RESUMEN
The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs.
Asunto(s)
Genoma Humano , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Células A549 , Sitios de Unión/efectos de los fármacos , Inmunoprecipitación de Cromatina , Dexametasona/metabolismo , Dexametasona/farmacología , Genes Reporteros , Glucocorticoides/farmacología , Humanos , Unión Proteica/efectos de los fármacos , Elementos de RespuestaRESUMEN
The inactive X chromosome's (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.
Asunto(s)
Silenciador del Gen , Elementos Reguladores de la Transcripción , Inactivación del Cromosoma X , Animales , Factor de Unión a CCCTC , Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Código de Histonas , Elementos de Nucleótido Esparcido Largo , Ratones , Proteínas del Grupo Polycomb/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trofoblastos/citologíaRESUMEN
Over a thousand different transcription factors (TFs) bind with varying occupancy across the human genome. Chromatin immunoprecipitation (ChIP) can assay occupancy genome-wide, but only one TF at a time, limiting our ability to comprehensively observe the TF occupancy landscape, let alone quantify how it changes across conditions. We developed TF occupancy profiler (TOP), a Bayesian hierarchical regression framework, to profile genome-wide quantitative occupancy of numerous TFs using data from a single chromatin accessibility experiment (DNase- or ATAC-seq). TOP is supervised, and its hierarchical structure allows it to predict the occupancy of any sequence-specific TF, even those never assayed with ChIP. We used TOP to profile the quantitative occupancy of hundreds of sequence-specific TFs at sites throughout the genome and examined how their occupancies changed in multiple contexts: in approximately 200 human cell types, through 12 h of exposure to different hormones, and across the genetic backgrounds of 70 individuals. TOP enables cost-effective exploration of quantitative changes in the landscape of TF binding.
Asunto(s)
Cromatina , Factores de Transcripción , Teorema de Bayes , Sitios de Unión/genética , Cromatina/genética , Genoma Humano , Humanos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, which are defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with five out of six elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in larval hearts. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration. This article has an associated 'The people behind the papers' interview.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Corazón/fisiología , Pericardio/metabolismo , Regeneración/fisiología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Cromatina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Regulación de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Pericardio/citología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.
Asunto(s)
Aberraciones Cromosómicas , Análisis Citogenético/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Genoma Humano , Mutación , Variaciones en el Número de Copia de ADN , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cariotipificación , Masculino , Análisis de Secuencia de ADNRESUMEN
To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.
Asunto(s)
Aletas de Animales/metabolismo , Elementos de Facilitación Genéticos/genética , Regeneración/fisiología , Pez Cebra/metabolismo , Aletas de Animales/fisiología , Animales , Animales Modificados Genéticamente/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Conexina 43/genética , Conexina 43/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Leptina/genética , Leptina/metabolismo , Midkina/genética , Midkina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Mapping DNase I hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. We employed high-throughput sequencing and whole-genome tiled array strategies to identify DNase I HS sites within human primary CD4+ T cells. Combining these two technologies, we have created a comprehensive and accurate genome-wide open chromatin map. Surprisingly, only 16%-21% of the identified 94,925 DNase I HS sites are found in promoters or first exons of known genes, but nearly half of the most open sites are in these regions. In conjunction with expression, motif, and chromatin immunoprecipitation data, we find evidence of cell-type-specific characteristics, including the ability to identify transcription start sites and locations of different chromatin marks utilized in these cells. In addition, and unexpectedly, our analyses have uncovered detailed features of nucleosome structure.
