Understanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutic and public health strategies. Viral-host interactions can guide discovery of disease regulators, and protein ...structure function analysis points to several immune pathways, including complement and coagulation, as targets of coronaviruses. To determine whether conditions associated with dysregulated complement or coagulation systems impact disease, we performed a retrospective observational study and found that history of macular degeneration (a proxy for complement-activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis and hemorrhage) are risk factors for SARS-CoV-2-associated morbidity and mortality-effects that are independent of age, sex or history of smoking. Transcriptional profiling of nasopharyngeal swabs demonstrated that in addition to type-I interferon and interleukin-6-dependent inflammatory responses, infection results in robust engagement of the complement and coagulation pathways. Finally, in a candidate-driven genetic association study of severe SARS-CoV-2 disease, we identified putative complement and coagulation-associated loci including missense, eQTL and sQTL variants of critical complement and coagulation regulators. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multimodal analytical approach to reveal determinants and predictors of immunity, susceptibility and clinical outcome associated with infection.
Recent studies have provided insights into the pathology of and immune response to COVID-19
. However, a thorough investigation of the interplay between infected cells and the immune system at sites ...of infection has been lacking. Here we use high-parameter imaging mass cytometry
that targets the expression of 36 proteins to investigate the cellular composition and spatial architecture of acute lung injury in humans (including injuries derived from SARS-CoV-2 infection) at single-cell resolution. These spatially resolved single-cell data unravel the disordered structure of the infected and injured lung, alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyperinflammatory cell state that is associated with lung damage. We leverage the temporal range of fatal outcomes of COVID-19 in relation to the onset of symptoms, which reveals increased macrophage extravasation and increased numbers of mesenchymal cells and fibroblasts concomitant with increased proximity between these cell types as the disease progresses-possibly as a result of attempts to repair the damaged lung tissue. Our data enable us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. We use this landscape to characterize the pathophysiology of the human lung from its macroscopic presentation to the single-cell level, which provides an important basis for understanding COVID-19 and lung pathology in general.
lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response ...to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.
H1 linker histones are the most abundant chromatin-binding proteins
. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes ...to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood
. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8
T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.
Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in ...Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2−/−;Flt3ITD progenitors (LSK CD48+CD150−) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3ITD mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.
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•Tet2−/− and Flt3ITD alleles cooperate to induce AML•LSK MPP cells propagate the disease and are resistant to anti-leukemic therapies•Tet2−/− and Flt3ITD cooperatively induce DNA hypermethylation and expression changes•Reactivation of Gata2 induces differentiation and inhibits leukemogenesis
Shih et al. show that combined mutations in Tet2 and Flt3 alter DNA methylation and gene expression to an extent not seen with either mutation alone and cause fully penetrant, lethal acute myeloid leukemia (AML) in mice. Similar characteristics are observed in human AML with combined TET2 and FLT3 mutations.
Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin ...mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function.
Telomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, and whose lengths can be correlated with age and adverse health ...risk factors. Yet, given their length and repetitive nature, telomeric regions are not easily reconstructed from short-read sequencing, thus making telomere sequencing, mapping, and variant resolution challenging problems. Recently, long-read sequencing, with read lengths measuring in hundreds of kilobase pairs, has made it possible to routinely read into telomeric regions and inspect their sequence structure. Here, we describe a framework for extracting telomeric reads from whole-genome single-molecule sequencing experiments, including de novo identification of telomere repeat motifs and repeat types, and also describe their sequence variation. We find that long, complex telomeric stretches and repeats can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize noncanonical telomere sequence motifs (both previously reported, as well as novel), and validate them in short-read sequence data. These data reveal extensive intra- and inter-population diversity of repeats in telomeric haplotypes, reveal higher paternal inheritance of telomeric variants, and represent the first motif composition maps of multi-kilobase-pair human telomeric haplotypes across three distinct ancestries (Ashkenazi, Chinese, and Utah), which can aid in future studies of genetic variation, aging, and genome biology.
The hematologic and metabolic benefits of high altitude exposure have been extensively studied in athletes due to their promising performance enhancing effects. However, despite the increased ...research and development of various high altitude protocols for achieving peak performance, the reproducibility of the results at the individual level remains sparse. To systematically address this limitation and establish a more effective method to achieve consistent results at the individual level, we conducted a multi-dimensional study of one elite endurance athlete in two Phases. In Phase 1, we applied the standard protocol of LHTH (Live-High-Train-High) using a commercially available, at-home, normobaric, high altitude simulation tent under the SHTL (Sleep-High-Train-Low) model. Then, we developed the athlete's personalized protocol for peak hematologic parameters during their off-season. This protocol determined the exact total high altitude exposure time required to achieve peak hematologic parameters, which in the case of this athlete, amounted to 45 nights with approximately 8hrs per night. In Phase 2, we replicated the Phase 1 protocol during the athlete's in-season and observed the same or even higher hematologic and metabolic benefits compared to Phase 1. During both phases, we collected thousands of multi-dimensional data points to ensure that the athlete's lifestyle and environmental factors remained stable, and to increase the likelihood that physiological changes resulted primarily from the high altitude exposure. The data trends in both Phases validated that, for this athlete, hematologic measures such as red blood cell count, hematocrit, and hemoglobin, as well as electrolyte content, body weight and gut microbiome composition improved to their personal best values after a total of approximately 15 days of high altitude exposure (45 nights with roughly 8hrs per night totaling 360hrs or 15days). These improvements did not occur after the 21 days recommended by the LHTH protocol highlighting the significance of personalization in high altitude protocols that are designed for peak performance parameters. Therefore, to maximize the benefits in hematologic and other metabolic values and thus increase muscle oxygen supply and peak aerobic capacity through high altitude exposure, each athlete may require a unique total duration of high altitude exposure tailored to their individual physiology. This duration must be determined by their specific response in hematologic peaking. Therefore, initially establishing a personalized protocol for an athlete by determining their required total duration of high altitude exposure for peak hematologic values during their off-season and applying this protocol during their in-season phase may lead to more successful and reproducible benefits compared to following a generalized protocol alone.
Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase ...paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.
Abstract
While early investigations into the physiological effects of spaceflight suggest the body's ability to reversibly adapt, the corresponding effects of long-term spaceflight (>6 months) are ...much less conclusive. Prolonged exposure to microgravity and radiation yields profound effects on the cardiovascular system, including a massive cephalad fluid translocation and altered arterial pressure, which attenuate blood pressure regulatory mechanisms and increase cardiac output. Also, central venous pressure decreases as a result of the loss of venous compression. The stimulation of baroreceptors by the cephalad shift results in an approximately 10%–15% reduction in plasma volume, with fluid translocating from the vascular lumen to the interstitium. Despite possible increases in cardiac workload, myocyte atrophy and notable, yet unexplained, alterations in hematocrit have been observed. Atrophy is postulated to result from shunting of protein synthesis from the endoplasmic reticulum to the mitochondria via mortalin-mediated action. While data are scarce regarding their causative agents, arrhythmias have been frequently reported, albeit sublethal, during both Russian and American expeditions, with QT interval prolongation observed in long, but not short duration, spaceflight. Exposure of the heart to the proton and heavy ion radiation of deep space has also been shown to result in coronary artery degeneration, aortic stiffness, carotid intima thickening via collagen-mediated action, accelerated atherosclerosis, and induction of a pro-inflammatory state. Upon return, long-term spaceflight frequently results in orthostatic intolerance and altered sympathetic responses, which can prove hazardous should any rapid mobilization or evacuation be required, and indicates that these cardiac risks should be especially monitored for future missions.
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