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1.
Mol Neurodegener ; 16(1): 58, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429139

RESUMO

BACKGROUND: In the post-GWAS era, there is an unmet need to decode the underpinning genetic etiologies of late-onset Alzheimer's disease (LOAD) and translate the associations to causation. METHODS: We conducted ATAC-seq profiling using NeuN sorted-nuclei from 40 frozen brain tissues to determine LOAD-specific changes in chromatin accessibility landscape in a cell-type specific manner. RESULTS: We identified 211 LOAD-specific differential chromatin accessibility sites in neuronal-nuclei, four of which overlapped with LOAD-GWAS regions (±100 kb of SNP). While the non-neuronal nuclei did not show LOAD-specific differences, stratification by sex identified 842 LOAD-specific chromatin accessibility sites in females. Seven of these sex-dependent sites in the non-neuronal samples overlapped LOAD-GWAS regions including APOE. LOAD loci were functionally validated using single-nuclei RNA-seq datasets. CONCLUSIONS: Using brain sorted-nuclei enabled the identification of sex-dependent cell type-specific LOAD alterations in chromatin structure. These findings enhance the interpretation of LOAD-GWAS discoveries, provide potential pathomechanisms, and suggest novel LOAD-loci.

2.
Am J Hum Genet ; 108(8): 1436-1449, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216551

RESUMO

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.


Assuntos
Aberrações Cromossômicas , Análise Citogenética/métodos , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Genoma Humano , Mutação , Variações do Número de Cópias de DNA , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Masculino , Análise de Sequência de DNA
3.
PLoS Genet ; 16(1): e1008537, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961859

RESUMO

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.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Locos de Características Quantitativas , Animais , Cromatina/genética , Cromatina/metabolismo , Rim/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo
4.
Genome Biol Evol ; 11(10): 3035-3053, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31599933

RESUMO

Changes in transcriptional regulation are thought to be a major contributor to the evolution of phenotypic traits, but the contribution of changes in chromatin accessibility to the evolution of gene expression remains almost entirely unknown. To address this important gap in knowledge, we developed a new method to identify DNase I Hypersensitive (DHS) sites with differential chromatin accessibility between species using a joint modeling approach. Our method overcomes several limitations inherent to conventional threshold-based pairwise comparisons that become increasingly apparent as the number of species analyzed rises. Our approach employs a single quantitative test which is more sensitive than existing pairwise methods. To illustrate, we applied our joint approach to DHS sites in fibroblast cells from five primates (human, chimpanzee, gorilla, orangutan, and rhesus macaque). We identified 89,744 DHS sites, of which 41% are identified as differential between species using the joint model compared with 33% using the conventional pairwise approach. The joint model provides a principled approach to distinguishing single from multiple chromatin accessibility changes among species. We found that nondifferential DHS sites are enriched for nucleotide conservation. Differential DHS sites with decreased chromatin accessibility relative to rhesus macaque occur more commonly near transcription start sites (TSS), while those with increased chromatin accessibility occur more commonly distal to TSS. Further, differential DHS sites near TSS are less cell type-specific than more distal regulatory elements. Taken together, these results point to distinct classes of DHS sites, each with distinct characteristics of selection, genomic location, and cell type specificity.


Assuntos
Cromatina/química , Evolução Molecular , Animais , Linhagem Celular , Desoxirribonuclease I , Genômica , Gorilla gorilla/genética , Humanos , Macaca mulatta/genética , Modelos Genéticos , Pan troglodytes/genética , Pongo/genética , Sítio de Iniciação de Transcrição
5.
G3 (Bethesda) ; 9(8): 2521-2533, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31186305

RESUMO

Identifying the regulatory mechanisms of genome-wide association study (GWAS) loci affecting adipose tissue has been restricted due to limited characterization of adipose transcriptional regulatory elements. We profiled chromatin accessibility in three frozen human subcutaneous adipose tissue needle biopsies and preadipocytes and adipocytes from the Simpson Golabi-Behmel Syndrome (SGBS) cell strain using an assay for transposase-accessible chromatin (ATAC-seq). We identified 68,571 representative accessible chromatin regions (peaks) across adipose tissue samples (FDR < 5%). GWAS loci for eight cardiometabolic traits were enriched in these peaks (P < 0.005), with the strongest enrichment for waist-hip ratio. Of 110 recently described cardiometabolic GWAS loci colocalized with adipose tissue eQTLs, 59 loci had one or more variants overlapping an adipose tissue peak. Annotated variants at the SNX10 waist-hip ratio locus and the ATP2A1-SH2B1 body mass index locus showed allelic differences in regulatory assays. These adipose tissue accessible chromatin regions elucidate genetic variants that may alter adipose tissue function to impact cardiometabolic traits.


