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Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo , upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes. [Display omitted] • Profiling 3D genome architecture reveals a boundary between Il22 and Ifng enhancers • Deletion of a 17 bp CTCF-binding motif dysregulates Il22 and Ifng expression in Th1 cells • The mutation does not impair Ifng production in NK cells • The mutant mice are susceptible to Toxoplasma gondii infection Multilayered gene regulation orchestrates cell-type-specific cytokine expression. Liu et al. examine the impact of chromatin loop formation on the expression of IFN-γ and IL-22, which are cytokine loci closely located in the genome, and identify an inducible topologically associating domain required for proper IFN-γ production in Th1 cells. Deleting a CTCF-binding site within this domain leads to lineage-specific cytokine dysregulation and increased susceptibility to pathogens. [ABSTRACT FROM AUTHOR]

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1–4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences. Engineered variants of DNA base editors have reduced RNA off-target editing while retaining DNA on-target efficiency. [ABSTRACT FROM AUTHOR]

Summary Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution. [ABSTRACT FROM AUTHOR]

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5′ end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5′ ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production. [Display omitted] • SND1 is required for nascent SARS-CoV-2 RNA synthesis early during infection • SND1 directly interacts with NSP9 and both proteins bind negative-sense viral RNA • NSP9 is covalently linked to viral RNA at initiation sites, indicating protein priming • SND1 modulates the covalent linkage of NSP9 to positive- and negative-sense viral RNA Mapping of subgenome-resolved SARS-CoV-2 RNA-protein interactions reveals that the host protein SND1 binds negative-sense SARS-CoV-2 RNA and promotes viral RNA synthesis by recruiting NSP9, which likely serves as a protein primer for RNA production. [ABSTRACT FROM AUTHOR]

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis -chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype. [Display omitted] • Cohesin-SA2 occupies enhancer and PRC2-marked regulatory regions in Ewing sarcoma • STAG2 depletion alters a subset of EWS/FLI1 anchored enhancer-promoter interactions • Loss of STAG2 modifies oncogenic and developmental transcriptional programs • STAG2 loss enhances the migratory and metastatic potential of Ewing sarcoma cells Adane et al. demonstrate that deletion of STAG2 changes the distribution of cohesin complexes and leads to reprograming of cis -chromatin interactions in Ewing sarcoma. STAG2 loss attenuated EWS/FLI1-driven oncogenic programs and disrupted PRC2-regulated developmental processes to enhance the metastatic potential of Ewing sarcoma cells. [ABSTRACT FROM AUTHOR]

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance. [Display omitted] • Enhancers activated at Foxp3-CNS0 and -CNS3 are indispensable for thymic Treg generation • CNS0 and CNS3 interact with other enhancers and Foxp3 promoter during Treg development • Deletion of both CNS0 and CNS3 causes lethal autoimmunity • CNS0 is responsible for IL-2-dependent induction and maintenance of Foxp3 expression. The Foxp3+ Treg generation process in the thymus has not been fully understood, especially in the perspective of genomic enhancer coordination. Kawakami et al. reveal that two independently activated enhancers, Foxp3-CNS0 and Foxp3-CNS3, cooperatively induce and maintain Foxp3 expression for the establishment of self-tolerance. [ABSTRACT FROM AUTHOR]

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis- regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues. • Cell states marked by chromatin and gene expression are correlated but distinct • Lineage-determining genes are marked by domains of regulatory chromatin (DORCs) • DORCs are accessible prior to gene expression, foreshadowing lineage choice • Chromatin accessibility lineage-priming predicts cell fate decisions SHARE-seq, a highly scalable approach to measure chromatin accessibility and gene expression in the same single cell, links distal regulatory elements to key lineage-specifying genes and finds that chromatin accessibility foreshadows gene expression. [ABSTRACT FROM AUTHOR]

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic. • Hierarchical layers of nuclear architecture are altered in colorectal tumors • An intermediate genome compartment is defined in primary tissues • Compartmental reorganization and hypomethylation occur in tumors and aging cells • Reorganization is associated with tumor-suppressive transcriptional programs Integrated analyses of genome topological, epigenetic, and transcriptional features of colorectal tumors highlight substantial genome compartmental reorganization associated with tumor-suppressive rather than oncogenic transcriptional outcomes. [ABSTRACT FROM AUTHOR]

Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression. • Cancer progression is marked by a continuum of heterogeneous epigenomic states • Lung cancer cells adopt features of other cell identities across tumor evolution • RUNX2 transcription factor activity is associated with a pre-metastatic cell state • Regulatory programs defined from mouse models are useful predictive biomarkers Using a generalizable framework to leverage single-cell chromatin accessibility data to investigate cell state transitions across tumor evolution in a mouse model of lung adenocarcinoma, LaFave et al. elucidate a pre-metastatic cell state that arises in primary tumors prior to metastasis. [ABSTRACT FROM AUTHOR]

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing identifies rare populations that express specific marker transcript combinations, traditional flow sorting requires cell surface markers with high-fidelity antibodies, limiting our ability to interrogate these populations. In addition, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. In the present report, we addressed these limitations by developing Programmable Enrichment via RNA FlowFISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations defined by the abundance of specific RNA transcripts. Across immune populations (n = 184,126 cells) and fresh-frozen and formalin-fixed, paraffin-embedded brain tissue (n = 33,145 nuclei), we demonstrated that programmable sorting logic via RNA-based cytometry can isolate rare cell populations and uncover phenotypic heterogeneity via downstream, high-throughput, single-cell genomics analyses., Competing Interests: Competing interests: A.T.S. is a founder of Immunai, Cartography Biosciences, Santa Ana Bio and Prox Biosciences, an advisor to Wing Venture Capital and receives research funding from Astellas and Merck Research Laboratories. R.R.S., L.S.L. and C.A.L. are consultants to Cartography Biosciences. R.C. is a consultant for Sanavia Oncology, S2 Genomics and LevitasBio. The other authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Patients treated with chimeric antigen receptor T-cell (CAR-T) therapy are subject to profound immune suppression. Dynamics of immune reconstitution (IR) and impacts of IR on outcomes following infusion across CAR-T products are not well understood. Here, we profiled IR in 263 patients with relapsed/refractory large B-cell lymphoma receiving CAR-T therapy (axicabtagene ciloleucel 44.9%, lisocabtagene maraleucel 30.4%, tisagenlecleucel 24.7%). Following infusion, patients remain persistently immunosuppressed, with 48.1% having CD4+ T cell counts <200/µL and median CD3-19+ B cell counts remaining zero through 1 year after CAR-T. IR differences exist by product, with fastest CD4+ T cell recovery seen for tisagenlecleucel, driven primarily by more rapid recovery of the CD4+CCR7-45RA- effector memory subset. Natural killer cell, but not CD4+ T cell, recovery is significantly associated with favorable progression-free (HR: 0.647; 95% CI: 0.476-0.880) and overall survival (HR: 0.637; 95% CI: 0.441-0.920) and inversely correlated with inflammatory markers measured at time of infusion.

Targeted antibody-based therapy for oncology represents a highly efficacious approach that has demonstrated robust responses against single tumor-associated antigen (TAA) targets. However, tumor heterogeneity presents a major obstacle for targeting most solid tumors due to a lack of single targets that possess the right on-tumor/off-tumor expression profile required for adequate therapeutic index. Multi-targeting antibodies that engage two TAAs simultaneously may address this challenge through Boolean logic-gating function by improving both therapeutic specificity and efficacy. In addition to the complex engineering of multi-targeting antibodies for ideal logic-gate function, selecting optimal TAA combinations ab initio is the critical step to initiate preclinical development but remains largely unexplored with modern data-generation platforms. Here, we propose that single-cell atlases of both primary tumor and normal tissues are uniquely positioned to unveil optimal target combinations for multi-targeting antibody therapeutics. We review the most recent progress in multi-targeting antibody clinical development, as well as the designs of current TAA combinations currently exploited. Ultimately, we describe how multi-targeting antibodies tuned to target pairs nominated through a data-driven process are poised to revolutionize therapeutic safety and efficacy, particularly for difficult-to-treat solid tumors., Competing Interests: MN, JV, and DK are employees and equity holders of Cartography Biosciences. CL is an equity holder of Cartography Biosciences., (Copyright © 2024 Nix, Lareau, Verboon and Kugler.)

