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1.
Cell ; 166(5): 1269-1281.e19, 2016 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-27565349

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 Respuesta
2.
Cell ; 151(5): 951-63, 2012 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-23178118

RESUMEN

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/citología
3.
Proc Natl Acad Sci U S A ; 121(11): e2317430121, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38437540

RESUMEN

Brown-and-white giant pandas (hereafter brown pandas) are distinct coat color mutants found exclusively in the Qinling Mountains, Shaanxi, China. However, its genetic mechanism has remained unclear since their discovery in 1985. Here, we identified the genetic basis for this coat color variation using a combination of field ecological data, population genomic data, and a CRISPR-Cas9 knockout mouse model. We de novo assembled a long-read-based giant panda genome and resequenced the genomes of 35 giant pandas, including two brown pandas and two family trios associated with a brown panda. We identified a homozygous 25-bp deletion in the first exon of Bace2, a gene encoding amyloid precursor protein cleaving enzyme, as the most likely genetic basis for brown-and-white coat color. This deletion was further validated using PCR and Sanger sequencing of another 192 black giant pandas and CRISPR-Cas9 edited knockout mice. Our investigation revealed that this mutation reduced the number and size of melanosomes of the hairs in knockout mice and possibly in the brown panda, further leading to the hypopigmentation. These findings provide unique insights into the genetic basis of coat color variation in wild animals.


Asunto(s)
Ursidae , Animales , Ratones , Ursidae/genética , Péptido Hidrolasas , Precursor de Proteína beta-Amiloide , Animales Salvajes , Ratones Noqueados
4.
Genome Res ; 32(6): 1183-1198, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35609992

RESUMEN

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/metabolismo
5.
Development ; 149(4)2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35179181

RESUMEN

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/metabolismo
6.
Clin Lab ; 70(8)2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39193968

RESUMEN

BACKGROUND: Krüppel-like 11 factor (KLF11) gene mutation has been implicated in the pathogenesis of maturity onset diabetes of the young type 7 (MODY7). Recently, this potential correlation has been questioned, suggesting the need for more comprehensive diagnostic approaches. METHODS: The proband is a 30-years-old male who underwent next-generation sequencing (NGS). This was followed by whole-exon sequencing of the proband and his parents to screen for KLF11 variants. RESULTS: A heterozygous KLF11 mutation c.793G>A (p.Glu265Lys) was identified in the proband and his non-diabetic mother. CONCLUSIONS: The novel KLF11 mutation documented in this study might exhibit incomplete penetrance in relation to impaired glucose tolerance, which could also contribute to the argument against the necessity of including KLF11 genetic testing for MODY diagnosis.


Asunto(s)
Diabetes Mellitus Tipo 2 , Proteínas Represoras , Adulto , Femenino , Humanos , Masculino , Proteínas Reguladoras de la Apoptosis , China , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/diagnóstico , Pueblos del Este de Asia/genética , Predisposición Genética a la Enfermedad , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Linaje , Proteínas Represoras/genética
7.
Development ; 147(14)2020 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-32665240

RESUMEN

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/metabolismo
8.
BMC Infect Dis ; 23(1): 300, 2023 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-37158831

RESUMEN

BACKGROUND: Standard treatment for drug-susceptible tuberculosis (DS-TB) includes a multidrug regimen requiring at least 6 months of treatment, and this lengthy treatment easily leads to poor adherence. There is an urgent need to simplify and shorten treatment regimens to reduce interruption and adverse event rates, improve compliance, and reduce costs. METHODS: ORIENT is a multicenter, randomized controlled, open-label, phase II/III, non-inferiority trial involving DS-TB patients to evaluate the safety and efficacy of short-term regimens compared with the standardized six-month treatment regimen. In stage 1, corresponding to a phase II trial, a total of 400 patients are randomly divided into four arms, stratified by site and the presence of lung cavitation. Investigational arms include 3 short-term regimens with rifapentine 10 mg/kg, 15 mg/kg, and 20 mg/kg, while the control arm uses the standardized six-month treatment regimen. A combination of rifapentine, isoniazid, pyrazinamide, and moxifloxacin is administered for 17 or 26 weeks in rifapentine arms, while a 26-week regimen containing rifampicin, isoniazid, pyrazinamide, and ethambutol is applied in the control arm. After the safety and preliminary effectiveness analysis of patients in stage 1, the control arm and the investigational arm meeting the conditions will enter into stage 2, which is equivalent to a phase III trial and will be expanded to recruit DS-TB patients. If all investigational arms do not meet the safety conditions, stage 2 will be canceled. In stage 1, the primary safety endpoint is permanent regimen discontinuation at 8 weeks after the first dose. The primary efficacy endpoint is the proportion of favorable outcomes at 78 weeks after the first dose for both two stages. DISCUSSION: This trial will contribute to the optimal dose of rifapentine in the Chinese population and suggest the feasibility of the short-course treatment regimen containing high-dose rifapentine and moxifloxacin for DS-TB. TRIAL REGISTRATION: The trial has been registered on ClinicalTrials.gov on 28 May 2022 with the identifier NCT05401071.


Asunto(s)
Rifampin , Tuberculosis , Humanos , Rifampin/efectos adversos , Isoniazida/efectos adversos , Pirazinamida , Moxifloxacino/uso terapéutico , Tuberculosis/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios Multicéntricos como Asunto , Ensayos Clínicos Fase II como Asunto
9.
Genome Res ; 28(10): 1577-1588, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30139769

RESUMEN

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 Órganos
10.
Genome Res ; 28(9): 1272-1284, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30097539

RESUMEN

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/metabolismo
11.
Genome Res ; 27(7): 1195-1206, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28385711

RESUMEN

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/microbiología
12.
Development ; 144(4): 720-730, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28087634

RESUMEN

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 , Transgenes
13.
Nucleic Acids Res ; 45(20): 11684-11699, 2017 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-28977539

RESUMEN

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/citología
14.
Genome Res ; 25(8): 1158-69, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26025803

RESUMEN

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/química
15.
Nat Methods ; 12(12): 1143-9, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26501517

RESUMEN

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.


Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Epigénesis Genética , Epigenómica/métodos , Elementos Reguladores de la Transcripción/genética , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Globinas/genética , Células HEK293 , Humanos , Células K562 , Lentivirus/genética , ARN Guía de Kinetoplastida/genética , Proteínas Represoras/genética , Proteínas Virales/genética
16.
Nature ; 489(7414): 75-82, 2012 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-22955617

RESUMEN

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.


Asunto(s)
Cromatina/genética , Cromatina/metabolismo , ADN/genética , Enciclopedias como Asunto , Genoma Humano/genética , Anotación de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Huella de ADN , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Evolución Molecular , Genómica , Humanos , Tasa de Mutación , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética
17.
PLoS Genet ; 11(1): e1004885, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25569532

RESUMEN

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.


Asunto(s)
Cromatina/genética , Elementos de Respuesta/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Animales , Sitios de Unión , Cromatina/efectos de los fármacos , Estructuras Cromosómicas/efectos de los fármacos , Estructuras Cromosómicas/genética , Elementos Transponibles de ADN , Doxorrubicina/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina , Humanos , Nucleosomas/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteína p53 Supresora de Tumor/metabolismo
18.
Hum Mol Genet ; 24(19): 5433-50, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26206884

RESUMEN

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Melanocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Transcripción SOXE/metabolismo , Animales , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Epigenómica/métodos , Melanocitos/citología , Ratones , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/genética
19.
Hum Mol Genet ; 24(22): 6552-63, 2015 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-26307087

RESUMEN

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a well-defined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis.


Asunto(s)
Aminoácido Oxidorreductasas/genética , Síndrome de Exfoliación/genética , ARN Largo no Codificante/genética , Anciano , Alelos , Estudios de Casos y Controles , Síndrome de Exfoliación/metabolismo , Femenino , Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Masculino , Estrés Oxidativo/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas
20.
BMC Genomics ; 17(1): 887, 2016 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-27821050

RESUMEN

BACKGROUND: The transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination. RESULTS: We developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function. CONCLUSIONS: Our computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the computational and functional datasets we present here will be valuable for the study of transcriptional regulation, SOX protein function, and glial cell biology.


Asunto(s)
Diferenciación Celular , Neuroglía/citología , Neuroglía/metabolismo , Factores de Transcripción SOXE/metabolismo , Secuencia de Bases , Diferenciación Celular/genética , Secuencia de Consenso , Secuencia Conservada , Exones , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Regiones Promotoras Genéticas , Elementos Reguladores de la Transcripción , Elementos de Respuesta , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/genética , Células de Schwann/metabolismo
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