RESUMEN
Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is being increasingly used to study gene regulation. However, major analytical gaps limit its utility in studying gene regulatory programs in complex diseases. In response, MOCHA (Model-based single cell Open CHromatin Analysis) presents major advances over existing analysis tools, including: 1) improving identification of sample-specific open chromatin, 2) statistical modeling of technical drop-out with zero-inflated methods, 3) mitigation of false positives in single cell analysis, 4) identification of alternative transcription-starting-site regulation, and 5) modules for inferring temporal gene regulatory networks from longitudinal data. These advances, in addition to open chromatin analyses, provide a robust framework after quality control and cell labeling to study gene regulatory programs in human disease. We benchmark MOCHA with four state-of-the-art tools to demonstrate its advances. We also construct cross-sectional and longitudinal gene regulatory networks, identifying potential mechanisms of COVID-19 response. MOCHA provides researchers with a robust analytical tool for functional genomic inference from scATAC-seq data.
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COVID-19 , Cromatina , Redes Reguladoras de Genes , Genómica , Modelos Estadísticos , Análisis de la Célula Individual , Humanos , COVID-19/genética , COVID-19/virología , Análisis de la Célula Individual/métodos , Genómica/métodos , Cromatina/genética , Cromatina/metabolismo , SARS-CoV-2/genética , Transposasas/metabolismo , Transposasas/genética , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Estudios de Cohortes , Regulación de la Expresión GénicaRESUMEN
Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.
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Subgrupos de Linfocitos T , Transcriptoma , Niño , Humanos , Anciano , Envejecimiento/genética , Epítopos/metabolismo , Análisis de la Célula IndividualRESUMEN
Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.
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COVID-19 , Síndrome Post Agudo de COVID-19 , Humanos , SARS-CoV-2 , Proteínas Sanguíneas , Progresión de la Enfermedad , InflamaciónRESUMEN
Longitudinal bulk and single-cell omics data is increasingly generated for biological and clinical research but is challenging to analyze due to its many intrinsic types of variations. We present PALMO ( https://github.com/aifimmunology/PALMO ), a platform that contains five analytical modules to examine longitudinal bulk and single-cell multi-omics data from multiple perspectives, including decomposition of sources of variations within the data, collection of stable or variable features across timepoints and participants, identification of up- or down-regulated markers across timepoints of individual participants, and investigation on samples of same participants for possible outlier events. We have tested PALMO performance on a complex longitudinal multi-omics dataset of five data modalities on the same samples and six external datasets of diverse background. Both PALMO and our longitudinal multi-omics dataset can be valuable resources to the scientific community.
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Multiómica , Humanos , Programas InformáticosRESUMEN
BACKGROUND: Barcode-based multiplexing methods can be used to increase throughput and reduce batch effects in large single-cell genomics studies. Despite advantages in flexibility of sample collection and scale, there are additional complications in the data deconvolution steps required to assign each cell to their originating samples. RESULTS: To meet computational needs for efficient sample deconvolution, we developed the tools BarCounter and BarMixer that compute barcode counts and deconvolute mixed single-cell data into sample-specific files, respectively. Together, these tools are implemented as the BarWare pipeline to support demultiplexing from large sequencing projects with many wells of hashed 10x Genomics scRNA-seq data. CONCLUSIONS: BarWare is a modular set of tools linked by shell scripting: BarCounter, a computationally efficient barcode sequence quantification tool implemented in C; and BarMixer, an R package for identification of barcoded populations, merging barcoded data from multiple wells, and quality-control reporting related to scRNA-seq data. These tools and a self-contained implementation of the pipeline are freely available for non-commercial use at https://github.com/AllenInstitute/BarWare-pipeline .
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Genómica , Programas Informáticos , Procesamiento Automatizado de Datos , Genómica/métodos , Control de CalidadRESUMEN
Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).
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Citometría de Flujo/métodos , Leucocitos Mononucleares , Monitorización Inmunológica/métodos , Análisis de la Célula Individual/métodos , Biología Computacional , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/clasificación , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Análisis de Secuencia de ARNRESUMEN
SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).
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Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.
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Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.
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Cromatina/metabolismo , Epigenómica/métodos , Epítopos/metabolismo , Regulación de la Expresión Génica , Transcriptoma , Humanos , Análisis de la Célula IndividualRESUMEN
The DUX4 transcription factor is briefly expressed in the early cleavage-stage embryo, where it induces an early wave of zygotic gene transcription, whereas its mis-expression in skeletal muscle causes the muscular dystrophy facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4 induces the expression of the histone variants H3.X and H3.Y. We have used a myoblast cell line with doxycycline-inducible DUX4 to show that these histone variants are incorporated throughout the body of DUX4-induced genes. Following a brief pulse of DUX4, these histones contribute to greater perdurance and to enhanced re-activation of DUX4 target gene expression. These findings provide a model for H3.X/Y as a chromatin mechanism that facilitates the expression of DUX4 target genes subsequent to a brief pulse of DUX4 expression.
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Regulación de la Expresión Génica , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/metabolismo , Línea Celular , Histonas/genética , Proteínas de Homeodominio/genética , Humanos , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patologíaRESUMEN
Cleavage under targets and release using nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used chromatin immunoprecipitation (ChIP) protocols in resolution, signal-to-noise ratio and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here, we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data when starting with only 100 cells for a histone modification and 1,000 cells for a transcription factor. From cells to purified DNA, CUT&RUN requires less than a day at the laboratory bench and requires no specialized skills.
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Cromatina/química , Proteínas de Unión al ADN/análisis , ADN/análisis , Epigenómica/métodos , Animales , Línea Celular , HumanosRESUMEN
Transcriptional changes occur presymptomatically and throughout Huntington's disease (HD), motivating the study of transcriptional regulatory networks (TRNs) in HD We reconstructed a genome-scale model for the target genes of 718 transcription factors (TFs) in the mouse striatum by integrating a model of genomic binding sites with transcriptome profiling of striatal tissue from HD mouse models. We identified 48 differentially expressed TF-target gene modules associated with age- and CAG repeat length-dependent gene expression changes in Htt CAG knock-in mouse striatum and replicated many of these associations in independent transcriptomic and proteomic datasets. Thirteen of 48 of these predicted TF-target gene modules were also differentially expressed in striatal tissue from human disease. We experimentally validated a specific model prediction that SMAD3 regulates HD-related gene expression changes using chromatin immunoprecipitation and deep sequencing (ChIP-seq) of mouse striatum. We found CAG repeat length-dependent changes in the genomic occupancy of SMAD3 and confirmed our model's prediction that many SMAD3 target genes are downregulated early in HD.
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Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Enfermedad de Huntington/genética , Proteína smad3/genética , Animales , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Enfermedad de Huntington/metabolismo , Ratones , Mapas de Interacción de Proteínas , Proteómica , Proteína smad3/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.
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Sitios de Unión , ADN/metabolismo , Biología Molecular/métodos , Factores de Transcripción/metabolismo , Humanos , Nucleasa Microcócica/metabolismo , Unión Proteica , Saccharomyces cerevisiaeRESUMEN
Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously, we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here, we build upon this work and compare X-ChIP-seq to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity, to fragment the chromatin and thereby generate precise protein-DNA footprints, high-resolution X-ChIP-seq achieves single base-pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.
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Sitios de Unión , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Inmunoprecipitación de Cromatina , Nucleasa Microcócica/metabolismo , Unión ProteicaRESUMEN
RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency. DOI: http://dx.doi.org/10.7554/eLife.02042.001.
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Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Most mammalian gene promoters are embedded within genomic regions called CpG islands, characterized by elevated levels of nonmethylated CpG dinucleotides. Here, we describe recent work demonstrating that CpG islands act as specific nucleation sites for the zinc finger CxxC domain-containing proteins CFP1 and KDM2A. Importantly, both CFP1 and KDM2A are associated with enzymatic activities that modulate specific histone lysine methylation marks. The action of these zinc finger CxxC domain proteins therefore imposes a defined chromatin architecture on CpG islands that distinguishes these important regulatory elements from the surrounding genome. The functional consequence of this CpG island-directed chromatin environment is discussed.
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Cromatina/metabolismo , Islas de CpG , Histona Demetilasas con Dominio de Jumonji/metabolismo , Transactivadores/metabolismo , Animales , Metilación de ADN , Genómica , Ratones , Modelos Genéticos , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Most histones are assembled into nucleosomes during replication to package genomic DNA. However, several variant histones are deposited independently of replication at particular regions of chromosomes. Such histone variants include cenH3, which forms the nucleosomal foundation for the centromere, and H3.3, which replaces histones that are lost during dynamic processes that disrupt nucleosomes. Furthermore, various H2A variants participate in DNA repair, gene regulation and other processes that are, as yet, not fully understood. Here, we review recent studies that have implicated histone variants in maintaining pluripotency and as causal factors in cancer and other diseases.
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Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Neoplasias/metabolismo , Nucleosomas/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Reprogramación Celular , Cromatina/genética , Cromatina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Progresión de la Enfermedad , Células Madre Embrionarias/patología , Epigénesis Genética , Histonas/genética , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.