scholarly journals Epigenetic Landscapes of Single-Cell Chromatin Accessibility and Transcriptomic Immune Profiles of T Cells in COVID-19 Patients

2021 ◽  
Vol 12 ◽  
Author(s):  
Shun Li ◽  
Bin Wu ◽  
Yun Ling ◽  
Mingquan Guo ◽  
Boyin Qin ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Signaling Pathway ◽  
Single Cells ◽  
Critical Role ◽  
Regulatory Elements ◽  
Mapk Signaling ◽  
Chromatin Accessibility ◽  
Integrated Analysis ◽  
Activation Markers

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

scholarly journals NEAT-seq: Simultaneous profiling of intra-nuclear proteins, chromatin accessibility, and gene expression in single cells

2021 ◽  
Author(s):  
Amy F Chen ◽  
Benjamin Parks ◽  
Arwa Kathiria ◽  
Benjamin Ober-Reynolds ◽  
Jorg Goronzy ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Nuclear Protein ◽  
Single Cells ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
T Cell Subsets ◽  
Protein Abundance ◽  
Joint Measurement

Oligonucleotide-conjugated antibodies have allowed for joint measurement of surface protein abundance and the transcriptome in single cells using high-throughput sequencing. Extending these measurements to gene regulatory proteins in the nucleus would provide a powerful means to link changes in abundance of trans-acting TFs to changes in activity of cis-acting elements and expression of target genes. Here, we introduce Nuclear protein Epitope, chromatin Accessibility, and Transcriptome sequencing (NEAT-seq), a technique to simultaneously measure nuclear protein abundance, chromatin accessibility, and the transcriptome in single cells. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive distinct helper T cell subsets and regulatory T cells (Tregs) and identify examples of TFs with regulatory activity gated by three distinct mechanisms: transcription, translation, and regulation of chromatin binding. Furthermore, we identify regulatory elements and target genes associated with each TF, which we use to link a non-coding GWAS SNP within a GATA motif to both strong allele-specific chromatin accessibility in cells expressing high levels of GATA3 protein, and a putative target gene.

scholarly journals Single-cell regulatory landscape and disease vulnerability map of adult Macaque cortex

2020 ◽  
Author(s):  
Ying Lei ◽  
Mengnan Cheng ◽  
Zihao Li ◽  
Zhenkun Zhuang ◽  
Liang Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Primary Motor Cortex ◽  
Neurological Diseases ◽  
Single Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Nucleotide Polymorphisms ◽  
Cell Type

Non-human primates (NHP) provide a unique opportunity to study human neurological diseases, yet detailed characterization of the cell types and transcriptional regulatory features in the NHP brain is lacking. We applied a combinatorial indexing assay, sci-ATAC-seq, as well as single-nuclei RNA-seq, to profile chromatin accessibility in 43,793 single cells and transcriptomics in 11,477 cells, respectively, from prefrontal cortex, primary motor cortex and the primary visual cortex of adult cynomolgus monkey Macaca fascularis. Integrative analysis of these two datasets, resolved regulatory elements and transcription factors that specify cell type distinctions, and discovered area-specific diversity in chromatin accessibility and gene expression within excitatory neurons. We also constructed the dynamic landscape of chromatin accessibility and gene expression of oligodendrocyte maturation to characterize adult remyelination. Furthermore, we identified cell type-specific enrichment of differentially spliced gene isoforms and disease-associated single nucleotide polymorphisms. Our datasets permit integrative exploration of complex regulatory dynamics in macaque brain tissue at single-cell resolution.

scholarly journals Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

2019 ◽  
Author(s):  
Dominik Trzupek ◽  
Melanie Dunstan ◽  
Antony J. Cutler ◽  
Mercede Lee ◽  
Leila Godfrey ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cost Effective ◽  
Activated T Cells ◽  
Activation Markers ◽  
High Resolution Map ◽  
Treg Differentiation

AbstractThe transcriptomic and proteomic characterisation of CD4+ T cells at the single-cell level has been performed traditionally by two largely exclusive types of technologies: single cell RNA-sequencing (scRNA-seq) technologies and antibody-based cytometry. Here we demonstrate that the simultaneous targeted quantification of mRNA and protein expression in single-cells provides a high-resolution map of human primary CD4+ T cells, and identified precise trajectories of Th1, Th17 and regulatory T-cell (Treg) differentiation in blood and tissue. Furthermore, the sensitivity provided by this massively-parallel multi-omics approach revealed novel insight into the mechanism of expression of CD80 and CD86 on the surface of activated CD4+ Tregs and demonstrate their potential to identify recently activated T cells in circulation. This transcriptomic and proteomic hybrid technology provides a cost-effective solution to dissect the heterogeneity of immune cell populations, including more precise and detailed descriptions of the differentiation and activation of circulating and tissue-resident cells in response to therapies and in stratification of patients.

scholarly journals A combinatorial indexing strategy for epigenomic profiling of plant single cells

2021 ◽  
Author(s):  
Xiaoyu Tu ◽  
Alexandre P Marand ◽  
Robert J. Schmitz ◽  
Silin Zhong
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Arabidopsis Root ◽  
Indexing Method ◽  
Specialized Equipment ◽  
Cell Type Specific ◽  
Accessible Chromatin ◽  
Insight Into

Understanding how cis-regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we profiled the Arabidopsis root single-cell epigenome using a combinatorial index and dual PCR barcode strategy without the need of any specialized equipment. We generated chromatin accessibility profiles for 13,576 Arabidopsis thaliana root nuclei with an average of 12,784 unique Tn5 integrations per cell and 85% of the Tn5 insertions localizing to discrete accessible chromatin regions. Comparison with data generated from a commercial microfluidic platform revealed that our method is capable of unbiased identification of cell type-specific chromatin accessibility with improved throughput, quality, and efficiency. We anticipate that by removing cost, instrument, and other technical obstacles, this combinatorial indexing method will be a valuable tool for routine investigation of single-cell epigenomes and usher new insight into plant growth, development and their interactions with the environment.

scholarly journals DC3 is a method for deconvolution and coupled clustering from bulk and single-cell genomics data

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Wanwen Zeng ◽  
Xi Chen ◽  
Zhana Duren ◽  
Yong Wang ◽  
Rui Jiang ◽  
...  
Keyword(s):  
Single Cell ◽  
Target Genes ◽  
Single Cells ◽  
Regulatory System ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Cellular Level ◽  
Joint Analysis ◽  
Cell Assays ◽  
Cell Expression

Abstract Characterizing and interpreting heterogeneous mixtures at the cellular level is a critical problem in genomics. Single-cell assays offer an opportunity to resolve cellular level heterogeneity, e.g., scRNA-seq enables single-cell expression profiling, and scATAC-seq identifies active regulatory elements. Furthermore, while scHi-C can measure the chromatin contacts (i.e., loops) between active regulatory elements to target genes in single cells, bulk HiChIP can measure such contacts in a higher resolution. In this work, we introduce DC3 (De-Convolution and Coupled-Clustering) as a method for the joint analysis of various bulk and single-cell data such as HiChIP, RNA-seq and ATAC-seq from the same heterogeneous cell population. DC3 can simultaneously identify distinct subpopulations, assign single cells to the subpopulations (i.e., clustering) and de-convolve the bulk data into subpopulation-specific data. The subpopulation-specific profiles of gene expression, chromatin accessibility and enhancer-promoter contact obtained by DC3 provide a comprehensive characterization of the gene regulatory system in each subpopulation.

scholarly journals Profiling of transcribed cis-regulatory elements in single cells

2021 ◽  
Author(s):  
Jonathan Moody ◽  
Tsukasa Kouno ◽  
Akari Suzuki ◽  
Youtaro Shibayama ◽  
Chikashi Terao ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Cell Analysis ◽  
Cell Type ◽  
Simultaneous Quantification ◽  
Promoter Usage

Profiling of cis-regulatory elements (CREs, mostly promoters and enhancers) in single cells allows the interrogation of the cell-type and -state specific contexts of gene regulation and genetic predisposition to diseases. Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay with no extra cost. We show enhancer RNAs can be effectively detected using sc-end5-seq methods with either random or oligo(dT) priming. To analyze tCREs in single cells, we developed SCAFE (Single Cell Analysis of Five-prime Ends) to identify genuine tCREs and analyze their activities (https://github.com/chung-lab/scafe). As compared to accessible CRE (aCRE, based on chromatin accessibility), tCREs are more accurate in predicting CRE interactions by co-activity, more sensitive in detecting shifts in alternative promoter usage and more enriched in diseases heritability. Our results highlight additional dimensions within sc-end5-seq data which can be used for interrogating gene regulation and disease heritability.

scholarly journals Single cell transcriptomic analysis indentifies Langerhans cells immunocompetency is critical for IDO1- dependent ability to induce tolerogenic T cells

2019 ◽  
Author(s):  
James Davies ◽  
Sofia Sirvent ◽  
Andres F. Vallejo ◽  
Kalum Clayton ◽  
Gemma Porter ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Langerhans Cells ◽  
Cell Activation ◽  
Single Cells ◽  
Therapeutic Interventions ◽  
Dendritic Cell Activation ◽  
Activation Markers ◽  
Efficient Induction ◽  
Regulatory Feedback Loop

AbstractHuman epidermal Langerhans cells (LCs) can coordinate both immunogenic and tolerogenic immune responses, creating an attractive opportunity for immunomodulation strategies. To investigate transcriptional determinants of human primary LC tolerance we applied single cells RNA-sequencing combined with extensive functional analysis. Unsupervised clustering of single cell transcriptomes indicated that steady-state LC populations exist in a spectrum of immune activation between two states: immunocompetent and immature, distinguishable by high or low CD86 expression, respectively. Suprisingly, LC immunompetency was critical for the efficient induction of regulatory T cells during co-culture assays with naïve CD4+ T cells and expansion of autologous memory T cells. Consistently, LC tolerogenic potential was significantly enhanced upon migration from the epidermis. Transcriptional programmes underpinning LC immunocompetency, with increased expression of dendritic cell activation markers (CD83, HLA-DRA and CCR7), were complemented with expression of tolerogenic markers (IDO1, LGALS1 and AHR) in migrated LC. Using protein expression analysis and perturbation with inhibitors, we confirmed the role of IDO1 as a key regulator of LC tolerogenic responses induced during LC migration, identified AHR as a potential component of IDO1-regulatory feedback loop, and demonstrated LC-mediated tolerance can be modulated through treatment with dexamethasone, indicating an opportunity for targeted therapeutic interventions in inflammatory skin disease.

scholarly journals Direct identification of neoantigen-specific TCRs from tumor specimens by high-throughput single-cell sequencing

2021 ◽  
Vol 9 (7) ◽  
pp. e002595
Author(s):  
Yong-Chen Lu ◽  
Zhili Zheng ◽  
Frank J Lowery ◽  
Jared J Gartner ◽  
Todd D Prickett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
High Throughput ◽  
Cell Activation ◽  
Single Cells ◽  
Interleukin 2 ◽  
Activation Markers ◽  
Single Cell Sequencing

BackgroundRecognition of neoantigens by T cells plays a major role in cancer immunotherapy. Identification of neoantigen-specific T-cell receptors (TCRs) has become a critical research tool for studying T cell-mediated responses after immunotherapy. In addition, neoantigen-specific TCRs can be used to modify the specificity of T cells for T cell-based therapies targeting tumor-specific mutations. Although several techniques have been developed to identify TCR sequences, these techniques still require a significant amount of labor, making them impractical in the clinical setting.MethodsThanks to the availability of high-throughput single-cell sequencing, we developed a new process to isolate neoantigen-specific TCR sequences. This process included the isolation of tumor-infiltrating T cells from a tumor specimen and the stimulation of T cells by neoantigen-loaded dendritic cells, followed by single-cell sequencing for TCR and T-cell activation markers, interferon-γ and interleukin-2.ResultsIn this study, potential neoantigen-specific TCRs were isolated from three melanoma and three colorectal tumor specimens. These TCRs were then synthesized and transduced into autologous T cells, followed by testing the recognition of neoantigens. A total of 28 neoantigen-specific TCRs were identified by this process. If identical TCR sequences were detected from two or more single cells, this approach was highly reliable (100%, 19 out of 19 TCRs).ConclusionThis single-cell approach provides an efficient process to isolate antigen-specific TCRs for research and clinical applications.

scholarly journals Single cell transcriptome atlas of mouse mammary epithelial cells across development

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Bhupinder Pal ◽  
Yunshun Chen ◽  
Michael J. G. Milevskiy ◽  
François Vaillant ◽  
Lexie Prokopuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Developmental Stages ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Mammary Epithelial ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

scholarly journals RA3 is a reference-guided approach for epigenetic characterization of single cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shengquan Chen ◽  
Guanao Yan ◽  
Wenyu Zhang ◽  
Jinzhao Li ◽  
Rui Jiang ◽  
...  
Keyword(s):  
Single Cell ◽  
Computational Analysis ◽  
Reference Data ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Biological Variation ◽  
Superior Performance ◽  
High Dimensionality ◽  
High Degree

AbstractThe recent advancements in single-cell technologies, including single-cell chromatin accessibility sequencing (scCAS), have enabled profiling the epigenetic landscapes for thousands of individual cells. However, the characteristics of scCAS data, including high dimensionality, high degree of sparsity and high technical variation, make the computational analysis challenging. Reference-guided approaches, which utilize the information in existing datasets, may facilitate the analysis of scCAS data. Here, we present RA3 (Reference-guided Approach for the Analysis of single-cell chromatin Accessibility data), which utilizes the information in massive existing bulk chromatin accessibility and annotated scCAS data. RA3 simultaneously models (1) the shared biological variation among scCAS data and the reference data, and (2) the unique biological variation in scCAS data that identifies distinct subpopulations. We show that RA3 achieves superior performance when used on several scCAS datasets, and on references constructed using various approaches. Altogether, these analyses demonstrate the wide applicability of RA3 in analyzing scCAS data.

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