ispartof: location:England status: published

B-lymphocyte development and selection is central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR)-signaling and occur in bone marrow, an environment with variable hypoxia, but whether Hypoxia-inducible factor (HIF) is involved is unknown. We show high HIF-activity in human bone marrow pro/preB cells that decreased at the immature B cell stage. Stage-specific HIF-suppression was required for B cell development, since constitutive HIF-1α-activation in murine B cells led to immature B cell developmental-arrest, limited BCR-editing and reduced repertoire-diversity. Disruption of HIF-1α-regulation reduced surface BCR and CD19 and increased expression of pro-apoptotic BIM. Transient administration of a clinical HIF-activator reduced immature and transitional B cells, confirming the on-going importance of this axis for the maintenance of the established B cell-compartment. Our work identifies a critical role for HIF-1α in tuning BCR-signal strength and demonstrates that dynamic HIF-regulation is essential for normal B cell development, with significant clinical implications.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

Pediatric and adult kidney tumors differUnderstanding tumor origins and the similarities and differences between organ-specific cancers is important for determining treatment options. Younget al.generated more than 72,000 single-cell transcriptomes from healthy and cancerous human kidneys. From these data, they determined that Wilms tumor, a pediatric kidney cancer, originates from aberrant fetal cells, whereas adult kidney cancers are likely derived from a specific subtype of proximal convoluted tubular cell.Science, this issue p.594

Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general. publisher: Elsevier articletitle: HIF-1α Promotes Glutamine-Mediated Redox Homeostasis and Glycogen-Dependent Bioenergetics to Support Postimplantation Bone Cell Survival journaltitle: Cell Metabolism articlelink: http://dx.doi.org/10.1016/j.cmet.2016.01.002 content_type: article copyright: Copyright © 2016 Elsevier Inc. All rights reserved. ispartof: Cell Metabolism vol:23 issue:2 pages:265-79 ispartof: location:United States status: published

SLC10A1 codes for the sodium-taurocholate cotransporting polypeptide (NTCP), which is a hepatocellular transporter for bile acids (BAs) and the receptor for hepatitis B and D viruses. NTCP is also a target of multiple drugs. We aimed to evaluate the medical consequences of the loss of function mutation p.Ser267Phe in SLC10A1. We identified eight individuals with homozygous p.Ser267Phe mutation in SLC10A1 and followed up for 8–90 months. We compared their total serum BAs and 6 species of BAs with 170 wild-type and 107 heterozygous healthy individuals. We performed in-depth medical examinations and exome sequencing in the homozygous individuals. All homozygous individuals had persistent hypercholanemia (P = 5.8 × 10–29). Exome sequencing excluded the involvement of other BA metabolism-associated genes in the hypercholanemia. Although asymptomatic, all individuals had low vitamin D levels. Of six adults that were subjected to bone mineral density analysis, three presented with osteoporosis/osteopenia. Sex hormones and blood lipids were deviated in all subjects. Homozygosity of p.Ser267Phe in SLC10A1 is associated with asymptomatic hypercholanemia. Individuals with homozygous p.Ser267Phe in SLC10A1 are prone to vitamin D deficiency, deviated sex hormones and blood lipids. Surveillance of these parameters may also be needed in patients treated with drugs targeting NTCP.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease. ispartof: Journal of Clinical Investigation vol:127 issue:9 pages:3407-3420 ispartof: location:United States status: published

A variety of mild stresses have been shown to extend lifespan in diverse species through hormesis, which is a beneficial response to a stress or toxin that would cause a negative response at a higher exposure. Whether particular stresses induce hormesis can vary with genotype for a given species, and the underlying mechanisms of lifespan extension are only partly understood in most cases. We show that low doses of the DNA damaging or replication stress agents hydroxyurea, methyl methanesulfonate, 4-nitroquinoline 1-oxide, or Zeocin (a phleomycin derivative) lengthened chronological lifespan in Saccharomyces cerevisiae if cells were exposed during growth, but not if they were exposed during stationary phase. Treatment with these agents did not change mitochondrial activity, increase resistance to acetic acid, ethanol, or heat stress, and three of four treatments did not increase resistance to hydrogen peroxide. Stationary phase yeast populations consist of both quiescent and nonquiescent cells, and all four treatments increased the proportion of quiescent cells. Several mutant strains with deletions in genes that influence quiescence prevented Zeocin treatment from extending lifespan and from increasing the proportion of quiescent stationary phase cells. These data indicate that mild DNA damage stress can extend lifespan by promoting quiescence in the absence of mitohormesis or improved general stress responses that have been frequently associated with improved longevity in other cases of hormesis. Further study of the underlying mechanism may yield new insights into quiescence regulation that will be relevant to healthy aging.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Oxygen‐dependent regulation of the erythropoietin gene is mediated by the hypoxia‐inducible factor (HIF) family of transcription factors. When oxygen is plentiful, HIF undergoes hydroxylation by a family of oxygen‐dependent prolyl hydroxylase domain (PHD) proteins, promoting its association with the von Hippel‐Lindau (VHL) ubiquitin E3 ligase and subsequent proteosomal degradation. When oxygen is scarce, the PHD enzymes are inactivated, leading to HIF accumulation and upregulation not only of erythropoietin expression, but also the expression of hundreds of other genes, including those coordinating cardiovascular and ventilatory adaptation to hypoxia. Nevertheless, despite the identification of over 50 mutations in the PHD‐HIF‐VHL pathway in patients with previously unexplained congenital erythrocytosis, there are very few reports of associated cardiopulmonary abnormalities. We now report exaggerated pulmonary vascular and ventilatory responses to acute hypoxia in a 35‐year‐old man with erythrocytosis secondary to heterozygous mutation in PHD2, the most abundant of the PHD isoforms. We compare this phenotype with that reported in patients with the archetypal disorder of cellular oxygen sensing, Chuvash polycythemia, and discuss the possible clinical implications of our findings, particularly in the light of the emerging role for small molecule PHD inhibitors in clinical practice.