Asunto(s)
Cromatina/genética , Genoma Humano/genética , Algoritmos , Área Bajo la Curva , Sitios de Unión , Linfocitos T CD4-Positivos/citología , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Mapeo Cromosómico/métodos , Cromosomas Humanos , Desoxirribonucleasa I/química , Desoxirribonucleasa I/farmacología , Genoma Humano/inmunología , Histonas/química , Humanos , Nucleosomas/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Curva ROC , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Gene transcription profiles across tissues are largely defined by the activity of regulatory elements, most of which correspond to regions of accessible chromatin. Regulatory element activity is in turn modulated by genetic variation, resulting in variable transcription rates across individuals. The interplay of these factors, however, is poorly understood. Here we characterize expression and chromatin state dynamics across three tissues-liver, lung, and kidney-in 47 strains of the Collaborative Cross (CC) mouse population, examining the regulation of these dynamics by expression quantitative trait loci (eQTL) and chromatin QTL (cQTL). QTL whose allelic effects were consistent across tissues were detected for 1,101 genes and 133 chromatin regions. Also detected were eQTL and cQTL whose allelic effects differed across tissues, including local-eQTL for Pik3c2g detected in all three tissues but with distinct allelic effects. Leveraging overlapping measurements of gene expression and chromatin accessibility on the same mice from multiple tissues, we used mediation analysis to identify chromatin and gene expression intermediates of eQTL effects. Based on QTL and mediation analyses over multiple tissues, we propose a causal model for the distal genetic regulation of Akr1e1, a gene involved in glycogen metabolism, through the zinc finger transcription factor Zfp985 and chromatin intermediates. This analysis demonstrates the complexity of transcriptional and chromatin dynamics and their regulation over multiple tissues, as well as the value of the CC and related genetic resource populations for identifying specific regulatory mechanisms within cells and tissues.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/química , Sitios de Carácter Cuantitativo , Animales , Cromatina/genética , Cromatina/metabolismo , Riñón/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Especificidad de Órganos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Cis-regulatory elements (CRE), short DNA sequences through which transcription factors (TFs) exert regulatory control on gene expression, are postulated to be the major sites of causal sequence variation underlying the genetics of complex traits and diseases. We present integrative analyses, combining high-throughput genomic and epigenomic data with sequence-based computations, to identify the causal transcriptional components in a given tissue. We use data on adult human hearts to demonstrate that (1) sequence-based predictions detect numerous, active, tissue-specific CREs missed by experimental observations, (2) learned sequence features identify the cognate TFs, (3) CRE variants are specifically associated with cardiac gene expression, and (4) a significant fraction of the heritability of exemplar cardiac traits (QT interval, blood pressure, pulse rate) is attributable to these variants. This general systems approach can thus identify candidate causal variants and the components of gene regulatory networks (GRN) to enable understanding of the mechanisms of complex disorders on a tissue- or cell-type basis.
Asunto(s)
Miocardio/metabolismo , Elementos Reguladores de la Transcripción , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genética , Adulto , Epigenómica , Expresión Génica , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Especificidad de ÓrganosRESUMEN
Glucocorticoids are potent steroid hormones that regulate immunity and metabolism by activating the transcription factor (TF) activity of glucocorticoid receptor (GR). Previous models have proposed that DNA binding motifs and sites of chromatin accessibility predetermine GR binding and activity. However, there are vast excesses of both features relative to the number of GR binding sites. Thus, these features alone are unlikely to account for the specificity of GR binding and activity. To identify genomic and epigenetic contributions to GR binding specificity and the downstream changes resultant from GR binding, we performed hundreds of genome-wide measurements of TF binding, epigenetic state, and gene expression across a 12-h time course of glucocorticoid exposure. We found that glucocorticoid treatment induces GR to bind to nearly all pre-established enhancers within minutes. However, GR binds to only a small fraction of the set of accessible sites that lack enhancer marks. Once GR is bound to enhancers, a combination of enhancer motif composition and interactions between enhancers then determines the strength and persistence of GR binding, which consequently correlates with dramatic shifts in enhancer activation. Over the course of several hours, highly coordinated changes in TF binding and histone modification occupancy occur specifically within enhancers, and these changes correlate with changes in the expression of nearby genes. Following GR binding, changes in the binding of other TFs precede changes in chromatin accessibility, suggesting that other TFs are also sensitive to genomic features beyond that of accessibility.