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations coevolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Furthermore, detection of mtDNA mutations via single-cell assay for transposase-accessible chromatin with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells but also their phenotype at frequencies of 0.1% to 1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh insights into residual donor-derived engraftment and short-term clonal evolution following therapy for post-transplant leukemia relapse. As throughput evolves, we envision future development of single-cell sequencing-based post-transplant monitoring as a powerful approach for guiding clinical decision-making. Significance: mtDNA mutations enable single-cell tracking of leukemic clonal evolution and donor-recipient origin following allogeneic HSCT. This provides unprecedented insight into chimeric cellular phenotypes of early immune reconstitution, incipient relapse, and quality of donor engraftment with immediate translational potential for future clinical post-transplant monitoring and decision-making., (©2024 American Association for Cancer Research.)

Single cell sequencing technologies have become a fixture in the molecular profiling of cells due to their ease, flexibility, and commercial availability. In particular, partitioning individual cells inside oil droplets via microfluidic reactions enables transcriptomic or multi-omic measurements for thousands of cells in parallel. Complementing the multitude of biological discoveries from genomics analyses, the past decade has brought new capabilities from assay baselines to enable a deeper understanding of the complex data from single cell multi-omics. Here, we highlight four innovations that have improved the reliability and understanding of droplet microfluidic assays. We emphasize new developments that further orient principles of technology development and guidelines for the design, benchmarking, and implementation of new droplet-based methodologies., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease., (© 2024. Springer Nature America, Inc.)

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics. The majority of these variants have individually weak effects and lie in non-coding gene-regulatory elements where we lack a complete understanding of how single nucleotide alterations modulate transcriptional processes to affect human phenotypes. To address this, we measured the activity of 221,412 trait-associated variants that had been statistically fine-mapped using a Massively Parallel Reporter Assay (MPRA) in 5 diverse cell-types. We show that MPRA is able to discriminate between likely causal variants and controls, identifying 12,025 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% can plausibly be explained by the disruption of a known transcription factor (TF) binding motif. We dissect the mechanisms of 136 variants using saturation mutagenesis and assign impacted TFs for 91% of variants without a clear canonical mechanism. Finally, we provide evidence that epistasis is prevalent for variants in close proximity and identify multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants underlying complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk., Competing Interests: Competing Interests PCS is a co-founder of and consultant to Sherlock Biosciences and Board Member of Danaher Corporation. PCS and RT hold patents related to the application of MPRA. JCU and FA are employees of Illumina. QSW is an employee of Calico Life Sciences LLC. ZRM is an employee of insitro.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo 1 . The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy 2-6 , suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states., (© 2024. The Author(s).)

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations ( n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue ( n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts., Competing Interests: Competing interests A.T.S. is a founder of Immunai, Cartography Biosciences, and Prox Biosciences, an advisor to Zafrens, Pallando Therapeutics, and Wing Venture Capital, and receives research funding from Merck Research Laboratories. R.R.S., L.S.L., and C.A.L. are consultants to Cartography Biosciences. R.C. is a consultant for Sanavia Oncology, S2 Genomics, and LevitasBio.

