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1.
Genome Biol ; 22(1): 252, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34465366

RESUMO

Detecting multiplets in single nucleus (sn)ATAC-seq data is challenging due to data sparsity and limited dynamic range. AMULET (ATAC-seq MULtiplet Estimation Tool) enumerates regions with greater than two uniquely aligned reads across the genome to effectively detect multiplets. We evaluate the method by generating snATAC-seq data in the human blood and pancreatic islet samples. AMULET has high precision, estimated via donor-based multiplexing, and high recall, estimated via simulated multiplets, compared to alternatives and identifies multiplets most effectively when a certain read depth of 25K median valid reads per nucleus is achieved.

2.
Nat Commun ; 12(1): 5242, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475398

RESUMO

Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) at >250 loci in the human genome to type 2 diabetes (T2D) risk. For each locus, identifying the functional variant(s) among multiple SNPs in high linkage disequilibrium is critical to understand molecular mechanisms underlying T2D genetic risk. Using massively parallel reporter assays (MPRA), we test the cis-regulatory effects of SNPs associated with T2D and altered in vivo islet chromatin accessibility in MIN6 ß cells under steady state and pathophysiologic endoplasmic reticulum (ER) stress conditions. We identify 1,982/6,621 (29.9%) SNP-containing elements that activate transcription in MIN6 and 879 SNP alleles that modulate MPRA activity. Multiple T2D-associated SNPs alter the activity of short interspersed nuclear element (SINE)-containing elements that are strongly induced by ER stress. We identify 220 functional variants at 104 T2D association signals, narrowing 54 signals to a single candidate SNP. Together, this study identifies elements driving ß cell steady state and ER stress-responsive transcriptional activation, nominates causal T2D SNPs, and uncovers potential roles for repetitive elements in ß cell transcriptional stress response and T2D genetics.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/patologia , Polimorfismo de Nucleotídeo Único , Ativação Transcricional/genética , Alelos , Animais , Linhagem Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Locos de Características Quantitativas , Elementos Nucleotídeos Curtos e Dispersos/genética
3.
Front Immunol ; 12: 636720, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815388

RESUMO

Immune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) but reduced monocyte numbers. Activated T and NK cells expressed senescence and effector molecules, whereas activated B cells transcriptionally resembled autoimmune disease- or age-associated B cells (e.g., CD11c, T-bet). In contrast, LPS specifically targeted monocytes and induced two main states: early activation characterized by the expression of chemoattractants and a later pro-inflammatory state characterized by expression of effector molecules. These data provide a foundation for future immune activation studies with single cell technologies (https://czi-pbmc-cite-seq.jax.org/).

4.
Immun Ageing ; 17: 13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32457811

RESUMO

While Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is disrupting lives across the globe for everyone, it has a more devastating impact on the health of older adults, especially that of older men. This pandemic has highlighted the crucial importance of considering an individual's age and biological sex in the clinic in addition to other confounding diseases (Kuchel, G.A, J Am Geriatr Soc, 67, 203, 2019, Tannenbaum, C., Nature, 575 451-458, 2009) As an interdisciplinary team of scientists in immunology, hematology, genomics, bioinformatics, and geriatrics, we have been studying how age and sex shape the human immune system. Herein we reflect on how our recent findings on the alterations of the immune system in aging might contribute to our current understanding of COVID-19 infection rate and disease risk.

5.
Nat Immunol ; 21(6): 684-694, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231301

RESUMO

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Senescência Celular/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citotoxicidade Imunológica , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais , Febre Amarela/genética , Febre Amarela/imunologia , Febre Amarela/metabolismo , Febre Amarela/virologia , Vírus da Febre Amarela/imunologia
6.
Bioinformatics ; 36(11): 3582-3584, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119082

RESUMO

SUMMARY: Single-cell RNA-sequencing (scRNA-seq) technology enables studying gene expression programs from individual cells. However, these data are subject to diverse sources of variation, including 'unwanted' variation that needs to be removed in downstream analyses (e.g. batch effects) and 'wanted' or biological sources of variation (e.g. variation associated with a cell type) that needs to be precisely described. Surrogate variable analysis (SVA)-based algorithms, are commonly used for batch correction and more recently for studying 'wanted' variation in scRNA-seq data. However, interpreting whether these variables are biologically meaningful or stemming from technical reasons remains a challenge. To facilitate the interpretation of surrogate variables detected by algorithms including IA-SVA, SVA or ZINB-WaVE, we developed an R Shiny application [Visual Surrogate Variable Analysis (V-SVA)] that provides a web-browser interface for the identification and annotation of hidden sources of variation in scRNA-seq data. This interactive framework includes tools for discovery of genes associated with detected sources of variation, gene annotation using publicly available databases and gene sets, and data visualization using dimension reduction methods. AVAILABILITY AND IMPLEMENTATION: The V-SVA Shiny application is publicly hosted at https://vsva.jax.org/ and the source code is freely available at https://github.com/nlawlor/V-SVA. CONTACT: leed13@miamioh.edu or duygu.ucar@jax.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Perfilação da Expressão Gênica , RNA-Seq , Análise de Sequência de RNA , Análise de Célula Única , Software
7.
Nat Commun ; 11(1): 751, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029736

RESUMO

Differences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22-93 years of age using ATAC-seq, RNA-seq, and flow cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. Impact of age and sex on immune phenotypes can be visualized at https://immune-aging.jax.org to provide insights into future studies.


Assuntos
Envelhecimento/imunologia , Caracteres Sexuais , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Linfócitos B/imunologia , Sequenciamento de Cromatina por Imunoprecipitação , Epigênese Genética , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/classificação , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Imunológicos , Monócitos/imunologia , RNA-Seq , Transcriptoma , Adulto Jovem
8.
Cell Rep ; 26(3): 788-801.e6, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650367

RESUMO

EndoC-ßH1 is emerging as a critical human ß cell model to study the genetic and environmental etiologies of ß cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-ßH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) ß cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or ß cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-ßH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing ß cell identity and (dys)function in diabetes.


Assuntos
Redes Reguladoras de Genes/genética , Células Secretoras de Insulina/metabolismo , Linhagem Celular , Humanos
9.
Nucleic Acids Res ; 47(2): e11, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30428075

RESUMO

Transcription factor (TF) footprinting uncovers putative protein-DNA binding via combined analyses of chromatin accessibility patterns and their underlying TF sequence motifs. TF footprints are frequently used to identify TFs that regulate activities of cell/condition-specific genomic regions (target loci) in comparison to control regions (background loci) using standard enrichment tests. However, there is a strong association between the chromatin accessibility level and the GC content of a locus and the number and types of TF footprints that can be detected at this site. Traditional enrichment tests (e.g. hypergeometric) do not account for this bias and inflate false positive associations. Therefore, we developed a novel post-processing method, Bias-free Footprint Enrichment Test (BiFET), that corrects for the biases arising from the differences in chromatin accessibility levels and GC contents between target and background loci in footprint enrichment analyses. We applied BiFET on TF footprint calls obtained from EndoC-ßH1 ATAC-seq samples using three different algorithms (CENTIPEDE, HINT-BC and PIQ) and showed BiFET's ability to increase power and reduce false positive rate when compared to hypergeometric test. Furthermore, we used BiFET to study TF footprints from human PBMC and pancreatic islet ATAC-seq samples to show its utility to identify putative TFs associated with cell-type-specific loci.


Assuntos
Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Algoritmos , Composição de Bases , Viés , Linhagem Celular , DNA/química , Humanos , Motivos de Nucleotídeos , Software
10.
Genetics ; 211(2): 549-562, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30593493

RESUMO

Epigenomic signatures from histone marks and transcription factor (TF)-binding sites have been used to annotate putative gene regulatory regions. However, a direct comparison of these diverse annotations is missing, and it is unclear how genetic variation within these annotations affects gene expression. Here, we compare five widely used annotations of active regulatory elements that represent high densities of one or more relevant epigenomic marks-"super" and "typical" (nonsuper) enhancers, stretch enhancers, high-occupancy target (HOT) regions, and broad domains-across the four matched human cell types for which they are available. We observe that stretch and super enhancers cover cell type-specific enhancer "chromatin states," whereas HOT regions and broad domains comprise more ubiquitous promoter states. Expression quantitative trait loci (eQTL) in stretch enhancers have significantly smaller effect sizes compared to those in HOT regions. Strikingly, chromatin accessibility QTL in stretch enhancers have significantly larger effect sizes compared to those in HOT regions. These observations suggest that stretch enhancers could harbor genetically primed chromatin to enable changes in TF binding, possibly to drive cell type-specific responses to environmental stimuli. Our results suggest that current eQTL studies are relatively underpowered or could lack the appropriate environmental context to detect genetic effects in the most cell type-specific "regulatory annotations," which likely contributes to infrequent colocalization of eQTL with genome-wide association study signals.


Assuntos
Elementos Facilitadores Genéticos , Locos de Características Quantitativas , Ativação Transcricional , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Hep G2 , Humanos , Especificidade de Órgãos , Fatores de Transcrição/metabolismo
11.
Nat Med ; 25(1): 75-81, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478422

RESUMO

Understanding the mechanisms underlying autoantibody development will accelerate therapeutic target identification in autoimmune diseases such as systemic lupus erythematosus (SLE)1. Follicular helper T cells (TFH cells) have long been implicated in SLE pathogenesis. Yet a fraction of autoantibodies in individuals with SLE are unmutated, supporting that autoreactive B cells also differentiate outside germinal centers2. Here, we describe a CXCR5-CXCR3+ programmed death 1 (PD1)hiCD4+ helper T cell population distinct from TFH cells and expanded in both SLE blood and the tubulointerstitial areas of individuals with proliferative lupus nephritis. These cells produce interleukin-10 (IL-10) and accumulate mitochondrial reactive oxygen species as the result of reverse electron transport fueled by succinate. Furthermore, they provide B cell help, independently of IL-21, through IL-10 and succinate. Similar cells are generated in vitro upon priming naive CD4+ T cells with plasmacytoid dendritic cells activated with oxidized mitochondrial DNA, a distinct class of interferogenic toll-like receptor 9 ligand3. Targeting this pathway might blunt the initiation and/or perpetuation of extrafollicular humoral responses in SLE.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-10/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Ácido Succínico/metabolismo , Proliferação de Células , DNA Mitocondrial/genética , Células Dendríticas/metabolismo , Humanos , Memória Imunológica , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/imunologia , Oxirredução
12.
Sci Rep ; 8(1): 17040, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451954

RESUMO

Single cell RNA-sequencing (scRNA-seq) precisely characterizes gene expression levels and dissects variation in expression associated with the state (technical or biological) and the type of the cell, which is averaged out in bulk measurements. Multiple and correlated sources contribute to gene expression variation in single cells, which makes their estimation difficult with the existing methods developed for batch correction (e.g., surrogate variable analysis (SVA)) that estimate orthogonal transformations of these sources. We developed iteratively adjusted surrogate variable analysis (IA-SVA) that can estimate hidden factors even when they are correlated with other sources of variation by identifying a set of genes associated with each hidden factor in an iterative manner. Analysis of scRNA-seq data from human cells showed that IA-SVA could accurately capture hidden variation arising from technical (e.g., stacked doublet cells) or biological sources (e.g., cell type or cell-cycle stage). Furthermore, IA-SVA delivers a set of genes associated with the detected hidden source to be used in downstream data analyses. As a proof of concept, IA-SVA recapitulated known marker genes for islet cell subsets (e.g., alpha, beta), which improved the grouping of subsets into distinct clusters. Taken together, IA-SVA is an effective and novel method to dissect multiple and correlated sources of variation in scRNA-seq data.


Assuntos
Análise de Célula Única , Transcriptoma , Algoritmos , Ciclo Celular , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Análise de Sequência de RNA/métodos
13.
Sci Rep ; 8(1): 16048, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375457

RESUMO

Enhancers are cis-acting sequences that regulate transcription rates of their target genes in a cell-specific manner and harbor disease-associated sequence variants in cognate cell types. Many complex diseases are associated with enhancer malfunction, necessitating the discovery and study of enhancers from clinical samples. Assay for Transposase Accessible Chromatin (ATAC-seq) technology can interrogate chromatin accessibility from small cell numbers and facilitate studying enhancers in pathologies. However, on average, ~35% of open chromatin regions (OCRs) from ATAC-seq samples map to enhancers. We developed a neural network-based model, Predicting Enhancers from ATAC-Seq data (PEAS), to effectively infer enhancers from clinical ATAC-seq samples by extracting ATAC-seq data features and integrating these with sequence-related features (e.g., GC ratio). PEAS recapitulated ChromHMM-defined enhancers in CD14+ monocytes, CD4+ T cells, GM12878, peripheral blood mononuclear cells, and pancreatic islets. PEAS models trained on these 5 cell types effectively predicted enhancers in four cell types that are not used in model training (EndoC-ßH1, naïve CD8+ T, MCF7, and K562 cells). Finally, PEAS inferred individual-specific enhancers from 19 islet ATAC-seq samples and revealed variability in enhancer activity across individuals, including those driven by genetic differences. PEAS is an easy-to-use tool developed to study enhancers in pathologies by taking advantage of the increasing number of clinical epigenomes.


Assuntos
Sítios de Ligação , Elementos Facilitadores Genéticos , Redes Neurais de Computação , Transposases/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Curva ROC , Sensibilidade e Especificidade , Análise de Sequência de DNA , Transcriptoma , Transposases/química
14.
Diabetes ; 67(11): 2466-2477, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30181159

RESUMO

Type 2 diabetes (T2D) is a complex disorder in which both genetic and environmental risk factors contribute to islet dysfunction and failure. Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs), most of which are noncoding, in >200 loci to islet dysfunction and T2D. Identification of the putative causal variants and their target genes and whether they lead to gain or loss of function remains challenging. Here, we profiled chromatin accessibility in pancreatic islet samples from 19 genotyped individuals and identified 2,949 SNPs associated with in vivo cis-regulatory element use (i.e., chromatin accessibility quantitative trait loci [caQTL]). Among the caQTLs tested (n = 13) using luciferase reporter assays in MIN6 ß-cells, more than half exhibited effects on enhancer activity that were consistent with in vivo chromatin accessibility changes. Importantly, islet caQTL analysis nominated putative causal SNPs in 13 T2D-associated GWAS loci, linking 7 and 6 T2D risk alleles, respectively, to gain or loss of in vivo chromatin accessibility. By investigating the effect of genetic variants on chromatin accessibility in islets, this study is an important step forward in translating T2D-associated GWAS SNP into functional molecular consequences.


Assuntos
Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Ilhotas Pancreáticas/metabolismo , Alelos , Cromatina/genética , Diabetes Mellitus Tipo 2/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos
15.
Am J Hum Genet ; 102(4): 620-635, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625024

RESUMO

Genome-wide association studies (GWASs) and functional genomics approaches implicate enhancer disruption in islet dysfunction and type 2 diabetes (T2D) risk. We applied genetic fine-mapping and functional (epi)genomic approaches to a T2D- and proinsulin-associated 15q22.2 locus to identify a most likely causal variant, determine its direction of effect, and elucidate plausible target genes. Fine-mapping and conditional analyses of proinsulin levels of 8,635 non-diabetic individuals from the METSIM study support a single association signal represented by a cluster of 16 strongly associated (p < 10-17) variants in high linkage disequilibrium (r2 > 0.8) with the GWAS index SNP rs7172432. These variants reside in an evolutionarily and functionally conserved islet and ß cell stretch or super enhancer; the most strongly associated variant (rs7163757, p = 3 × 10-19) overlaps a conserved islet open chromatin site. DNA sequence containing the rs7163757 risk allele displayed 2-fold higher enhancer activity than the non-risk allele in reporter assays (p < 0.01) and was differentially bound by ß cell nuclear extract proteins. Transcription factor NFAT specifically potentiated risk-allele enhancer activity and altered patterns of nuclear protein binding to the risk allele in vitro, suggesting that it could be a factor mediating risk-allele effects. Finally, the rs7163757 proinsulin-raising and T2D risk allele (C) was associated with increased expression of C2CD4B, and possibly C2CD4A, both of which were induced by inflammatory cytokines, in human islets. Together, these data suggest that rs7163757 contributes to genetic risk of islet dysfunction and T2D by increasing NFAT-mediated islet enhancer activity and modulating C2CD4B, and possibly C2CD4A, expression in (patho)physiologic states.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Ilhotas Pancreáticas/patologia , Mutação/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Idoso , Alelos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Cromatina/metabolismo , Cromossomos Humanos Par 15/genética , Citocinas/metabolismo , DNA Intergênico/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/metabolismo , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único/genética , Proinsulina/metabolismo , Ratos , Fatores de Risco
16.
Sci Rep ; 7(1): 14466, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29089515