Asunto(s)
Elementos de Facilitación Genéticos , Código de Histonas , Motivos de Nucleótidos , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional , Línea Celular Tumoral , Epigénesis Genética , Humanos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Most human transcription factors bind a small subset of potential genomic sites and often use different subsets in different cell types. To identify mechanisms that govern cell-type-specific transcription factor binding, we used an integrative approach to study estrogen receptor α (ER). We found that ER exhibits two distinct modes of binding. Shared sites, bound in multiple cell types, are characterized by high-affinity estrogen response elements (EREs), inaccessible chromatin, and a lack of DNA methylation, while cell-specific sites are characterized by a lack of EREs, co-occurrence with other transcription factors, and cell-type-specific chromatin accessibility and DNA methylation. These observations enabled accurate quantitative models of ER binding that suggest tethering of ER to one-third of cell-specific sites. The distinct properties of cell-specific binding were also observed with glucocorticoid receptor and for ER in primary mouse tissues, representing an elegant genomic encoding scheme for generating cell-type-specific gene regulation.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Secuencia Conservada , Metilación de ADN , Estradiol/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Estrógenos/farmacología , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas/efectos de los fármacos , Interferencia de ARN , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta , Termodinámica , Factores de Transcripción/genética , TransfecciónRESUMEN
Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.
Asunto(s)
Microbioma Gastrointestinal , Regulación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/biosíntesis , Enfermedades Inflamatorias del Intestino , Proteínas de Pez Cebra/biosíntesis , Pez Cebra , Animales , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Especificidad de la Especie , Pez Cebra/metabolismo , Pez Cebra/microbiologíaRESUMEN
A current goal of molecular biology is to identify transcriptional networks that regulate cell differentiation. However, identifying functional gene regulatory elements has been challenging in the context of developing tissues where material is limited and cell types are mixed. To identify regulatory sites during sex determination, we subjected Sertoli cells from mouse fetal testes to DNaseI-seq and ChIP-seq for H3K27ac. DNaseI-seq identified putative regulatory sites around genes enriched in Sertoli and pregranulosa cells; however, active enhancers marked by H3K27ac were enriched proximal to only Sertoli-enriched genes. Sequence analysis identified putative binding sites of known and novel transcription factors likely controlling Sertoli cell differentiation. As a validation of this approach, we identified a novel Sertoli cell enhancer upstream of Wt1, and used it to drive expression of a transgenic reporter in Sertoli cells. This work furthers our understanding of the complex genetic network that underlies sex determination and identifies regions that potentially harbor non-coding mutations underlying disorders of sexual development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Elementos Reguladores de la Transcripción , Células de Sertoli/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Desoxirribonucleasa I/metabolismo , Elementos de Facilitación Genéticos , Genes Reporteros , Genoma , Histonas/metabolismo , Homocigoto , Masculino , Ratones , Mutación , Regiones Promotoras Genéticas , Procesos de Determinación del Sexo , Testículo/embriología , TransgenesRESUMEN
Interleukin (IL)-1ß plays a critical role in IL-6ß- and transforming growth factor ß (TGFß)-initiated Th17 differentiation and induction of Th17-mediated autoimmunity. However, the means by which IL-1 regulates various aspects of Th17 development remain poorly understood. We recently reported that IL-1ß enhances STAT3 phosphorylation via NF-κB-mediated repression of SOCS3 to facilitate Il17 transcription and Th17 differentiation, identifying an effect of IL-1 signaling on proximal events of STAT3 signaling. Here, we show that IL-1ß promotes STAT3 binding to key cis-elements that control IL-17 expression. Additionally, we demonstrate that the IL-1-induced NF-κB factor RelA directly regulates the Il17a/f loci in cooperation with STAT3. Our findings reveal that IL-1 impacts both proximal signaling events and downstream interactions between transcription factors and cis-regulatory elements to promote Il17a/f transcription and Th17 differentiation.
Asunto(s)
Interleucina-17/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , ADN/química , ADN/genética , Interleucina-17/genética , Ratones Endogámicos C57BL , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , Células Th17 , Factor de Transcripción ReIA/genética , Activación TranscripcionalRESUMEN
Although deoxyribonuclease I (DNase I) was used to probe the structure of the nucleosome in the 1960s and 1970s, in the current high-throughput sequencing era, DNase I has mainly been used to study genomic regions devoid of nucleosomes. Here, we reveal for the first time that DNase I can be used to precisely map the (translational) positions of in vivo nucleosomes genome-wide. Specifically, exploiting a distinctive DNase I cleavage profile within nucleosome-associated DNA--including a signature 10.3 base pair oscillation that corresponds to accessibility of the minor groove as DNA winds around the nucleosome--we develop a Bayes-factor-based method that can be used to map nucleosome positions along the genome. Compared to methods that require genetically modified histones, our DNase-based approach is easily applied in any organism, which we demonstrate by producing maps in yeast and human. Compared to micrococcal nuclease (MNase)-based methods that map nucleosomes based on cuts in linker regions, we utilize DNase I cuts both outside and within nucleosomal DNA; the oscillatory nature of the DNase I cleavage profile within nucleosomal DNA enables us to identify translational positioning details not apparent in MNase digestion of linker DNA. Because the oscillatory pattern corresponds to nucleosome rotational positioning, it also reveals the rotational context of transcription factor (TF) binding sites. We show that potential binding sites within nucleosome-associated DNA are often centered preferentially on an exposed major or minor groove. This preferential localization may modulate TF interaction with nucleosome-associated DNA as TFs search for binding sites.
Asunto(s)
Mapeo Cromosómico , ADN/genética , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Nucleosomas/metabolismo , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Biología Computacional/métodos , Genoma Fúngico , Genoma Humano , Genómica/métodos , Humanos , Motivos de Nucleótidos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-kappaB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-kappaB therefore cooperate at multiple cis elements to enable NF-kappaB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8(+) T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Factor de Transcripción STAT4/metabolismo , Proteínas de Dominio T Box/metabolismo , Células TH1/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-18/inmunología , Interleucina-18/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Elementos Reguladores de la Transcripción/genética , Factor de Transcripción STAT4/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Células TH1/inmunología , Células TH1/patología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Activación TranscripcionalRESUMEN
Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. We identified epigenetic modifications across the clustered Il17a and Il17f and the Ifng loci before and after differential IL-12 or TGF-beta cytokine signaling, which induce divergent fates of Th17 cell precursors. We found that Th17 cell precursors had substantial remodeling of the Ifng locus, but underwent critical additional modifications to enable high expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus were amplified by TGF-beta signaling in Th17 cells, but were rapidly reversed downstream of IL-12-induced silencing of the Rorc gene by the transcription factors STAT4 and T-bet. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 cell lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 cell lineage factors.
Asunto(s)
Epigénesis Genética , Inestabilidad Genómica , Interleucina-17 , Factor de Transcripción STAT4 , Proteínas de Dominio T Box , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linaje de la Célula , Células Cultivadas , Citometría de Flujo , Silenciador del Gen , Sitios Genéticos , Immunoblotting , Interleucina-17/genética , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genéticaRESUMEN
Our current understanding of cellular transdifferentiation systems is limited. It is oftentimes unknown, at a genome-wide scale, how much transdifferentiated cells differ quantitatively from both the starting cells and the target cells. Focusing on transdifferentiation of primary human skin fibroblasts by forced expression of myogenic transcription factor MyoD, we performed quantitative analyses of gene expression and chromatin accessibility profiles of transdifferentiated cells compared to fibroblasts and myoblasts. In this system, we find that while many of the early muscle marker genes are reprogrammed, global gene expression and accessibility changes are still incomplete when compared to myoblasts. In addition, we find evidence of epigenetic memory in the transdifferentiated cells, with reminiscent features of fibroblasts being visible both in chromatin accessibility and gene expression. Quantitative analyses revealed a continuum of changes in chromatin accessibility induced by MyoD, and a strong correlation between chromatin-remodeling deficiencies and incomplete gene expression reprogramming. Classification analyses identified genetic and epigenetic features that distinguish reprogrammed from non-reprogrammed sites, and suggested ways to potentially improve transdifferentiation efficiency. Our approach for combining gene expression, DNA accessibility, and protein-DNA binding data to quantify and characterize the efficiency of cellular transdifferentiation on a genome-wide scale can be applied to any transdifferentiation system.
Asunto(s)
Transdiferenciación Celular/genética , Reprogramación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Proteína MioD/genética , Western Blotting , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Células HEK293 , Humanos , Microscopía Confocal , Proteína MioD/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citologíaRESUMEN
Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.
Asunto(s)
Sistemas CRISPR-Cas , Cromatina/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Ensamble y Desensamble de Cromatina , ADN/química , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Ingeniería Genética/métodos , Genoma Humano , Células HEK293 , Humanos , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Factores de Transcripción/químicaRESUMEN
Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements.