Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.140
Filtrar
1.
Nat Commun ; 11(1): 5061, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033262

RESUMO

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.


Assuntos
Reprogramação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Catálise , Proliferação de Células , Senescência Celular , Desmetilação , Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
2.
Nat Commun ; 11(1): 4544, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917861

RESUMO

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Ativação Transcricional , Acetilação , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Eritroblastos , Feminino , Feto , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq
3.
Nat Commun ; 11(1): 3419, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647123

RESUMO

The development and function of the brain require tight control of gene expression. Genome architecture is thought to play a critical regulatory role in gene expression, but the mechanisms governing genome architecture in the brain in vivo remain poorly understood. Here, we report that conditional knockout of the chromatin remodeling enzyme Chd4 in granule neurons of the mouse cerebellum increases accessibility of gene regulatory sites genome-wide in vivo. Conditional knockout of Chd4 promotes recruitment of the architectural protein complex cohesin preferentially to gene enhancers in granule neurons in vivo. Importantly, in vivo profiling of genome architecture reveals that conditional knockout of Chd4 strengthens interactions among developmentally repressed contact domains as well as genomic loops in a manner that tightly correlates with increased accessibility, enhancer activity, and cohesin occupancy at these sites. Collectively, our findings define a role for chromatin remodeling in the control of genome architecture organization in the mammalian brain.


Assuntos
Encéfalo/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Genoma , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/metabolismo , DNA Helicases/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Camundongos Knockout , Modelos Genéticos , Ligação Proteica
4.
Nat Commun ; 11(1): 2799, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493979

RESUMO

Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.


Assuntos
Canais Iônicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Acetiltransferases , Animais , Biocatálise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Fluorescência , Genoma Humano , Células HEK293 , Humanos , Ativação do Canal Iônico , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/química
5.
Nature ; 583(7816): 447-452, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499651

RESUMO

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.


Assuntos
Cromossomos Humanos Par 11/genética , Colite/genética , Colite/imunologia , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Linfócitos T Reguladores/imunologia , Acetilação , Alelos , Animais , Cromossomos de Mamíferos/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Sintenia/genética
6.
Nat Commun ; 11(1): 2722, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483172

RESUMO

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.


Assuntos
Diferenciação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismo , Animais , Células Cultivadas , Cromatina/genética , Cromossomos de Mamíferos/genética , Feminino , Perfilação da Expressão Gênica/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco/citologia
7.
Nature ; 582(7812): 432-437, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499643

RESUMO

Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/genética , Análise de Sequência de RNA/métodos , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos/genética , Elementos Facilitadores Genéticos/genética , Genes myc/genética , Genes de RNAr/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes , Transcrição Genética
8.
PLoS One ; 15(6): e0235343, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584896

RESUMO

Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFß pseudo-receptor BAMBI.


Assuntos
Elementos Facilitadores Genéticos/genética , Oncogenes/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Edição de Genes , Regulação Neoplásica da Expressão Gênica , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RNA Guia/metabolismo , Neoplasias de Mama Triplo Negativas/genética
9.
J Vis Exp ; (160)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32568241

RESUMO

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to "loss-of-function" approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct "gain-of-function" studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.


Assuntos
Elementos Facilitadores Genéticos/genética , RNA/genética , Transcrição Genética/genética , Humanos
10.
Nucleic Acids Res ; 48(12): 6597-6610, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479598

RESUMO

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of this enhancer cooperativity. We generated genetic deletions of four estrogen receptor α (ER) bound enhancers that regulate two genes and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences, we found that the genomic location of a full ERE strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing permissible chromatin environment can also be needed for estrogen-driven gene regulation to occur.


Assuntos
Elementos Facilitadores Genéticos/genética , Receptor alfa de Estrogênio/genética , Motivos de Nucleotídeos/genética , Transcrição Genética , Acetilação , Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genética
11.
Nat Commun ; 11(1): 2082, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350257

RESUMO

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.