3-dimensional (3D) genome conformation is central to gene expression regulation, yet our understanding of its contribution to rapid transcriptional responses, signal integration, and memory in immune cells is limited. Here, we study the molecular regulation of the inflammatory response in primary macrophages using integrated transcriptomic, epigenomic, and chromosome conformation data, including base pair-resolution Micro-Capture C. We demonstrate that interleukin-4 (IL-4) primes the inflammatory response in macrophages by stably rewiring 3D genome conformation, juxtaposing endotoxin-, interferon-gamma-, and dexamethasone-responsive enhancers in close proximity to their cognate gene promoters. CRISPR-based perturbations of enhancer-promoter contacts or CCCTC-binding factor (CTCF) boundary elements demonstrated that IL-4-driven conformation changes are indispensable for enhanced and synergistic endotoxin-induced transcriptional responses, as well as transcriptional memory following stimulus removal. Moreover, transcriptional memory mediated by changes in chromosome conformation often occurred in the absence of changes in chromatin accessibility or histone modifications. Collectively, these findings demonstrate that rapid and memory transcriptional responses to immunological stimuli are encoded in the 3D genome., Competing Interests: A.T.S. is a founder of Immunai, Cartography Biosciences, and Prox Biosciences, an advisor to Zafrens, Pallando Therapeutics, and Wing Venture Capital, and receives research funding from Merck Research Laboratories. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, Cartography Biosciences, Orbital Therapeutics, and an advisor of 10x Genomics, Arsenal Biosciences, Chroma Medicine, Exai Bio, and Spring Discovery. B.D. is an employee of Genentech. K.S. is an employee of Cartography Biosciences. K.E.Y. is a consultant for Cartography Biosciences. C.A.L. is a consultant of Cartography Biosciences.

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6) 1 . Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4 + T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration 2 or are in clinical studies 3-5 , we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials 1,6-8 and may influence the design and production of autologous and allogeneic cell therapies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12 + dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19 + IL4I1 + fibroblasts, and clonally expanded IL13 + IL22 + IL26 + T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13 + IL22 + IL26 + T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy., Competing Interests: Declaration of Interests R.M. is on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, and 858 Therapeutics and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. R.M. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.A.L. is a consultant for Cartography Biosciences.

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called 'mitochondrial single-cell assay for transposase-accessible chromatin with sequencing' (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3-4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery., (© 2023. Springer Nature Limited.)

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize murine cancer models., Significance: mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities., (©2022 American Association for Cancer Research.)

Mitochondria are unique organelles of eukaryotic cells that carry their own multicopy number and circular genome. In most mammals, including humans and mice, the size of the chromosome is ~16,000 base pairs and unlike nuclear DNA, mitochondrial DNA (mtDNA) is not densely compacted. This results in mtDNA to be highly accessible for enzymes such as the Tn5 transposase, commonly used for accessible chromatin profiling of nuclear chromatinized DNA. Here, we describe a method for the concomitant sequencing of mtDNA and accessible chromatin in thousands of individual cells via the mitochondrial single-cell assay for transposase accessible chromatin by sequencing (mtscATAC-seq). Our approach extends the utility of existing scATAC-seq products and protocols as we (Nam et al, Nat Rev Genet 22:3-18, 2021) fix cells using formaldehyde to retain mitochondria and its mtDNA within its originating cell, (Buenrostro et al, Nat Methods 10:1213-1218, 2013) modify lysis conditions to permeabilize cells and mitochondria, and (Corces et al, Nat Methods 14:959-962, 2017) optimize bioinformatic processing protocols to collectively increase mitochondrial genome coverage for downstream analysis. Here, we discuss the essentials for the experimental and computational methodologies to generate and analyze thousands of multiomic profiles of single cells over the course of a few days, enabling the profiling of accessible chromatin and mtDNA genotypes to reconstruct clonal relationships and studies of mitochondrial genetics and disease., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

