ABSTRACT
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.
Subject(s)
T-Lymphocyte Subsets , Transcriptome , Child , Humans , Aged , Aging/genetics , Epitopes/metabolism , Single-Cell AnalysisABSTRACT
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 .
Subject(s)
Genomics , Software , Electronic Data Processing , Genomics/methods , Quality ControlABSTRACT
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.
Subject(s)
Gene Expression Profiling/methods , Gene Regulatory Networks , Huntington Disease/genetics , Smad3 Protein/genetics , Animals , Corpus Striatum/metabolism , Disease Models, Animal , Gene Expression Regulation , Humans , Huntington Disease/metabolism , Mice , Protein Interaction Maps , Proteomics , Smad3 Protein/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions, and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner.
Subject(s)
Chromatin/metabolism , Histones/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Neurons/metabolism , Protein Multimerization , Animals , Brain/cytology , Brain/metabolism , Cell Nucleus/metabolism , CpG Islands , DNA Methylation , Genome , Methyl-CpG-Binding Protein 2/deficiency , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/cytology , Nucleosomes/metabolism , Protein Binding , Transcription, GeneticABSTRACT
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.
Subject(s)
Embryonic Stem Cells/metabolism , Histones/metabolism , Neoplasms/metabolism , Nucleosomes/metabolism , Pluripotent Stem Cells/metabolism , Animals , Cellular Reprogramming , Chromatin/genetics , Chromatin/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Disease Progression , Embryonic Stem Cells/pathology , Epigenesis, Genetic , Histones/genetics , Humans , Neoplasms/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , X-linked Nuclear ProteinABSTRACT
CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides. Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro. Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/genetics , Chromatin/metabolism , CpG Islands/genetics , Trans-Activators/metabolism , Alleles , Animals , Brain/cytology , Cell Line , Chromatin Immunoprecipitation , DNA Methylation , Genome/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/chemistry , Histones/metabolism , Methylation , Mice , NIH 3T3 Cells , Promoter Regions, Genetic , Trans-Activators/chemistry , Trans-Activators/deficiency , Trans-Activators/genetics , Zinc FingersABSTRACT
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.
Subject(s)
COVID-19 , Chromatin , Gene Regulatory Networks , Genomics , Models, Statistical , Single-Cell Analysis , Humans , COVID-19/genetics , COVID-19/virology , Single-Cell Analysis/methods , Genomics/methods , Chromatin/genetics , Chromatin/metabolism , SARS-CoV-2/genetics , Transposases/metabolism , Transposases/genetics , Chromatin Immunoprecipitation Sequencing/methods , Cohort Studies , Gene Expression RegulationABSTRACT
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.
Subject(s)
Cell Nucleus/physiology , Cellular Reprogramming/physiology , Chromatin Assembly and Disassembly/physiology , Chromatin/physiology , Induced Pluripotent Stem Cells/physiology , Gene Expression Regulation , HistonesABSTRACT
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.
Subject(s)
Multiomics , Humans , SoftwareABSTRACT
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.
Subject(s)
COVID-19 , Post-Acute COVID-19 Syndrome , Humans , SARS-CoV-2 , Blood Proteins , Disease Progression , InflammationABSTRACT
Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5' side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by approximately 70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.
Subject(s)
Peptide Chain Initiation, Translational , RNA, Viral/metabolism , Rhinovirus/physiology , Ribosomes/metabolism , Virus Replication , Cell Extracts , Codon, Initiator/genetics , Epithelial Cells , HeLa Cells , Humans , Nucleic Acid Conformation , Point Mutation , RNA, Viral/genetics , ReticulocytesABSTRACT
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).
Subject(s)
Flow Cytometry/methods , Leukocytes, Mononuclear , Monitoring, Immunologic/methods , Single-Cell Analysis/methods , Computational Biology , Humans , Leukocytes, Mononuclear/chemistry , Leukocytes, Mononuclear/classification , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Sequence Analysis, RNAABSTRACT
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.
Subject(s)
Chromatin/metabolism , Epigenomics/methods , Epitopes/metabolism , Gene Expression Regulation , Transcriptome , Humans , Single-Cell AnalysisABSTRACT
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.
ABSTRACT
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).
ABSTRACT
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.
Subject(s)
Gene Expression Regulation , Histones/metabolism , Homeodomain Proteins/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Facioscapulohumeral/metabolism , Cell Line , Histones/genetics , Homeodomain Proteins/genetics , Humans , Muscle, Skeletal/pathology , Muscular Dystrophy, Facioscapulohumeral/genetics , Muscular Dystrophy, Facioscapulohumeral/pathologyABSTRACT
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.
Subject(s)
Chromatin/chemistry , DNA-Binding Proteins/analysis , DNA/analysis , Epigenomics/methods , Animals , Cell Line , HumansABSTRACT
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.
Subject(s)
Binding Sites , DNA/metabolism , Molecular Biology/methods , Transcription Factors/metabolism , Humans , Micrococcal Nuclease/metabolism , Protein Binding , Saccharomyces cerevisiaeABSTRACT
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.