ABSTRACT
Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.
Subject(s)
Basic-Leucine Zipper Transcription Factors , Chromatin , Th17 Cells , Th17 Cells/immunology , Th17 Cells/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Animals , Chromatin/metabolism , Mice , Mice, Inbred C57BL , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Mice, Knockout , Th1 Cells/immunology , Humans , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Inflammation/immunology , Inflammation/genetics , Single-Cell Analysis , Multiple Sclerosis/immunology , Multiple Sclerosis/genetics , FemaleABSTRACT
Hematopoietic stem cell (HSC) mutations can result in clonal hematopoiesis (CH) with heterogeneous clinical outcomes. Here, we investigate how the cell state preceding Tet2 mutation impacts the pre-malignant phenotype. Using an inducible system for clonal analysis of myeloid progenitors, we find that the epigenetic features of clones at similar differentiation status are highly heterogeneous and functionally respond differently to Tet2 mutation. Cell differentiation stage also influences Tet2 mutation response indicating that the cell of origin's epigenome modulates clone-specific behaviors in CH. Molecular features associated with higher risk outcomes include Sox4 that sensitizes cells to Tet2 inactivation, inducing dedifferentiation, altered metabolism and increasing the in vivo clonal output of mutant cells, as confirmed in primary GMP and HSC models. Our findings validate the hypothesis that epigenetic features can predispose specific clones for dominance, explaining why identical genetic mutations can result in different phenotypes.
Subject(s)
DNA-Binding Proteins , Dioxygenases , Epigenesis, Genetic , Hematopoietic Stem Cells , Mutation , Proto-Oncogene Proteins , Dioxygenases/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Animals , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Humans , Hematopoiesis/genetics , Mice , Cell Differentiation/geneticsABSTRACT
Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.
ABSTRACT
Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.
Subject(s)
COVID-19 , Epigenetic Memory , Post-Acute COVID-19 Syndrome , Animals , Humans , Mice , Cell Differentiation , COVID-19/immunology , Disease Models, Animal , Hematopoietic Stem Cells , Inflammation/genetics , Trained Immunity , Monocytes/immunology , Post-Acute COVID-19 Syndrome/genetics , Post-Acute COVID-19 Syndrome/immunology , Post-Acute COVID-19 Syndrome/pathologyABSTRACT
Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.
Subject(s)
Gene Expression Profiling , Transcriptome , Transcriptome/genetics , Kinetics , Organoids , PhysicsABSTRACT
Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance1-3. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer4,5, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.
Subject(s)
Chromosomal Instability , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Micronuclei, Chromosome-Defective , Neoplasms , Transcription, Genetic , Humans , Chromatin/genetics , Chromatin/metabolism , Chromosomes/genetics , Clone Cells/metabolism , DNA Damage/genetics , Neoplasms/genetics , Neoplasms/pathology , Single-Cell Gene Expression AnalysisABSTRACT
The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.
Subject(s)
Chromatin Assembly and Disassembly , Neoplasms , Animals , Humans , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Chromatin/genetics , Mammals/metabolismABSTRACT
In biological systems, spatial organization and function are interconnected. Here we present photoselective sequencing, a new method for genomic and epigenomic profiling within morphologically distinct regions. Starting with an intact biological specimen, photoselective sequencing uses targeted illumination to selectively unblock a photocaged fragment library, restricting the sequencing-based readout to microscopically identified spatial regions. We validate photoselective sequencing by measuring the chromatin accessibility profiles of fluorescently labeled cell types within the mouse brain and comparing with published data. Furthermore, by combining photoselective sequencing with a computational strategy for decomposing bulk accessibility profiles, we find that the oligodendrocyte-lineage-cell population is relatively enriched for oligodendrocyte-progenitor cells in the cortex versus the corpus callosum. Finally, we leverage photoselective sequencing at the subcellular scale to identify features of chromatin that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that photoselective sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.
Subject(s)
Chromatin , Genomics , Mice , Animals , Cell Nucleus , Epigenomics/methods , High-Throughput Nucleotide Sequencing/methodsABSTRACT
Cis-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time1-3. Here we sought to connect the structural changes at cis-regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate cis-regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.
ABSTRACT
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
Subject(s)
Cell Differentiation , Transcription Factors , Humans , Chromatin , Gene Expression Regulation , Human Embryonic Stem Cells/metabolism , Transcription Factors/metabolism , Atlases as TopicABSTRACT
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.
ABSTRACT
Mammalian neocortical neurons span one of the most diverse cell type spectra of any tissue. Cortical neurons are born during embryonic development, and their maturation extends into postnatal life. The regulatory strategies underlying progressive neuronal development and maturation remain unclear. Here we present an integrated single-cell epigenomic and transcriptional analysis of individual mouse and marmoset cortical neuron classes, spanning both early postmitotic stages of identity acquisition and later stages of neuronal plasticity and circuit integration. We found that, in both species, the regulatory strategies controlling early and late stages of pan-neuronal development diverge. Early postmitotic neurons use more widely shared and evolutionarily conserved molecular regulatory programs. In contrast, programs active during later neuronal maturation are more brain- and neuron-specific and more evolutionarily divergent. Our work uncovers a temporal shift in regulatory choices during neuronal diversification and maturation in both mice and marmosets, which likely reflects unique evolutionary constraints on distinct events of neuronal development in the neocortex.
Subject(s)
Neocortex , Animals , Callithrix , Mammals , Mice , Neurogenesis/physiology , Neuronal Plasticity , Neurons/physiologyABSTRACT
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 aberrations1-4 as well as the makeup of the tumour microenvironment5,6. 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.
Subject(s)
Clone Cells/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Genomics/methods , Animals , Clone Cells/pathology , DNA Copy Number Variations/genetics , Humans , Mice , Phenotype , RNA-Seq , Sequence Analysis, DNA , Transcription, Genetic , TranscriptomeABSTRACT
SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein-positive cells within the lung in a cell type-dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. SIGNIFICANCE: We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution.This article is highlighted in the In This Issue feature, p. 275.
Subject(s)
Adenocarcinoma of Lung/genetics , Cell Transformation, Neoplastic , DNA Helicases/genetics , Lung Neoplasms/genetics , Neoplasm Metastasis , Nuclear Proteins/genetics , Transcription Factors/genetics , Adenocarcinoma of Lung/secondary , Animals , Disease Models, Animal , Humans , Lung Neoplasms/pathology , MiceABSTRACT
To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.
Subject(s)
Gene Expression Profiling , Immune System/metabolism , MicroRNAs/genetics , Promoter Regions, Genetic , Transcriptome , Animals , Cells, Cultured , Chromatin Immunoprecipitation , Computational Biology , Gene Expression Regulation, Developmental , Immune System/cytology , Immune System/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/metabolism , RNA-SeqABSTRACT
Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples. For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020).
Subject(s)
Adenocarcinoma of Lung/genetics , Lung Neoplasms/genetics , Single-Cell Analysis/methods , Animals , Computational Biology/methods , Epigenesis, Genetic , MiceABSTRACT
Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.
Subject(s)
Chromatin Immunoprecipitation Sequencing , GATA2 Deficiency , Animals , GATA2 Transcription Factor , Lymphopoiesis , Transcription Factors/genetics , Xenopus Proteins , ZebrafishABSTRACT
Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here we report that the stress hormone corticosterone-which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans-regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.