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Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from Metabolic dysfunction-associated steatotic liver (MASL) to Metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Activation of Hepatic Stellate Cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. We compared transcriptome and chromatin accessibility of human HSCs from NORMAL, MASL, and MASH livers at single cell resolution. We aimed to identify genes that are upregulated in activated HSCs and to determine which of these genes are key in the pathogenesis of MASH fibrosis., Methods: 18 human livers were profiled using single-nucleus (sn)RNA-seq and snATAC-seq. High priority targets were identified, then tested in 2D human HSC cultures, 3D human liver spheroids, and HSC-specific gene knockout mice., Results: MASH-enriched activated (A) HSC subclusters are the major source of extracellular matrix proteins. We identified a set of concurrently upregulated and more accessible core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive activation of (A) HSC subclusters. Expression of these genes was regulated via crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors. The pathological relevance of the selected targets, such as SERPINE1 (PAI-1), was demonstrated using dsiRNA-based HSC-specific gene knockdown or pharmacological inhibition of PAI-1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice., Conclusion: This study identified novel gene targets and regulatory mechanisms underlying activation of MASH fibrogenic HSCs and demonstrated that genetic or pharmacological inhibition of select genes suppressed liver fibrosis., Impact and Implications: Here we present snRNA-seq and snATAC-seq analysis of human HSCs from NORMAL, MASL, and MASH livers. We identified additional subclusters that were not detected by previous studies and characterized the mechanism by which HSCs activate in the MASH livers, including the transcriptional machinery that activates HSCs into myofibroblasts. For the first time, we described the pathogenic role of activated HSC-derived PAI-1 (a product of SERPINE1 gene) in the development of MASH liver fibrosis. Targeting of RUNX1/2-SERPINE1 axis may provide a novel strategy for treatment of liver fibrosis in patients., Competing Interests: Declaration of Competing Interest Nothing to declare, (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Physiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs for cis regulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk., Competing Interests: Conflicts of interest K.J.G. has done consulting for Genentech, received honoraria from Pfizer, and is a shareholder of Neurocrine biosciences. M.I.M. is currently an employee of Genentech, and a holder of Roche stock.

Biological sex is an important risk factor in cancer, but the underlying cell types and mechanisms remain obscure. Since tumor development is regulated by the immune system, we hypothesize that sex-biased immune interactions underpin sex differences in cancer. The male-biased glioblastoma multiforme (GBM) is an aggressive and treatment-refractory tumor in urgent need of more innovative approaches, such as considering sex differences, to improve outcomes. GBM arises in the specialized brain immune environment dominated by microglia, so we explored sex differences in this immune cell type. We isolated adult human TAM-MGs (tumor-associated macrophages enriched for microglia) and control microglia and found sex-biased inflammatory signatures in GBM and lower-grade tumors associated with pro-tumorigenic activity in males and anti-tumorigenic activity in females. We demonstrated that genes expressed or modulated by the inactive X chromosome facilitate this bias. Together, our results implicate TAM-MGs, specifically their sex chromosomes, as drivers of male bias in GBM., Competing Interests: Declaration of interests The authors declare no competing interests.

Background: Catecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved., Results: C57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment., Conclusions: The results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment., (© 2024. The Author(s).)

Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development., (© 2024. The Author(s).)

Background: Although aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs., Methods and Results: Lower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20-24 months) compared to young mice (2-3 months). In old mice, chondroitin sulfate proteoglycan 4 (CSPG4) was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice., Conclusions: On the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2023. Published by Elsevier Ltd.)

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete 1-4 . Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains., (© 2023. The Author(s).)

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV ( n  = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes., Competing Interests: Declaration of interests M.B.-J. is a co-inventor of patent application WO/2018/160496, related to the differentiation of pluripotent stem cells into microglia and co-founder of NovoGlia, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have linked hundreds of thousands of sequence variants in the human genome to common traits and diseases. However, translating this knowledge into a mechanistic understanding of disease-relevant biology remains challenging, largely because such variants are predominantly in non-protein-coding sequences that still lack functional annotation at cell-type resolution. Recent advances in single-cell epigenomics assays have enabled the generation of cell type-, subtype- and state-resolved maps of the epigenome in heterogeneous human tissues. These maps have facilitated cell type-specific annotation of candidate cis-regulatory elements and their gene targets in the human genome, enhancing our ability to interpret the genetic basis of common traits and diseases., (© 2023. Springer Nature Limited.)

Single-cell sequencing could help to solve the fundamental challenge of linking millions of cell-type-specific enhancers with their target genes. However, this task is confounded by patterns of gene co-expression in much the same way that genetic correlation due to linkage disequilibrium confounds fine-mapping in genome-wide association studies (GWAS). We developed a non-parametric permutation-based procedure to establish stringent statistical criteria to control the risk of false-positive associations in enhancer-gene association studies (EGAS). We applied our procedure to large-scale transcriptome and epigenome data from multiple tissues and species, including the mouse and human brain, to predict enhancer-gene associations genome wide. We tested the functional validity of our predictions by comparing them with chromatin conformation data and causal enhancer perturbation experiments. Our study shows how controlling for gene co-expression enables robust enhancer-gene linkage using single-cell sequencing data., Competing Interests: J.R.E. serves on the scientific advisory board of Zymo Research, Inc. B.R. is a shareholder of Arima Genomics, Inc. and Epigenome Technologies, Inc. H.Z. is on the scientific advisory board of MapLight Therapeutics, Inc., (© 2023 The Authors.)

Gene regulation is highly cell type-specific and understanding the function of non-coding genetic variants associated with complex traits requires molecular phenotyping at cell type resolution. In this study we performed single nucleus ATAC-seq (snATAC-seq) and genotyping in peripheral blood mononuclear cells from 13 individuals. Clustering chromatin accessibility profiles of 96,002 total nuclei identified 17 immune cell types and sub-types. We mapped chromatin accessibility QTLs (caQTLs) in each immune cell type and sub-type using individuals of European ancestry which identified 6,901 caQTLs at FDR < .10 and 4,220 caQTLs at FDR < .05, including those obscured from assays of bulk tissue such as with divergent effects on different cell types. For 3,941 caQTLs we further annotated putative target genes of variant activity using single cell co-accessibility, and caQTL variants were significantly correlated with the accessibility level of linked gene promoters. We fine-mapped loci associated with 16 complex immune traits and identified immune cell caQTLs at 622 candidate causal variants, including those with cell type-specific effects. At the 6q15 locus associated with type 1 diabetes, in line with previous reports, variant rs72928038 was a naïve CD4+ T cell caQTL linked to BACH2 and we validated the allelic effects of this variant on regulatory activity in Jurkat T cells. These results highlight the utility of snATAC-seq for mapping genetic effects on accessible chromatin in specific cell types., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Dr. Gaulton has done consulting for Genentech and holds stock in Neurocrine Biosciences. Dr. Benaglio is an employee of Shoreline Bioscience. Dr. Chiou is an employee and shareholder of Pfizer. These affiliations have no competing interest related to the submitted work. The other authors have no competing interests to disclose. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright: © 2023 Benaglio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dysfunctional pancreatic islet beta cells are a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of the underlying mechanisms, including gene dysregulation, is lacking. Here we integrate information from measurements of chromatin accessibility, gene expression and function in single beta cells with genetic association data to nominate disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 nondiabetic, pre-T2D and T2D donors, we identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift during T2D progression. Subtype-defining accessible chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both beta cell subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is probably induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for characterizing mechanisms of complex diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem-cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, serotonin-producing pre-β cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro β cell maturation and identify sex hormones as drivers of β cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem-cell-derived islets and a framework for manipulating cell identities and maturity., Competing Interests: Declaration of interests K.J.G. does consulting for Genentech and holds stock in Vertex Pharmaceuticals., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cell type-specific gene expression patterns and dynamics during development or in disease are controlled by cis-regulatory elements (CREs), such as promoters and enhancers. Distinct classes of CREs can be characterized by their epigenomic features, including DNA methylation, chromatin accessibility, combinations of histone modifications and conformation of local chromatin. Tremendous progress has been made in cataloguing CREs in the human genome using bulk transcriptomic and epigenomic methods. However, single-cell epigenomic and multi-omic technologies have the potential to provide deeper insight into cell type-specific gene regulatory programmes as well as into how they change during development, in response to environmental cues and through disease pathogenesis. Here, we highlight recent advances in single-cell epigenomic methods and analytical tools and discuss their readiness for human tissue profiling., (© 2022. Springer Nature Limited.)