Assuntos
Tecido Adiposo/metabolismo , Cromatina/genética , Perfilação da Expressão Gênica , Genoma Humano , Genômica , Locos de Características Quantitativas , Característica Quantitativa Herdável , Adipócitos/metabolismo , Idoso , Alelos , Desequilíbrio Alélico , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Mapeamento Cromossômico , Suscetibilidade a Doenças , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica
6.
Genome Biol Evol ; 11(7): 1997-2008, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31233101

RESUMO

Humans carry a much larger percentage of body fat than other primates. Despite the central role of adipose tissue in metabolism, little is known about the evolution of white adipose tissue in primates. Phenotypic divergence is often caused by genetic divergence in cis-regulatory regions. We examined the cis-regulatory landscape of fat during human origins by performing comparative analyses of chromatin accessibility in human and chimpanzee adipose tissue using rhesus macaque as an outgroup. We find that many regions that have decreased accessibility in humans are enriched for promoter and enhancer sequences, are depleted for signatures of negative selection, are located near genes involved with lipid metabolism, and contain a short sequence motif involved in the beigeing of fat, the process in which lipid-storing white adipocytes are transdifferentiated into thermogenic beige adipocytes. The collective closing of many putative regulatory regions associated with beigeing of fat suggests a mechanism that increases body fat in humans.


Assuntos
Tecido Adiposo Branco/metabolismo , Tecido Adiposo/metabolismo , Genômica/métodos , Animais , Humanos , Primatas
7.
Science ; 362(6420)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30545853

RESUMO

Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.


Assuntos
Córtex Cerebral/embriologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Modelos Neurológicos , Neurogênese/genética , Organoides/embriologia , Elementos Facilitadores Genéticos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Transcriptoma
8.
Genome Res ; 28(10): 1577-1588, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30139769

RESUMO

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.


Assuntos
Miocárdio/metabolismo , Elementos Reguladores de Transcrição , Análise de Sequência de DNA/métodos , Fatores de Transcrição/genética , Adulto , Epigenômica , Expressão Gênica , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Especificidade de Órgãos
9.
Nat Commun ; 9(1): 3121, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087329

RESUMO

Schizophrenia genome-wide association studies have identified >150 regions of the genome associated with disease risk, yet there is little evidence that coding mutations contribute to this disorder. To explore the mechanism of non-coding regulatory elements in schizophrenia, we performed ATAC-seq on adult prefrontal cortex brain samples from 135 individuals with schizophrenia and 137 controls, and identified 118,152 ATAC-seq peaks. These accessible chromatin regions in the brain are highly enriched for schizophrenia SNP heritability. Accessible chromatin regions that overlap evolutionarily conserved regions exhibit an even higher heritability enrichment, indicating that sequence conservation can further refine functional risk variants. We identify few differences in chromatin accessibility between cases and controls, in contrast to thousands of age-related differential accessible chromatin regions. Altogether, we characterize chromatin accessibility in the human prefrontal cortex, the effect of schizophrenia and age on chromatin accessibility, and provide evidence that our dataset will allow for fine mapping of risk variants.


Assuntos
Cromatina/química , Estudo de Associação Genômica Ampla , Córtex Pré-Frontal/metabolismo , Locos de Características Quantitativas , Esquizofrenia/genética , Esquizofrenia/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/fisiopatologia , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Risco , Análise de Sequência de DNA
10.
Genome Res ; 28(9): 1272-1284, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30097539

RESUMO

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.


Assuntos
Elementos Facilitadores Genéticos , Código das Histonas , Motivos de Nucleotídeos , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Ligação Proteica , Fatores de Transcrição/metabolismo
11.
Cell Syst ; 7(2): 146-160.e7, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30031775

RESUMO

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor involved in metabolic and anti-inflammatory gene expression responses. To investigate what controls interactions between GR binding sites and their target genes, we used in situ Hi-C to generate high-resolution, genome-wide maps of chromatin interactions before and after glucocorticoid treatment. We found that GR binding to the genome typically does not cause new chromatin interactions to target genes but instead acts through chromatin interactions that already exist prior to hormone treatment. Both glucocorticoid-induced and glucocorticoid-repressed genes increased interactions with distal GR binding sites. In addition, while glucocorticoid-induced genes increased interactions with transcriptionally active chromosome compartments, glucocorticoid-repressed genes increased interactions with transcriptionally silent compartments. Lastly, while the architectural DNA-binding proteins CTCF and RAD21 were bound to most chromatin interactions, we found that glucocorticoid-responsive chromatin interactions were depleted for CTCF binding but enriched for RAD21. Together, these findings offer new insights into the mechanisms underlying GC-mediated gene activation and repression.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/genética , Proteínas de Ligação a DNA , Genoma Humano , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica
12.
Genome Biol Evol ; 10(3): 826-839, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29608722

RESUMO

Humans experience higher rates of age-associated diseases than our closest living evolutionary relatives, chimpanzees. Environmental factors can explain many of these increases in disease risk, but species-specific genetic changes can also play a role. Alleles that confer increased disease susceptibility later in life can persist in a population in the absence of selective pressure if those changes confer positive adaptation early in life. One age-associated disease that disproportionately affects humans compared with chimpanzees is epithelial cancer. Here, we explored genetic differences between humans and chimpanzees in a well-defined experimental assay that mimics gene expression changes that happen during cancer progression: A fibroblast serum challenge. We used this assay with fibroblasts isolated from humans and chimpanzees to explore species-specific differences in gene expression and chromatin state with RNA-Seq and DNase-Seq. Our data reveal that human fibroblasts increase expression of genes associated with wound healing and cancer pathways; in contrast, chimpanzee gene expression changes are not concentrated around particular functional categories. Chromatin accessibility dramatically increases in human fibroblasts, yet decreases in chimpanzee cells during the serum response. Many regions of opening and closing chromatin are in close proximity to genes encoding transcription factors or genes involved in wound healing processes, further supporting the link between changes in activity of regulatory elements and changes in gene expression. Together, these expression and open chromatin data show that humans and chimpanzees have dramatically different responses to the same physiological stressor, and how a core physiological process can evolve quickly over relatively short evolutionary time scales.


Assuntos
Cromatina/genética , Evolução Molecular , Variação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Pan troglodytes/sangue , Pan troglodytes/genética , Regiões Promotoras Genéticas , Especificidade da Espécie , Fatores de Transcrição/genética
13.
Nat Genet ; 50(4): 538-548, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29632383

RESUMO

Genome-wide association studies (GWAS) have identified over 100 risk loci for schizophrenia, but the causal mechanisms remain largely unknown. We performed a transcriptome-wide association study (TWAS) integrating a schizophrenia GWAS of 79,845 individuals from the Psychiatric Genomics Consortium with expression data from brain, blood, and adipose tissues across 3,693 primarily control individuals. We identified 157 TWAS-significant genes, of which 35 did not overlap a known GWAS locus. Of these 157 genes, 42 were associated with specific chromatin features measured in independent samples, thus highlighting potential regulatory targets for follow-up. Suppression of one identified susceptibility gene, mapk3, in zebrafish showed a significant effect on neurodevelopmental phenotypes. Expression and splicing from the brain captured most of the TWAS effect across all genes. This large-scale connection of associations to target genes, tissues, and regulatory features is an essential step in moving toward a mechanistic understanding of GWAS.


Assuntos
Cromatina/genética , Esquizofrenia/etiologia , Esquizofrenia/genética , Animais , Encéfalo/metabolismo , Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Herança Multifatorial , Proteína Fosfatase 2/genética , Locos de Características Quantitativas , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
14.
Mamm Genome ; 29(1-2): 153-167, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29429127

RESUMO

Epigenetic effects of environmental chemicals are under intense investigation to fill existing knowledge gaps between environmental/occupational exposures and adverse health outcomes. Chromatin accessibility is one prominent mechanism of epigenetic control of transcription, and understanding of the chemical effects on both could inform the causal role of epigenetic alterations in disease mechanisms. In this study, we hypothesized that baseline variability in chromatin organization and transcription profiles among various tissues and mouse strains influence the outcome of exposure to the DNA damaging chemical 1,3-butadiene. To test this hypothesis, we evaluated DNA damage along with comprehensive quantification of RNA transcripts (RNA-seq), identification of accessible chromatin (ATAC-seq), and characterization of regions with histone modifications associated with active transcription (ChIP-seq for acetylation at histone 3 lysine 27, H3K27ac). We collected these data in the lung, liver, and kidney of mice from two genetically divergent strains, C57BL/6J and CAST/EiJ, that were exposed to clean air or to 1,3-butadiene (~600 ppm) for 2 weeks. We found that tissue effects dominate differences in both gene expression and chromatin states, followed by strain effects. At baseline, xenobiotic metabolism was consistently more active in CAST/EiJ, while immune system pathways were more active in C57BL/6J across tissues. Surprisingly, even though all three tissues in both strains harbored butadiene-induced DNA damage, little transcriptional effect of butadiene was observed in liver and kidney. Toxicologically relevant effects of butadiene in the lung were on the pathways of xenobiotic metabolism and inflammation. We also found that variability in chromatin accessibility across individuals (i.e., strains) only partially explains the variability in transcription. This study showed that variation in the basal states of epigenome and transcriptome may be useful indicators for individuals or tissues susceptible to genotoxic environmental chemicals.


Assuntos
Dano ao DNA/efeitos dos fármacos , Epigênese Genética , Transcrição Genética/genética , Transcriptoma/genética , Animais , Butadienos/toxicidade , Carcinógenos/toxicidade , Cromatina/efeitos dos fármacos , Histonas/genética , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Testes de Mutagenicidade , Especificidade de Órgãos/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
15.
Environ Health Perspect ; 125(10): 107006, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038090

RESUMO

BACKGROUND: The damaging effects of exposure to environmental toxicants differentially affect genetically distinct individuals, but the mechanisms contributing to these differences are poorly understood. Genetic variation affects the establishment of the gene regulatory landscape and thus gene expression, and we hypothesized that this contributes to the observed heterogeneity in individual responses to exogenous cellular insults. OBJECTIVES: We performed an in vivo study of how genetic variation and chromatin organization may dictate susceptibility to DNA damage, and influence the cellular response to such damage, caused by an environmental toxicant. MATERIALS AND METHODS: We measured DNA damage, messenger RNA (mRNA) and microRNA (miRNA) expression, and genome-wide chromatin accessibility in lung tissue from two genetically divergent inbred mouse strains, C57BL/6J and CAST/EiJ, both in unexposed mice and in mice exposed to a model DNA-damaging chemical, 1,3-butadiene. RESULTS: Our results showed that unexposed CAST/EiJ and C57BL/6J mice have very different chromatin organization and transcription profiles in the lung. Importantly, in unexposed CAST/EiJ mice, which acquired relatively less 1,3-butadiene-induced DNA damage, we observed increased transcription and a more accessible chromatin landscape around genes involved in detoxification pathways. Upon chemical exposure, chromatin was significantly remodeled in the lung of C57BL/6J mice, a strain that acquired higher levels of 1,3-butadiene-induced DNA damage, around the same genes, ultimately resembling the molecular profile of CAST/EiJ. CONCLUSIONS: These results suggest that strain-specific changes in chromatin and transcription in response to chemical exposure lead to a "compensation" for underlying genetic-driven interindividual differences in the baseline chromatin and transcriptional state. This work represents an example of how chemical and environmental exposures can be evaluated to better understand gene-by-environment interactions, and it demonstrates the important role of chromatin response in transcriptomic changes and, potentially, in deleterious effects of exposure. https://doi.org/10.1289/EHP1937.


Assuntos
Poluentes Atmosféricos/toxicidade , Butadienos/toxicidade , Dano ao DNA , Transcrição Genética/efeitos dos fármacos , Animais , Cromatina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos
16.
Nat Biotechnol ; 35(6): 561-568, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28369033

RESUMO

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the ß-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Elementos Reguladores de Transcrição/genética , Análise de Sequência de DNA/métodos , Mapeamento Cromossômico/métodos , Epigenômica/métodos , Humanos
17.
Cell ; 166(5): 1269-1281.e19, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-27565349

RESUMO

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.


Assuntos
Genoma Humano , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células A549 , Sítios de Ligação/efeitos dos fármacos , Imunoprecipitação da Cromatina , Dexametasona/metabolismo , Dexametasona/farmacologia , Genes Reporter , Glucocorticoides/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , Elementos de Resposta
18.
Nucleic Acids Res ; 44(7): 3082-94, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26673704

RESUMO

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTRTAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.


Assuntos
Cromatina/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Elementos Isolantes , Fator de Ligação a CCCTC , Células CACO-2 , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Loci Gênicos , Humanos , Proteínas Repressoras/metabolismo
19.
Nat Methods ; 12(12): 1143-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26501517

RESUMO

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.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Epigênese Genética , Epigenômica/métodos , Elementos Reguladores de Transcrição/genética , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Globinas/genética , Células HEK293 , Humanos , Células K562 , Lentivirus/genética , RNA Guia/genética , Proteínas Repressoras/genética , Proteínas Virais/genética
20.
Hum Mol Genet ; 24(22): 6552-63, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26307087

RESUMO

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.


Assuntos
Aminoácido Oxirredutases/genética , Síndrome de Exfoliação/genética , RNA Longo não Codificante/genética , Idoso , Alelos , Estudos de Casos e Controles , Síndrome de Exfoliação/metabolismo , Feminino , Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Estresse Oxidativo/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas
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