While methods such as the Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) enable a comprehensive characterization of regulatory DNA, additional measurements are required to characterize the multifaceted nature of eukaryotic cells. Here, we delineate the ATAC with Select Antigen Profiling by sequencing (ASAP-seq) protocol, a scalable approach to quantifying proteins via oligo-tagged antibodies alongside accessible DNA in thousands of single cells. Critically, our method utilizes a custom bridge oligo that enables the utilization of a variety of oligo-conjugated antibodies, enabling the utilization and repurposing of other commercial products. The ASAP-seq method can be completed with straightforward experimental and computational modifications existing single-cell ATAC-seq workflows but yields distinct modalities underlying complex cellular states, including estimation of protein abundance on the cell surface as well as intracellular and intranuclear factors., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clonal expansion of cells with somatically diversified receptors and their long-term maintenance as memory cells is a hallmark of adaptive immunity. Here, we studied pathogen-specific adaptation within the innate immune system, tracking natural killer (NK) cell memory to human cytomegalovirus (HCMV) infection. Leveraging single-cell multiomic maps of ex vivo NK cells and somatic mitochondrial DNA mutations as endogenous barcodes, we reveal substantial clonal expansion of adaptive NK cells in HCMV + individuals. NK cell clonotypes were characterized by a convergent inflammatory memory signature enriched for AP1 motifs superimposed on a private set of clone-specific accessible chromatin regions. NK cell clones were stably maintained in specific epigenetic states over time, revealing that clonal inheritance of chromatin accessibility shapes the epigenetic memory repertoire. Together, we identify clonal expansion and persistence within the human innate immune system, suggesting that these mechanisms have evolved independent of antigen-receptor diversification., (© 2022. The Author(s).)

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment 1,2 . The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (T reg ) and T follicular helper (T FH ) cells under homeostatic conditions, but induce inflammatory T helper 17 (T H 17) cells when induced T reg (iT reg ) cells are compromised 3,4 . How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iT reg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of T reg cells. These RORγt + cells-probably type 3 innate lymphoid cells and/or Janus cells 5 -require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFβ activator α v integrin. In the absence of any of these factors, there was expansion of pathogenic T H 17 cells instead of iT reg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cells require coordinated control over gene expression when responding to environmental stimuli. Here we apply scATAC-seq and single-cell RNA sequencing (scRNA-seq) in resting and stimulated human blood cells. Collectively, we generate ~91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of the immunological response across cell types, stimuli, and time. Advancing tools to integrate multi-omics data, we develop functional inference of gene regulation (FigR), a framework to computationally pair scA-TAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene-regulatory networks (GRNs) to identify candidate transcription factor (TF) regulators. Utilizing these paired multi-omics data, we define domains of regulatory chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility and gene expression at timescales of minutes. Construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues., Competing Interests: DECLARATION OF INTERESTS J.D.B. holds patents related to ATAC-seq and scATAC-seq and serves on the scientific advisory boards of CAMP4 Therapeutics, seqWell, and CelSee. J.G.C., Z.D.B., A.S.K., and R.L. are employees of Bio-Rad.

Tissue-resident memory T cells (T RM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8 + T cell tissue residency, its expression is repressed in CD4 + T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4 + T RM cells lacked the transforming growth factor (TGF)-β-responsive transcriptional network that underpins the tissue residency of epithelial CD8 + T RM cells. While CD4 + T RM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4 + T RM cells, was insufficient to engage the TGF-β-driven residency program. Ectopic expression of Runx3 in CD4 + T cells incited this TGF-β-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8 + and CD4 + T RM cell subsets that is attributable to divergent Runx3 activity., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation., (© 2022 by The American Society of Hematology.)

Multimodal measurements of single-cell profiles are proving increasingly useful for characterizing cell states and regulatory mechanisms. In the present study, we developed PHAGE-ATAC (Assay for Transposase-Accessible Chromatin), a massively parallel droplet-based method that uses phage displaying, engineered, camelid single-domain antibodies ('nanobodies') for simultaneous single-cell measurements of protein levels and chromatin accessibility profiles, and mitochondrial DNA-based clonal tracing. We use PHAGE-ATAC for multimodal analysis in primary human immune cells, sample multiplexing, intracellular protein analysis and the detection of SARS-CoV-2 spike protein in human cell populations. Finally, we construct a synthetic high-complexity phage library for selection of antigen-specific nanobodies that bind cells of particular molecular profiles, opening an avenue for protein detection, cell characterization and screening with single-cell genomics., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy., (© 2022. The Author(s).)

The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations 1-4 as well as the makeup of the tumour microenvironment 5,6 . Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7 + Aire-expressing migratory dendritic cells (AmDCs) and an Aire hi population coexpressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7 + migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium—the other principal Aire-expressing population and a key regulator of central tolerance—identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.

The recent development of experimental methods for measuring chromatin state at single-cell resolution has created a need for computational tools capable of analyzing these datasets. Here we developed Signac, a comprehensive toolkit for the analysis of single-cell chromatin data. Signac enables an end-to-end analysis of single-cell chromatin data, including peak calling, quantification, quality control, dimension reduction, clustering, integration with single-cell gene expression datasets, DNA motif analysis and interactive visualization. Through its seamless compatibility with the Seurat package, Signac facilitates the analysis of diverse multimodal single-cell chromatin data, including datasets that co-assay DNA accessibility with gene expression, protein abundance and mitochondrial genotype. We demonstrate scaling of the Signac framework to analyze datasets containing over 700,000 cells., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Natural mitochondrial DNA (mtDNA) mutations enable the inference of clonal relationships among cells. mtDNA can be profiled along with measures of cell state, but has not yet been combined with the massively parallel approaches needed to tackle the complexity of human tissue. Here, we introduce a high-throughput, droplet-based mitochondrial single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), a method that combines high-confidence mtDNA mutation calling in thousands of single cells with their concomitant high-quality accessible chromatin profile. This enables the inference of mtDNA heteroplasmy, clonal relationships, cell state and accessible chromatin variation in individual cells. We reveal single-cell variation in heteroplasmy of a pathologic mtDNA variant, which we associate with intra-individual chromatin variability and clonal evolution. We clonally trace thousands of cells from cancers, linking epigenomic variability to subclonal evolution, and infer cellular dynamics of differentiating hematopoietic cells in vitro and in vivo. Taken together, our approach enables the study of cellular population dynamics and clonal properties in vivo.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10 -9 , including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies., Competing Interests: Declaration of Interests Competing financial interests are declared in Table S1F., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation., Competing Interests: Declaration of Interests Adam Butterworth has received grants (outside of this work) from AstraZeneca, Biogen, BioMarin, Bioverativ, Merck, Novartis, and Sanofi; James Floyd has consulted for Shionogi; Qi Guo is a full-time employee of BenevolentAI; Joanna Howson is a full-time employee of Novo Nordisk. Parsa Akbari is a full-time employee of Regeneron Pharmaceuticals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies have associated thousands of genetic variants with complex traits and diseases, but pinpointing the causal variant(s) among those in tight linkage disequilibrium with each associated variant remains a major challenge. Here, we use seven experimental assays to characterize all common variants at the multiple disease-associated TNFAIP3 locus in five disease-relevant immune cell lines, based on a set of features related to regulatory potential. Trait/disease-associated variants are enriched among SNPs prioritized based on either: (1) residing within CRISPRi-sensitive regulatory regions, or (2) localizing in a chromatin accessible region while displaying allele-specific reporter activity. Of the 15 trait/disease-associated haplotypes at TNFAIP3, 9 have at least one variant meeting one or both of these criteria, 5 of which are further supported by genetic fine-mapping. Our work provides a comprehensive strategy to characterize genetic variation at important disease-associated loci, and aids in the effort to identify trait causal genetic variants.

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia 1 . BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development 2-7 . However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.

Our ability to track cellular dynamics in humans over time in vivo has been limited. Here, we demonstrate how somatic mutations in mitochondrial DNA (mtDNA) can be used to longitudinally track the dynamic output of hematopoietic stem and progenitor cells in humans. Over the course of 3 years of blood sampling in a single individual, our analyses reveal somatic mtDNA sequence variation and evolution reminiscent of models of hematopoiesis established by genetic labeling approaches. Furthermore, we observe fluctuations in mutation heteroplasmy, coinciding with specific clinical events, such as infections, and further identify lineage-specific somatic mtDNA mutations in longitudinally sampled circulating blood cell subsets in individuals with leukemia. Collectively, these observations indicate the significant potential of using tracking of somatic mtDNA sequence variation as a broadly applicable approach to systematically assess hematopoietic clonal dynamics in human health and disease., (© 2019 by The American Society of Hematology.)

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases 1-4 . Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types 5,6 . We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (10 5 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.