We combined functional genomics and human genetics to investigate processes that affect type 1 diabetes (T1D) risk by mediating beta cell survival in response to proinflammatory cytokines. We mapped 38,931 cytokine-responsive candidate cis- regulatory elements (cCREs) in beta cells using ATAC-seq and snATAC-seq and linked them to target genes using co-accessibility and HiChIP. Using a genome-wide CRISPR screen in EndoC-βH1 cells, we identified 867 genes affecting cytokine-induced survival, and genes promoting survival and up-regulated in cytokines were enriched at T1D risk loci. Using SNP-SELEX, we identified 2,229 variants in cytokine-responsive cCREs altering transcription factor (TF) binding, and variants altering binding of TFs regulating stress, inflammation, and apoptosis were enriched for T1D risk. At the 16p13 locus, a fine-mapped T1D variant altering TF binding in a cytokine-induced cCRE interacted with SOCS1 , which promoted survival in cytokine exposure. Our findings reveal processes and genes acting in beta cells during inflammation that modulate T1D risk., Competing Interests: K.J.G. is a consultant of Genentech and holds stock in Neurocrine Biosciences. B.R. is a consultant of Arima Genomics and a co-founder of Epigenome Technologies. P.B. is an employee of Shoreline Bioscience. N.N. is an employee of Guardant Health. E.B. is an employee and shareholder of Aetion. K.K. is an employee of Cartography Bio. Y.Q. is an employee of Sana Biotechnology. M.D. is an employee and shareholder of Seer. J.C. is an employee and shareholder of Pfizer., (© 2022.)

Loss of heterochromatin has been implicated as a cause of pre-mature aging and age-associated decline in organ functions in mammals; however, the specific cell types and gene loci affected by this type of epigenetic change have remained unclear. To address this knowledge gap, we probed chromatin accessibility at single-cell resolution in the brains, hearts, skeletal muscles, and bone marrows from young, middle-aged, and old mice, and assessed age-associated changes at 353,126 candidate cis-regulatory elements (cCREs) across 32 major cell types. Unexpectedly, we detected increased chromatin accessibility within specific heterochromatin domains in old mouse excitatory neurons. The gain of chromatin accessibility at these genomic loci was accompanied by the cell-type-specific loss of heterochromatin and activation of LINE1 elements. Immunostaining further confirmed the loss of the heterochromatin mark H3K9me3 in the excitatory neurons but not in inhibitory neurons or glial cells. Our results reveal the cell-type-specific changes in chromatin landscapes in old mice and shed light on the scope of heterochromatin loss in mammalian aging., (© 2022. The Author(s).)

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers., Competing Interests: Ethics declarations The authors declare no competing interests.

We assembled an ancestrally diverse collection of genome-wide association studies (GWAS) of type 2 diabetes (T2D) in 180,834 affected individuals and 1,159,055 controls (48.9% non-European descent) through the Diabetes Meta-Analysis of Trans-Ethnic association studies (DIAMANTE) Consortium. Multi-ancestry GWAS meta-analysis identified 237 loci attaining stringent genome-wide significance (P < 5 × 10 -9 ), which were delineated to 338 distinct association signals. Fine-mapping of these signals was enhanced by the increased sample size and expanded population diversity of the multi-ancestry meta-analysis, which localized 54.4% of T2D associations to a single variant with >50% posterior probability. This improved fine-mapping enabled systematic assessment of candidate causal genes and molecular mechanisms through which T2D associations are mediated, laying the foundations for functional investigations. Multi-ancestry genetic risk scores enhanced transferability of T2D prediction across diverse populations. Our study provides a step toward more effective clinical translation of T2D GWAS to improve global health for all, irrespective of genetic background., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases., Competing Interests: DECLARATION OF INTERESTS J.R.E. serves on the scientific advisory board of Zymo Research Inc.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for ∼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems., Competing Interests: Declaration of interests B.R. is a shareholder and consultant of Arima Genomics, Inc., and a co-founder of Epigenome Technologies, Inc. K.J.G. is a consultant of Genentech and a shareholder in Vertex Pharmaceuticals. These relationships have been disclosed to and approved by the UCSD Independent Review Committee., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures 1 . Recent surveys of mouse and human brains with single-cell transcriptomics 2-6 and high-throughput imaging technologies 7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans., (© 2021. The Author(s).)

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing 1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data 3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments 4 . By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum., (© 2021. The Author(s).)

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain 1-3 . With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions 4 . We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis., (© 2021. The Author(s).)

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals 1 . Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., (© 2021. The Author(s).)

Integrating large single-cell gene expression, chromatin accessibility and DNA methylation datasets requires general and scalable computational approaches. Here we describe online integrative non-negative matrix factorization (iNMF), an algorithm for integrating large, diverse and continually arriving single-cell datasets. Our approach scales to arbitrarily large numbers of cells using fixed memory, iteratively incorporates new datasets as they are generated and allows many users to simultaneously analyze a single copy of a large dataset by streaming it over the internet. Iterative data addition can also be used to map new data to a reference dataset. Comparisons with previous methods indicate that the improvements in efficiency do not sacrifice dataset alignment and cluster preservation performance. We demonstrate the effectiveness of online iNMF by integrating more than 1 million cells on a standard laptop, integrating large single-cell RNA sequencing and spatial transcriptomic datasets, and iteratively constructing a single-cell multi-omic atlas of the mouse motor cortex., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding 1 . Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types 2 . Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.

Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease-associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type-specific gene regulation in human hearts during health and disease., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.

Identification of the cis-regulatory elements controlling cell-type specific gene expression patterns is essential for understanding the origin of cellular diversity. Conventional assays to map regulatory elements via open chromatin analysis of primary tissues is hindered by sample heterogeneity. Single cell analysis of accessible chromatin (scATAC-seq) can overcome this limitation. However, the high-level noise of each single cell profile and the large volume of data pose unique computational challenges. Here, we introduce SnapATAC, a software package for analyzing scATAC-seq datasets. SnapATAC dissects cellular heterogeneity in an unbiased manner and map the trajectories of cellular states. Using the Nyström method, SnapATAC can process data from up to a million cells. Furthermore, SnapATAC incorporates existing tools into a comprehensive package for analyzing single cell ATAC-seq dataset. As demonstration of its utility, SnapATAC is applied to 55,592 single-nucleus ATAC-seq profiles from the mouse secondary motor cortex. The analysis reveals ~370,000 candidate regulatory elements in 31 distinct cell populations in this brain region and inferred candidate cell-type specific transcriptional regulators.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 ( NOVA1 ) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1 , which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis -regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20 , a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases., Competing Interests: AW, JC, OP, JB, MV, JV, XH, PK, SN, JN, MG, KZ, RY, JB, ES, RM, HH, LR, CP, JW, GP, YX, SP, XS No competing interests declared, DF employee of and holds stock in Vertex Pharmaceuticals, KG does consulting for Genentech, (© 2020, Wang et al.)

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.

Technological improvements enable single-cell epigenetic analyses of organ development. We reasoned that high-resolution single-cell chromatin accessibility mapping would provide needed insight into the epigenetic reprogramming and transcriptional regulators involved in normal mammary gland development. Here, we provide a single-cell resource of chromatin accessibility for murine mammary development from the peak of fetal mammary stem cell (fMaSC) functional activity in late embryogenesis to the differentiation of adult basal and luminal cells. We find that the chromatin landscape within individual cells predicts both gene accessibility and transcription factor activity. The ability of single-cell chromatin profiling to separate E18 fetal mammary cells into clusters exhibiting basal-like and luminal-like chromatin features is noteworthy. Such distinctions were not evident in analyses of droplet-based single-cell transcriptomic data. We present a web application as a scientific resource for facilitating future analyses of the gene regulatory networks involved in mammary development., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)