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1.
Nat Commun ; 10(1): 4955, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672989

RESUMO

Systemic sclerosis (SSc) is an autoimmune disease that shows one of the highest mortality rates among rheumatic diseases. We perform a large genome-wide association study (GWAS), and meta-analysis with previous GWASs, in 26,679 individuals and identify 27 independent genome-wide associated signals, including 13 new risk loci. The novel associations nearly double the number of genome-wide hits reported for SSc thus far. We define 95% credible sets of less than 5 likely causal variants in 12 loci. Additionally, we identify specific SSc subtype-associated signals. Functional analysis of high-priority variants shows the potential function of SSc signals, with the identification of 43 robust target genes through HiChIP. Our results point towards molecular pathways potentially involved in vasculopathy and fibrosis, two main hallmarks in SSc, and highlight the spectrum of critical cell types for the disease. This work supports a better understanding of the genetic basis of SSc and provides directions for future functional experiments.

2.
Nat Biotechnol ; 37(8): 925-936, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375813

RESUMO

Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.


Assuntos
Células da Medula Óssea/metabolismo , Cromatina/química , Análise de Célula Única/métodos , Linfócitos T/metabolismo , Linhagem Celular , Simulação por Computador , Regulação da Expressão Gênica , Hematopoese , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos Mononucleares , Fatores de Transcrição/metabolismo
3.
Nat Methods ; 16(6): 489-492, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133759

RESUMO

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).


Assuntos
Cromatina/química , Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , RNA/química , Telomerase/química , Animais , Células Cultivadas , Cromossomos , Células-Tronco Embrionárias/citologia , Genoma , Camundongos , Regiões Promotoras Genéticas , RNA/genética , Telomerase/genética
4.
Mol Cell ; 73(6): 1174-1190.e12, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30745086

RESUMO

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

5.
Immunity ; 50(2): 362-377.e6, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30709738

RESUMO

Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.


Assuntos
Fatores de Transcrição Forkhead/imunologia , Mutação , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Criança , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Poliendocrinopatias Autoimunes/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismo
6.
J Invest Dermatol ; 139(3): 605-614, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30315781

RESUMO

The vast majority of polymorphisms for human dermatologic diseases fall in noncoding DNA regions, leading to difficulty interpreting their functional significance. Recent work using chromosome conformation capture technology in combination with chromatin immunoprecipitation (ChIP) has provided a systematic means of linking noncoding variants in active enhancer loci to putative gene targets. Here, we apply H3K27ac HiChIP high-resolution contact maps, generated from primary human T-cell subsets (CD4+ naïve, T helper type 17, and regulatory T cells), to 21 dermatologic conditions associated with single nucleotide polymorphisms from 106 genome-wide association studies. This "enhancer connectome" identified 1,492 HiChIP gene targets from 542 noncoding SNPs (P ≤ 5.0 × 10-8). SNP-containing enhancers from inflammatory skin conditions were significantly enriched at the HLA locus and also targeted several key factors from the JAK-STAT signaling pathway, but nonimmune conditions were not. A focused profiling of systemic lupus erythematosus HiChIP genes identified enhancer interactions with factors important for effector CD4+ T-cell differentiation and function, including IRF8 and members of the Ikaros family of zinc-finger proteins. Our results show the ability of the enhancer connectome to nominate functionally relevant candidates from genome-wide association study-identified variants, representing a powerful tool to guide future studies into the genomic regulatory mechanisms underlying dermatologic diseases.

7.
Cell ; 176(1-2): 361-376.e17, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30580963

RESUMO

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.


Assuntos
Epigenômica/métodos , Redes Reguladoras de Genes/genética , Análise de Célula Única/métodos , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Redes Reguladoras de Genes/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo
8.
Nat Genet ; 50(12): 1658-1665, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397335

RESUMO

Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Queratinócitos/fisiologia , Fatores de Transcrição/fisiologia , Tretinoína/farmacologia , Proteínas Supressoras de Tumor/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Epiderme/efeitos dos fármacos , Epiderme/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
9.
Science ; 362(6413)2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30361341

RESUMO

We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.


Assuntos
Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Neoplasias/genética , Neoplasias/metabolismo , Sequências Reguladoras de Ácido Nucleico , Cromatina/genética , Pegada de DNA , Elementos Facilitadores Genéticos , Loci Gênicos , Humanos , Imunidade/genética , Fatores de Transcrição/metabolismo , Transposases/metabolismo
10.
Nature ; 559(7715): E13, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29899441

RESUMO

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

11.
Cell ; 173(6): 1398-1412.e22, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29731168

RESUMO

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genes myc , RNA Longo não Codificante/genética , Animais , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Cromatina , DNA de Neoplasias/genética , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Transcrição Genética
12.
Mod Pathol ; 31(9): 1479-1486, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29743654

RESUMO

Distinguishing classical dendritic cells from other myeloid cell types is complicated by the shared expression of cell surface markers. ZBTB46 is a zinc finger and BTB domain-containing transcription factor, which is expressed by dendritic cells and committed dendritic cell precursors, but not by plasmacytoid dendritic cells, monocytes, macrophages, or other immune cell populations. In this study, we demonstrate that expression of ZBTB46 identifies human dendritic cell neoplasms. We examined ZBTB46 expression in a range of benign and malignant histiocytic disorders and found that ZBTB46 is able to clearly define the dendritic cell identity of many previously unclassified histiocytic disease subtypes. In particular, all examined cases of Langerhans cell histiocytosis and histiocytic sarcoma expressed ZBTB46, while all cases of blastic plasmacytoid dendritic cell neoplasm, chronic myelomonocytic leukemia, juvenile xanthogranuloma, Rosai-Dorfman disease, and Erdheim-Chester disease failed to demonstrate expression of ZBTB46. Moreover, ZBTB46 expression clarified the identity of diagnostically challenging neoplasms, such as cases of indeterminate cell histiocytosis, classifying a fraction of these entities as dendritic cell malignancies. These findings clarify the lineage origins of human histiocytic disorders and distinguish dendritic cell disorders from all other myeloid neoplasms.


Assuntos
Células Dendríticas/metabolismo , Histiocitose/diagnóstico , Células Mieloides/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Células Dendríticas/patologia , Diagnóstico Diferencial , Feminino , Histiocitose/metabolismo , Histiocitose/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Células Mieloides/patologia , Adulto Jovem
13.
Nat Med ; 24(5): 580-590, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29686426

RESUMO

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.


Assuntos
Cromatina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transposases/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Transformada , Células Clonais , Epigenômica , Humanos , Imunidade , Células Jurkat , Leucemia/imunologia , Leucemia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única
14.
Nat Genet ; 49(11): 1602-1612, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28945252

RESUMO

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.


Assuntos
Doenças Autoimunes/genética , Doenças Cardiovasculares/genética , DNA Intergênico/genética , Elementos Facilitadores Genéticos , Mutação , Regiões Promotoras Genéticas , Alelos , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Diferenciação Celular , Cromatina , Imunoprecipitação da Cromatina/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Intergênico/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , Cultura Primária de Células , Locos de Características Quantitativas , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia
15.
Nature ; 549(7670): 111-115, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28854172

RESUMO

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.


Assuntos
Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Elementos Facilitadores Genéticos/genética , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular , Linhagem Celular , Cromatina/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/biossíntese , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Células Th17/citologia , Células Th17/imunologia
16.
Nat Genet ; 49(10): 1522-1528, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28805829

RESUMO

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.


Assuntos
Linhagem da Célula/genética , Cromossomos Humanos/ultraestrutura , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Queratinócitos/citologia , Regiões Promotoras Genéticas/genética , Acetilação , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Cromossomos Humanos/genética , Células Epidérmicas , Biblioteca Gênica , Código das Histonas , Histonas/metabolismo , Humanos , Queratinócitos/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , RNA/genética , Interferência de RNA , Fatores de Transcrição/metabolismo
17.
Nat Methods ; 14(10): 959-962, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28846090

RESUMO

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-µm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.


Assuntos
DNA/genética , Congelamento , Genoma , Manejo de Espécimes/métodos , Animais , Encéfalo , Linhagem Celular , Eritrócitos , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Queratinócitos , Camundongos , Replicação de Sequência Autossustentável , Neoplasias da Glândula Tireoide , Transposases/metabolismo
18.
Nat Methods ; 13(11): 919-922, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27643841

RESUMO

Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.


Assuntos
Proteínas de Ciclo Celular/química , Imunoprecipitação da Cromatina/métodos , Cromatina/química , Proteínas Cromossômicas não Histona/química , DNA/química , Genômica/métodos , Animais , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Mapeamento Cromossômico , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Nat Protoc ; 11(2): 273-90, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26766114

RESUMO

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.


Assuntos
Conformação de Ácido Nucleico , RNA Mensageiro/química , Transcriptoma , Biologia Computacional , DNA Complementar/química , DNA Complementar/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Transcrição Reversa , Fatores de Tempo
20.
PLoS Genet ; 11(4): e1004953, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25874869

RESUMO

Shade from neighboring plants limits light for photosynthesis; as a consequence, plants have a variety of strategies to avoid canopy shade and compete with their neighbors for light. Collectively the response to foliar shade is called the shade avoidance syndrome (SAS). The SAS includes elongation of a variety of organs, acceleration of flowering time, and additional physiological responses, which are seen throughout the plant life cycle. However, current mechanistic knowledge is mainly limited to shade-induced elongation of seedlings. Here we use phenotypic profiling of seedling, leaf, and flowering time traits to untangle complex SAS networks. We used over-representation analysis (ORA) of shade-responsive genes, combined with previous annotation, to logically select 59 known and candidate novel mutants for phenotyping. Our analysis reveals shared and separate pathways for each shade avoidance response. In particular, auxin pathway components were required for shade avoidance responses in hypocotyl, petiole, and flowering time, whereas jasmonic acid pathway components were only required for petiole and flowering time responses. Our phenotypic profiling allowed discovery of seventeen novel shade avoidance mutants. Our results demonstrate that logical selection of mutants increased success of phenotypic profiling to dissect complex traits and discover novel components.


Assuntos
Magnoliopsida/genética , Redes e Vias Metabólicas , Fenótipo , Fototropismo/genética , Ciclopentanos/metabolismo , Flores/fisiologia , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Magnoliopsida/metabolismo , Magnoliopsida/fisiologia , Mutação , Oxilipinas/metabolismo , Folhas de Planta/fisiologia , Sementes/fisiologia , Luz Solar
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