Assuntos
Células Endócrinas/citologia , Células Endócrinas/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Histona Desmetilases/metabolismo , Ilhotas Pancreáticas/citologia , Transdução de Sinais , Tretinoína/metabolismo , Adulto , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Células Endócrinas/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/embriologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Adulto Jovem
12.
Nat Commun ; 11(1): 2364, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398665

RESUMO

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.


Assuntos
Autorrenovação Celular/genética , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Linhagem Celular , Reprogramação Celular/genética , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Dedos de Zinco
13.
Am J Hum Genet ; 106(6): 748-763, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32442411

RESUMO

The identification of causal variants and mechanisms underlying complex disease traits in humans is important for the progress of human disease genetics; this requires finding strategies to detect functional regulatory variants in disease-relevant cell types. To achieve this, we collected genetic and transcriptomic data from the aortic endothelial cells of up to 157 donors and four epigenomic phenotypes in up to 44 human donors representing individuals of both sexes and three major ancestries. We found thousands of expression quantitative trait loci (eQTLs) at all ranges of effect sizes not detected by the Gene-Tissue Expression Project (GTEx) in human tissues, showing that novel biological relationships unique to endothelial cells (ECs) are enriched in this dataset. Epigenetic profiling enabled discovery of over 3,000 regulatory elements whose activity is modulated by genetic variants that most frequently mutated ETS, AP-1, and NF-kB binding motifs, implicating these motifs as governors of EC regulation. Using CRISPR interference (CRISPRi), allele-specific reporter assays, and chromatin conformation capture, we validated candidate enhancer variants located up to 750 kb from their target genes, VEGFC, FGD6, and KIF26B. Regulatory SNPs identified were enriched in coronary artery disease (CAD) loci, and this result has specific implications for PECAM-1, FES, and AXL. We also found significant roles for EC regulatory variants in modifying the traits pulse pressure, blood protein levels, and monocyte count. Lastly, we present two unlinked SNPs in the promoter of MFAP2 that exhibit pleiotropic effects on human disease traits. Together, this supports the possibility that genetic predisposition for complex disease is manifested through the endothelium.


Assuntos
Doença/genética , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Variação Genética/genética , Alelos , Epigênese Genética/genética , Feminino , Humanos , Cinesina/genética , Masculino , Mutação , NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Locos de Características Quantitativas/genética , Fator de Transcrição AP-1/metabolismo , Regulador Transcricional ERG/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética
14.
Sci China Life Sci ; 63(6): 835-844, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32249388

RESUMO

During development, gene expression is spatiotemporally regulated by long-distance chromatin interactions between distal enhancers and target promoters. However, how specificity of the interactions between enhancers and promoters is achieved remains largely unknown. As there are far more enhancers than promoters in mammalian genomes, the complexities of enhancer choice during gene regulation remain obscure. CTCF, the CCCTC-binding factor that directionally binds to a vast range of genomic sites known as CBSs (CTCF-binding sites), mediates oriented chromatin looping between a substantial set of distal enhancers and target promoters. To investigate mechanisms by which CTCF engages in enhancer choice, we used CRISPR/Cas9-based DNA-fragment editing to duplicate CBS-containing enhancers and promoters in the native genomic locus of the clustered Pcdhα genes. We found that the promoter is regulated by the proximal one among duplicated enhancers and that this choice is dependent on CTCF-mediated directional enhancer-promoter looping. In addition, gene expression is unaltered upon the switch of enhancers. Moreover, after promoter duplication, only the proximal promoter is chosen by CTCF-mediated directional chromatin looping to contact with the distal enhancer. Finally, we demonstrated that both enhancer activation and chromatin looping with the promoter are essential for gene expression. These findings have important implications regarding the role of CTCF in specific interactions between enhancers and promoters as well as developmental regulation of gene expression by enhancer switching.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Animais , Sítios de Ligação , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/química , Edição de Genes , Regulação da Expressão Gênica , Genômica , Humanos , Camundongos , Família Multigênica , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética
15.
Nat Genet ; 52(5): 505-515, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251373