RESUMO

Broad domain promoters and super enhancers are regulatory elements that govern cell-specific functions and harbor disease-associated sequence variants. These elements are characterized by distinct epigenomic profiles, such as expanded deposition of histone marks H3K27ac for super enhancers and H3K4me3 for broad domains, however little is known about how they interact with each other and the rest of the genome in three-dimensional chromatin space. Using network theory methods, we studied chromatin interactions between broad domains and super enhancers in three ENCODE cell lines (K562, MCF7, GM12878) obtained via ChIA-PET, Hi-C, and Hi-CHIP assays. In these networks, broad domains and super enhancers interact more frequently with each other compared to their typical counterparts. Network measures and graphlets revealed distinct connectivity patterns associated with these regulatory elements that are robust across cell types and alternative assays. Machine learning models showed that these connectivity patterns could effectively discriminate broad domains from typical promoters and super enhancers from typical enhancers. Finally, targets of broad domains in these networks were enriched in disease-causing SNPs of cognate cell types. Taken together these results suggest a robust and unique organization of the chromatin around broad domains and super enhancers: loci critical for pathologies and cell-specific functions.


Assuntos
Cromatina/fisiologia , Histonas/metabolismo , Histonas/fisiologia , Linhagem Celular , Imunoprecipitação da Cromatina , Conectoma/métodos , Elementos Facilitadores Genéticos , Epigenômica , Código das Histonas , Humanos , Regiões Promotoras Genéticas , Domínios Proteicos
17.
PeerJ ; 5: e4040, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29181276

RESUMO

Assay for Transposase Accessible Chromatin (ATAC-seq) is an open chromatin profiling assay that is adapted to interrogate chromatin accessibility from small cell numbers. ATAC-seq surmounted a major technical barrier and enabled epigenome profiling of clinical samples. With this advancement in technology, we are now accumulating ATAC-seq samples from clinical samples at an unprecedented rate. These epigenomic profiles hold the key to uncovering how transcriptional programs are established in diverse human cells and are disrupted by genetic or environmental factors. Thus, the barrier to deriving important clinical insights from clinical epigenomic samples is no longer one of data generation but of data analysis. Specifically, we are still missing easy-to-use software tools that will enable non-computational scientists to analyze their own ATAC-seq samples. To facilitate systematic pre-processing and management of ATAC-seq samples, we developed an interactive, cross-platform, user-friendly and customized desktop application: interactive-ATAC (I-ATAC). I-ATAC integrates command-line data processing tools (FASTQC, Trimmomatic, BWA, Picard, ATAC_BAM_shiftrt_gappedAlign.pl, Bedtools and Macs2) into an easy-to-use platform with user interface to automatically pre-process ATAC-seq samples with parallelized and customizable pipelines. Its performance has been tested using public ATAC-seq datasets in GM12878 and CD4+T cells and a feature-based comparison is performed with some available interactive LIMS (Galaxy, SMITH, SeqBench, Wasp, NG6, openBIS). I-ATAC is designed to empower non-computational scientists to process their own datasets and to break to exclusivity of data analyses to computational scientists. Additionally, I-ATAC is capable of processing WGS and ChIP-seq samples, and can be customized by the user for one-independent or multiple-sequential operations.

18.
J Exp Med ; 214(10): 3123-3144, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28904110

RESUMO

Aging is linked to deficiencies in immune responses and increased systemic inflammation. To unravel the regulatory programs behind these changes, we applied systems immunology approaches and profiled chromatin accessibility and the transcriptome in PBMCs and purified monocytes, B cells, and T cells. Analysis of samples from 77 young and elderly donors revealed a novel and robust aging signature in PBMCs, with simultaneous systematic chromatin closing at promoters and enhancers associated with T cell signaling and a potentially stochastic chromatin opening mostly found at quiescent and repressed sites. Combined analyses of chromatin accessibility and the transcriptome uncovered immune molecules activated/inactivated with aging and identified the silencing of the IL7R gene and the IL-7 signaling pathway genes as potential biomarkers. This signature is borne by memory CD8+ T cells, which exhibited an aging-related loss in binding of NF-κB and STAT factors. Thus, our study provides a unique and comprehensive approach to identifying candidate biomarkers and provides mechanistic insights into aging-associated immunodeficiency.


Assuntos
Envelhecimento/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Cromatina/fisiologia , Adulto , Idoso , Envelhecimento/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Epigênese Genética , Feminino , Humanos , Interleucina-7/fisiologia , Subunidade alfa de Receptor de Interleucina-7/fisiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/fisiologia , Masculino , Transdução de Sinais/fisiologia , Adulto Jovem
19.
Sci Rep ; 7(1): 11959, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931935

RESUMO

Alpha TC1 (αTC1) and Beta-TC-6 (ßTC6) mouse islet cell lines are cellular models of islet (dys)function and type 2 diabetes (T2D). However, genomic characteristics of these cells, and their similarities to primary islet alpha and beta cells, are undefined. Here, we report the epigenomic (ATAC-seq) and transcriptomic (RNA-seq) landscapes of αTC1 and ßTC6 cells. Each cell type exhibits hallmarks of its primary islet cell counterpart including cell-specific expression of beta (e.g., Pdx1) and alpha (e.g., Arx) cell transcription factors (TFs), and enrichment of binding motifs for these TFs in αTC1/ßTC6 cis-regulatory elements. αTC1/ßTC6 transcriptomes overlap significantly with the transcriptomes of primary mouse/human alpha and beta cells. Our data further indicate that ATAC-seq detects cell-specific regulatory elements for cell types comprising ≥ 20% of a mixed cell population. We identified αTC1/ßTC6 cis-regulatory elements orthologous to those containing type 2 diabetes (T2D)-associated SNPs in human islets for 33 loci, suggesting these cells' utility to dissect T2D molecular genetics in these regions. Together, these maps provide important insights into the conserved regulatory architecture between αTC1/ßTC6 and primary islet cells that can be leveraged in functional (epi)genomic approaches to dissect the genetic and molecular factors controlling islet cell identity and function.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ilhotas Pancreáticas/patologia , Animais , Células Cultivadas , Camundongos , Transcrição Genética
20.
Trends Genet ; 33(4): 244-255, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28245910

RESUMO

Pancreatic islet dysfunction and beta cell failure are hallmarks of type 2 diabetes mellitus (T2DM) pathogenesis. In this review, we discuss how genome-wide association studies (GWASs) and recent developments in islet (epi)genome and transcriptome profiling (particularly single cell analyses) are providing novel insights into the genetic, environmental, and cellular contributions to islet (dys)function and T2DM pathogenesis. Moving forward, study designs that interrogate and model genetic variation [e.g., allelic profiling and (epi)genome editing] will be critical to dissect the molecular genetics of T2DM pathogenesis, to build next-generation cellular and animal models, and to develop precision medicine approaches to detect, treat, and prevent islet (dys)function and T2DM.


Assuntos
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transcriptoma/genética , Diabetes Mellitus Tipo 2/metabolismo , Edição de Genes , Genoma Humano , Genômica , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Análise de Célula Única
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