Inherited causes of clonal haematopoiesis in 97,691 whole genomes

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown1. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer2,3,4 and coronary heart disease5—this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP)6. Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays. Keywords: single-cell RNA-seq; pooled screen; CRISPR; epistasis; genetic interaction

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex

Changes in Ventilatory Threshold at High Altitude: Effect of Antioxidants

Purpose: To investigate the effects of prolonged hypoxia and antioxidant supplementation on ventilatory threshold (VT) during high-altitude (HA) exposure (4300 m). Methods: Sixteen physically fit males (25 ± 5 yr; 77.8 ± 8.5 kg) performed an incremental test to maximal exertion on a cycle ergometer at sea level (SL). Subjects were then matched on VO2peak, ventilatory chemosensitivity, and body mass and assigned to either a placebo (PL) or antioxidant (AO) supplement group in a randomized, double-blind manner. PL or AO (12 mg of β-carotene, 180 mg of α-tocopherol acetate, 500 mg of ascorbic acid, 100 μg of selenium, and 30 mg of zinc daily) were taken 21 d prior to and for 14 d at HA. During HA, subjects participated in an exercise program designed to achieve an energy deficit of approximately 1400 kcald-1. VT was reassessed on the second and ninth days at HA (HA2, HA9). Results: Peak power output (W2peak) and VO2peak decreased (28%) in both groups upon acute altitude exposure (HA2) and were unchanged with acclimatization and exercise (HA9). Power output at VT (WVT) decreased from SL to HA2 by 41% in PL, but only 32% in AO (P \u3c 0.05). WVT increased in PL only during acclimatization (P \u3c 0.05) and matched AO at HA9. Similar results were found when VT was expressed in terms of % Wpeak and % VO2peak. Conclusions: VT decreases upon acute HA exposure but improves with acclimatization. Prior AO supplementation improves VT upon acute, but not chronic altitude exposure