RESUMO

Active enhancers are frequently transcribed, yet the regulatory role of enhancer transcription remains debated. Here, we depleted the RNA polymerase II pausing and elongation factor Spt5 in activated mouse B cells and found that approximately 50% of enhancer-gene pairs showed co-regulated transcription, consistent with a potential functional requirement for enhancer transcription. In particular, Spt5 depletion led to loss of super-enhancer-promoter physical interaction and gene expression at the immunoglobulin heavy-chain locus (Igh), abrogating antibody class switch recombination. This defect correlated strictly with loss of enhancer transcription but did not affect acetylation of histone H3 at lysine 27, chromatin accessibility and occupancy of Mediator and cohesin at the enhancer. Strikingly, CRISPRa-mediated rescue of enhancer transcription in Spt5-depleted cells restored Igh gene expression. Our work suggests that Spt5-mediated enhancer transcription underlies the physical and functional interaction between a subset of active enhancers and their target promoters.


Assuntos
Elementos Facilitadores Genéticos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética , Acetilação , Animais , Proteínas de Ciclo Celular/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Expressão Gênica/genética , Rearranjo Gênico/genética , Switching de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
16.
PLoS Genet ; 16(4): e1008663, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32243438

RESUMO

Previous studies have surveyed the potential impact of loss-of-function (LoF) variants and identified LoF-tolerant protein-coding genes. However, the tolerance of human genomes to losing enhancers has not yet been evaluated. Here we present the catalog of LoF-tolerant enhancers using structural variants from whole-genome sequences. Using a conservative approach, we estimate that individual human genomes possess at least 28 LoF-tolerant enhancers on average. We assessed the properties of LoF-tolerant enhancers in a unified regulatory network constructed by integrating tissue-specific enhancers and gene-gene interactions. We find that LoF-tolerant enhancers tend to be more tissue-specific and regulate fewer and more dispensable genes relative to other enhancers. They are enriched in immune-related cells while enhancers with low LoF-tolerance are enriched in kidney and brain/neuronal stem cells. We developed a supervised learning approach to predict the LoF-tolerance of all enhancers, which achieved an area under the receiver operating characteristics curve (AUROC) of 98%. We predict 3,519 more enhancers would be likely tolerant to LoF and 129 enhancers that would have low LoF-tolerance. Our predictions are supported by a known set of disease enhancers and novel deletions from PacBio sequencing. The LoF-tolerance scores provided here will serve as an important reference for disease studies.


Assuntos
Elementos Facilitadores Genéticos/genética , Genoma Humano/genética , Mutação com Perda de Função , Sequência Conservada , Doença/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Especificidade de Órgãos/genética , Curva ROC , Reprodutibilidade dos Testes , Aprendizado de Máquina Supervisionado
17.
Nat Commun ; 11(1): 1813, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286279

RESUMO

The oocyte cytoplasm can reprogram the somatic cell nucleus into a totipotent state, but with low efficiency. The spatiotemporal chromatin organization of somatic cell nuclear transfer (SCNT) embryos remains elusive. Here, we examine higher order chromatin structures of mouse SCNT embryos using a low-input Hi-C method. We find that donor cell chromatin transforms to the metaphase state rapidly after SCNT along with the dissolution of typical 3D chromatin structure. Intriguingly, the genome undergoes a mitotic metaphase-like to meiosis metaphase II-like transition following activation. Subsequently, weak chromatin compartments and topologically associating domains (TADs) emerge following metaphase exit. TADs are further removed until the 2-cell stage before being progressively reestablished. Obvious defects including stronger TAD boundaries, aberrant super-enhancer and promoter interactions are found in SCNT embryos. These defects are partially caused by inherited H3K9me3, and can be rescued by Kdm4d overexpression. These observations provide insight into chromatin architecture reorganization during SCNT embryo development.


Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Técnicas de Transferência Nuclear , Animais , Reprogramação Celular , Desenvolvimento Embrionário , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Metáfase , Metilação , Camundongos , Regiões Promotoras Genéticas/genética , Zigoto/metabolismo
18.
Nat Genet ; 52(3): 273-282, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32139906

RESUMO

Mutations in enzymes that modify histone H3 at lysine 4 (H3K4) or lysine 36 (H3K36) have been linked to human disease, yet the role of these residues in mammals is unclear. We mutated K4 or K36 to alanine in the histone variant H3.3 and showed that the K4A mutation in mouse embryonic stem cells (ESCs) impaired differentiation and induced widespread gene expression changes. K4A resulted in substantial H3.3 depletion, especially at ESC promoters; it was accompanied by reduced remodeler binding and increased RNA polymerase II (Pol II) activity. Regulatory regions depleted of H3.3K4A showed histone modification alterations and changes in enhancer activity that correlated with gene expression. In contrast, the K36A mutation did not alter H3.3 deposition and affected gene expression at the later stages of differentiation. Thus, H3K4 is required for nucleosome deposition, histone turnover and chromatin remodeler binding at regulatory regions, where tight regulation of Pol II activity is necessary for proper ESC differentiation.


Assuntos
Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Código das Histonas/genética , Histonas/genética , Lisina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Alanina/metabolismo , Animais , Elementos Facilitadores Genéticos/genética , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Genética
19.
Nat Commun ; 11(1): 1112, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111830

RESUMO

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation.


Assuntos
Ilhas de CpG , Metilação de DNA , Elementos Facilitadores Genéticos/genética , Células-Tronco Pluripotentes/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Complexo Mediador/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , RNA Polimerase II/metabolismo , Transcrição Genética
20.
Proc Natl Acad Sci U S A ; 117(14): 7905-7916, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193341

RESUMO

Transposable elements (TEs) compose nearly half of mammalian genomes and provide building blocks for cis-regulatory elements. Using high-throughput sequencing, we show that 84 TE subfamilies are overrepresented, and distributed in a lineage-specific fashion in core and boundary domains of CD8+ T cell enhancers. Endogenous retroviruses are most significantly enriched in core domains with accessible chromatin, and bear recognition motifs for immune-related transcription factors. In contrast, short interspersed elements (SINEs) are preferentially overrepresented in nucleosome-containing boundaries. A substantial proportion of these SINEs harbor a high density of the enhancer-specific histone mark H3K4me1 and carry sequences that match enhancer boundary nucleotide composition. Motifs with regulatory features are better preserved within enhancer-enriched TE copies compared to their subfamily equivalents located in gene deserts. TE-rich and TE-poor enhancers associate with both shared and unique gene groups and are enriched in overlapping functions related to lymphocyte and leukocyte biology. The majority of T cell enhancers are shared with other immune lineages and are accessible in common hematopoietic progenitors. A higher proportion of immune tissue-specific enhancers are TE-rich compared to enhancers specific to other tissues, correlating with higher TE occurrence in immune gene-associated genomic regions. Our results suggest that during evolution, TEs abundant in these regions and carrying motifs potentially beneficial for enhancer architecture and immune functions were particularly frequently incorporated by evolving enhancers. Their putative selection and regulatory cooption may have accelerated the evolution of immune regulatory networks.


Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Linfócitos T/imunologia , Animais , Cromatina/genética , Cromatina/imunologia , Elementos de DNA Transponíveis/imunologia , Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Elementos Facilitadores Genéticos/imunologia , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Genoma Humano/imunologia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Elementos Nucleotídeos Curtos e Dispersos/genética , Elementos Nucleotídeos Curtos e Dispersos/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA