Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
Mol Cell ; 81(15): 3082-3095.e6, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34197738

RESUMO

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos , Animais , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas , Coativador 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Coesinas
2.
Mol Cell ; 61(3): 461-473, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26833089

RESUMO

Detailed genomic contact maps have revealed that chromosomes are structurally organized in megabase-sized topologically associated domains (TADs) that encompass smaller subTADs. These domains segregate in the nuclear space to form active and inactive nuclear compartments, but cause and consequence of compartmentalization are largely unknown. Here, we combined lacO/lacR binding platforms with allele-specific 4C technologies to track their precise position in the three-dimensional genome upon recruitment of NANOG, SUV39H1, or EZH2. We observed locked genomic loci resistant to spatial repositioning and unlocked loci that could be repositioned to different nuclear subcompartments with distinct chromatin signatures. Focal protein recruitment caused the entire subTAD, but not surrounding regions, to engage in new genomic contacts. Compartment switching was found uncoupled from transcription changes, and the enzymatic modification of histones per se was insufficient for repositioning. Collectively, this suggests that trans-associated factors influence three-dimensional compartmentalization independent of their cis effect on local chromatin composition and activity.


Assuntos
Núcleo Celular/metabolismo , Segregação de Cromossomos , Células-Tronco Embrionárias/metabolismo , Loci Gênicos , Óperon Lac , Repressores Lac/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Repressores Lac/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transfecção
3.
Mol Cell ; 60(4): 676-84, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26527277

RESUMO

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.


Assuntos
Cromatina/química , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/citologia , Camundongos , Ligação Proteica , Coesinas
4.
Am J Hum Genet ; 101(3): 326-339, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28844486

RESUMO

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.


Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Biomarcadores/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Polimorfismo de Nucleotídeo Único , Diagnóstico Pré-Natal/métodos , Esteroide 21-Hidroxilase/genética , Hiperplasia Suprarrenal Congênita/sangue , Hiperplasia Suprarrenal Congênita/genética , Células Cultivadas , Fibrose Cística/sangue , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , DNA/sangue , DNA/genética , Feminino , Haplótipos , Humanos , Gravidez , Esteroide 21-Hidroxilase/sangue
5.
Nature ; 501(7466): 227-31, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-23883933

RESUMO

It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.


Assuntos
Cromatina/química , Cromatina/metabolismo , Posicionamento Cromossômico , Genoma/genética , Imageamento Tridimensional , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Imagem Molecular , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/metabolismo
6.
Nat Methods ; 9(10): 969-72, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22961246

RESUMO

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.


Assuntos
DNA/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regulação da Expressão Gênica , Região de Controle de Locus Gênico , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas
7.
Nucleic Acids Res ; 41(14): 6905-16, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23748562

RESUMO

In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells-but not in T cells-the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.


Assuntos
Alelos , Linfócitos B/imunologia , Genes de Cadeia Pesada de Imunoglobulina , Animais , Núcleo Celular/química , Cromatina/química , Cromossomos de Mamíferos , Loci Gênicos , Cadeias Pesadas de Imunoglobulinas/análise , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/genética , Camundongos , Recombinação Genética , Análise de Sequência de DNA , Baço/imunologia , Linfócitos T/imunologia , Transcrição Gênica
8.
Cell Rep ; 42(4): 112373, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37060567

RESUMO

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina , Elementos Facilitadores Genéticos/genética , Mutação , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
9.
Nat Struct Mol Biol ; 29(6): 563-574, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35710842

RESUMO

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , Coesinas
10.
Nat Commun ; 12(1): 3361, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099699

RESUMO

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma de Células B/genética , Linfoma não Hodgkin/genética , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Translocação Genética , Biologia Computacional/métodos , Rearranjo Gênico , Genes bcl-2/genética , Genes myc/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Linfoma de Células B/diagnóstico , Linfoma não Hodgkin/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-6/genética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade
11.
Nat Protoc ; 15(2): 364-397, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932773

RESUMO

We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.


Assuntos
Cromatina/química , Cromatina/genética , Análise de Sequência de DNA/métodos , Humanos , Células K562 , Conformação Molecular
12.
Nat Genet ; 50(8): 1151-1160, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988121

RESUMO

Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the ß-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos/genética , Alelos , Animais , Fator de Ligação a CCCTC/genética , Camundongos , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Globinas beta/genética
14.
Genome Biol ; 16: 289, 2015 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-26700097

RESUMO

BACKGROUND: Linker histone H1 is a core chromatin component that binds to nucleosome core particles and the linker DNA between nucleosomes. It has been implicated in chromatin compaction and gene regulation and is anticipated to play a role in higher-order genome structure. Here we have used a combination of genome-wide approaches including DNA methylation, histone modification and DNase I hypersensitivity profiling as well as Hi-C to investigate the impact of reduced cellular levels of histone H1 in embryonic stem cells on chromatin folding and function. RESULTS: We find that depletion of histone H1 changes the epigenetic signature of thousands of potential regulatory sites across the genome. Many of them show cooperative loss or gain of multiple chromatin marks. Epigenetic alterations cluster to gene-dense topologically associating domains (TADs) that already showed a high density of corresponding chromatin features. Genome organization at the three-dimensional level is largely intact, but we find changes in the structural segmentation of chromosomes specifically for the epigenetically most modified TADs. CONCLUSIONS: Our data show that cells require normal histone H1 levels to expose their proper regulatory landscape. Reducing the levels of histone H1 results in massive epigenetic changes and altered topological organization particularly at the most active chromosomal domains. Changes in TAD configuration coincide with epigenetic landscape changes but not with transcriptional output changes, supporting the emerging concept that transcriptional control and nuclear positioning of TADs are not causally related but independently controlled by the locally associated trans-acting factors.


Assuntos
Montagem e Desmontagem da Cromatina , Epigênese Genética , Histonas/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Histonas/genética , Camundongos
15.
Nat Biotechnol ; 32(10): 1019-25, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25129690

RESUMO

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.


Assuntos
Genômica/métodos , Haplótipos/genética , Modelos Genéticos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Fusão Gênica/genética , Genes BRCA1 , Genes BRCA2 , Loci Gênicos/genética , Humanos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA