Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 109
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Development ; 147(14)2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32665240

RESUMO

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.

2.
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
3.
Cell Rep ; 29(4): 889-903.e10, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31644911

RESUMO

Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins.

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.
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
6.
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
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.
Science ; 362(6420)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30545857

RESUMO

Despite progress in defining genetic risk for psychiatric disorders, their molecular mechanisms remain elusive. Addressing this, the PsychENCODE Consortium has generated a comprehensive online resource for the adult brain across 1866 individuals. The PsychENCODE resource contains ~79,000 brain-active enhancers, sets of Hi-C linkages, and topologically associating domains; single-cell expression profiles for many cell types; expression quantitative-trait loci (QTLs); and further QTLs associated with chromatin, splicing, and cell-type proportions. Integration shows that varying cell-type proportions largely account for the cross-population variation in expression (with >88% reconstruction accuracy). It also allows building of a gene regulatory network, linking genome-wide association study variants to genes (e.g., 321 for schizophrenia). We embed this network into an interpretable deep-learning model, which improves disease prediction by ~6-fold versus polygenic risk scores and identifies key genes and pathways in psychiatric disorders.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Transtornos Mentais/genética , Conjuntos de Dados como Assunto , Aprendizado Profundo , Elementos Facilitadores Genéticos , Epigênese Genética , Epigenômica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas , Análise de Célula Única , Transcriptoma
9.
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
10.
J Biol Chem ; 293(41): 15790-15800, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30093408

RESUMO

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.


Assuntos
Interleucina-17/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , DNA/química , DNA/genética , Interleucina-17/genética , Camundongos Endogâmicos C57BL , Sequências Reguladoras de Ácido Nucleico/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Células Th17 , Fator de Transcrição RelA/genética , Ativação Transcricional
11.
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
12.
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
13.
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
14.
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
15.
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
16.
Nat Genet ; 50(5): 668-681, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29700475

RESUMO

Major depressive disorder (MDD) is a common illness accompanied by considerable morbidity, mortality, costs, and heightened risk of suicide. We conducted a genome-wide association meta-analysis based in 135,458 cases and 344,901 controls and identified 44 independent and significant loci. The genetic findings were associated with clinical features of major depression and implicated brain regions exhibiting anatomical differences in cases. Targets of antidepressant medications and genes involved in gene splicing were enriched for smaller association signal. We found important relationships of genetic risk for major depression with educational attainment, body mass, and schizophrenia: lower educational attainment and higher body mass were putatively causal, whereas major depression and schizophrenia reflected a partly shared biological etiology. All humans carry lesser or greater numbers of genetic risk factors for major depression. These findings help refine the basis of major depression and imply that a continuous measure of risk underlies the clinical phenotype.


Assuntos
Transtorno Depressivo Maior/genética , Herança Multifatorial , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Esquizofrenia/genética
18.
Methods Mol Biol ; 1767: 447-480, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29524151

RESUMO

Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.


Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Edição de Genes/métodos , RNA Guia/genética , Clonagem Molecular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica/métodos , Técnicas de Introdução de Genes/métodos , Biblioteca Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células K562 , Proteínas Luminescentes/genética , Sequências Reguladoras de Ácido Nucleico
19.
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
20.
Curr Opin Toxicol ; 6: 50-59, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29276797

RESUMO

It is well established that genetic variability has a major impact on susceptibility to common diseases, responses to drugs and toxicants, and influences disease-related outcomes. The appreciation that epigenetic marks also vary across the population is growing with more data becoming available from studies in humans and model organisms. In addition, the links between genetic variability, toxicity outcomes and epigenetics are being actively explored. Recent studies demonstrate that gene-by-environment interactions involve both chromatin states and transcriptional regulation, and that epigenetics provides important mechanistic clues to connect expression-related quantitative trait loci (QTL) and disease outcomes. However, studies of Gene×Environment×Epigenetics further extend the complexity of the experimental designs and create a challenge for selecting the most informative epigenetic readouts that can be feasibly performed to interrogate multiple individuals, exposures, tissue types and toxicity phenotypes. We propose that among the many possible epigenetic experimental methodologies, assessment of chromatin accessibility coupled with total RNA levels provides a cost-effective and comprehensive option to sufficiently characterize the complexity of epigenetic and regulatory activity in the context of understanding the inter-individual variability in responses to toxicants.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA