ABSTRACT
Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi -omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi -omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.
ABSTRACT
Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.
Subject(s)
Skin Diseases , Transcriptome , Humans , Animals , Mice , Skin , Keratinocytes/metabolism , Epidermis/pathology , Skin Diseases/pathology , Cell CommunicationABSTRACT
Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.
Subject(s)
Epidermis/physiology , Models, Genetic , Regulatory Elements, Transcriptional , Cell Differentiation/genetics , Chromatin/genetics , Epigenome , Gene Expression Regulation , Genes, Reporter , Genome-Wide Association Study , Humans , Keratinocytes/cytology , Keratinocytes/physiology , Neural Networks, Computer , Skin Diseases/genetics , Transcription Factors/geneticsABSTRACT
To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Genomics/methods , Skin Neoplasms/metabolism , Animals , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , RNA-Seq , Single-Cell Analysis , Skin/metabolism , Skin Neoplasms/pathology , Transcriptome , Transplantation, HeterologousABSTRACT
Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).
Subject(s)
Chromatin/chemistry , Chromatin/genetics , Embryonic Stem Cells/metabolism , Gene Expression Regulation , RNA, Long Noncoding/genetics , RNA/chemistry , Telomerase/chemistry , Animals , Cells, Cultured , Chromosomes , Embryonic Stem Cells/cytology , Genome , Mice , Promoter Regions, Genetic , RNA/genetics , Telomerase/geneticsABSTRACT
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in â¼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
Subject(s)
Epigenomics/methods , Gene Regulatory Networks/genetics , Single-Cell Analysis/methods , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly/physiology , Clustered Regularly Interspaced Short Palindromic Repeats/physiology , Gene Regulatory Networks/physiology , High-Throughput Nucleotide Sequencing/methods , Humans , Sequence Analysis, DNA/methods , Transcription Factors/metabolismABSTRACT
Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.
Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation , Chromatin Assembly and Disassembly , Keratinocytes/physiology , Transcription Factors/physiology , Tretinoin/pharmacology , Tumor Suppressor Proteins/physiology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Chromatin/drug effects , Chromatin/metabolism , Chromatin Assembly and Disassembly/drug effects , Chromatin Assembly and Disassembly/genetics , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/physiology , Epidermis/drug effects , Epidermis/physiology , Gene Expression Regulation, Developmental/drug effects , Humans , Keratinocytes/drug effects , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.
Subject(s)
Chromatin/metabolism , High-Throughput Nucleotide Sequencing/methods , Transposases/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Line, Transformed , Clone Cells , Epigenomics , Humans , Immunity , Jurkat Cells , Leukemia/immunology , Leukemia/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Antigen, T-Cell/metabolism , Single-Cell AnalysisABSTRACT
The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.
Subject(s)
Autoimmune Diseases/genetics , Cardiovascular Diseases/genetics , DNA, Intergenic/genetics , Enhancer Elements, Genetic , Mutation , Promoter Regions, Genetic , Alleles , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/pathology , Cell Differentiation , Chromatin , Chromatin Immunoprecipitation/methods , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Intergenic/metabolism , Genome, Human , Histones/genetics , Histones/metabolism , Humans , K562 Cells , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/immunology , Primary Cell Culture , Quantitative Trait Loci , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunologyABSTRACT
Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.
Subject(s)
Casein Kinase Ialpha/metabolism , Cell Self Renewal/physiology , Epidermal Cells , Keratinocytes/cytology , Protein-Arginine N-Methyltransferases/physiology , Stem Cells/cytology , Animals , Cell Differentiation , Cells, Cultured , Epidermis/metabolism , Humans , Infant, Newborn , Keratinocytes/metabolism , Mice , Mice, Knockout , Phosphorylation , Stem Cells/metabolismABSTRACT
We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-µm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.
Subject(s)
DNA/genetics , Freezing , Genome , Specimen Handling/methods , Animals , Brain , Cell Line , Erythrocytes , Gene Expression Regulation, Enzymologic , Genome-Wide Association Study , Humans , Keratinocytes , Mice , Self-Sustained Sequence Replication , Thyroid Neoplasms , Transposases/metabolismABSTRACT
Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.
Subject(s)
Cell Lineage/genetics , Chromosomes, Human/ultrastructure , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Keratinocytes/cytology , Promoter Regions, Genetic/genetics , Acetylation , Calcium/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Chromosomes, Human/genetics , Epidermal Cells , Gene Library , Histone Code , Histones/metabolism , Humans , Keratinocytes/metabolism , Male , Protein Processing, Post-Translational , RNA/genetics , RNA Interference , Transcription Factors/metabolismABSTRACT
Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.
Subject(s)
Cell Cycle Proteins/chemistry , Chromatin Immunoprecipitation/methods , Chromatin/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA/chemistry , Genomics/methods , Animals , B-Lymphocytes/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Mapping , Embryonic Stem Cells/metabolism , Humans , Mice , Reproducibility of Results , Sensitivity and Specificity , CohesinsABSTRACT
RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells, and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex, recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers, enhancers and promoters.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation , Nucleosomes/metabolism , RNA, Small Nuclear/metabolism , Transcription, Genetic , Animals , Cell Line , Humans , MiceABSTRACT
BACKGROUND: Open chromatin regions are correlated with active regulatory elements in development and are dysregulated in diseases. The BAF (SWI/SNF) complex is essential for development, and has been demonstrated to remodel reconstituted chromatin in vitro and to control the accessibility of a few individual regions in vivo. However, it remains unclear where and how BAF controls the open chromatin landscape to regulate developmental processes, such as human epidermal differentiation. RESULTS: Using a novel "on-plate" ATAC-sequencing approach for profiling open chromatin landscapes with a low number of adherent cells, we demonstrate that the BAF complex is essential for maintaining 11.6 % of open chromatin regions in epidermal differentiation. These BAF-dependent open chromatin regions are highly cell-type-specific and are strongly enriched for binding sites for p63, a master epidermal transcription factor. The DNA sequences of p63 binding sites intrinsically favor nucleosome formation and are inaccessible in other cell types without p63 to prevent ectopic activation. In epidermal cells, BAF and p63 mutually recruit each other to maintain 14,853 open chromatin regions. We further demonstrate that BAF and p63 cooperatively position nucleosomes away from p63 binding sites and recruit transcriptional machinery to control tissue differentiation. CONCLUSIONS: BAF displays high specificity in controlling the open chromatin landscape during epidermal differentiation by cooperating with the master transcription factor p63 to maintain lineage-specific open chromatin regions.
Subject(s)
Cell Lineage , Chromatin Assembly and Disassembly , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Cell Differentiation , Cells, Cultured , Chromatin/genetics , Gene Expression Regulation, Developmental , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Sequence Analysis, DNAABSTRACT
We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5'- or 3'-sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community.
Subject(s)
Bacteria/genetics , Insulator Elements , Promoter Regions, Genetic , Gene Library , Genes, Reporter , Genetic Engineering , Genetic Loci , Open Reading Frames , Transcription, GeneticABSTRACT
BACKGROUND: The engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories. RESULTS: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by approximately 50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards. CONCLUSION: Relative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.