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1.
Nat Commun ; 12(1): 4897, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385432

RESUMO

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Elementos Reguladores de Transcrição/genética , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos
2.
Nat Genet ; 52(8): 819-827, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514123

RESUMO

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Oxigenases de Função Mista/genética , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/fisiologia , Humanos , Camundongos , Camundongos Knockout
3.
Nat Genet ; 51(12): 1664-1669, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784727

RESUMO

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases1-4. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types5,6. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Desacetilase 6 de Histona/genética , Humanos , Hibridização in Situ Fluorescente , Células K562 , Camundongos , Modelos Genéticos , RNA Guia
4.
Nat Commun ; 10(1): 1874, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015400

RESUMO

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities.


Assuntos
Linfócitos B/metabolismo , Cromatina/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Metilação de DNA , Evolução Molecular , Inativação Gênica , Genes de Cadeia Pesada de Imunoglobulina/genética , Voluntários Saudáveis , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas/genética , Análise de Sequência de RNA , Análise de Célula Única/métodos , Sequenciamento Completo do Exoma
6.
Cell ; 173(5): 1165-1178.e20, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29706548

RESUMO

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Genoma , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA , Humanos , Camundongos , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
7.
Nat Commun ; 9(1): 597, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29426832

RESUMO

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.


Assuntos
Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias/enzimologia , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Endonucleases , Edição de Genes , Humanos , Camundongos
8.
Nat Genet ; 50(2): 250-258, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29358654

RESUMO

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.


Assuntos
DNA/metabolismo , Epigênese Genética/fisiologia , Redes Reguladoras de Genes/fisiologia , Fatores de Transcrição/metabolismo , Células A549 , Sítios de Ligação/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Células Cultivadas , Biologia Computacional , DNA/genética , Epistasia Genética/fisiologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Genes de Troca , Células HEK293 , Células Hep G2 , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica
9.
Artigo em Inglês | MEDLINE | ID: mdl-27980681

RESUMO

BACKGROUND: The ability to measure DNA methylation precisely and efficiently continues to drive our understanding of this modification in development and disease. Whole genome bisulfite sequencing has the advantage of theoretically capturing all cytosines in the genome at single-nucleotide resolution, but it has a number of significant practical drawbacks that become amplified with increasing sample numbers. All other technologies capture only a fraction of the cytosines that show dynamic regulation across cell and tissue types. RESULTS: Here, we present a novel hybrid selection design focusing on loci with dynamic methylation that captures a large number of differentially methylated gene-regulatory elements. We benchmarked this assay against matched whole genome data and profiled 25 human tissue samples to explore its ability to detect differentially methylated regions. CONCLUSIONS: Our target capture design fills a major gap left by all other assays that exist to map DNA methylation. It maintains the ability to link cytosine methylation to genetic differences, the single-base resolution and the analysis of neighboring cytosines while notably reducing the cost per sample by focusing the sequencing effort on the most informative and relevant regions of the genome.


Assuntos
Metilação de DNA , DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sulfitos/química , Imunoprecipitação da Cromatina , Ilhas de CpG , DNA/química , DNA/isolamento & purificação , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos
10.
Proc Natl Acad Sci U S A ; 113(31): E4504-12, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27432957

RESUMO

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.


Assuntos
Cromossomos Humanos X/genética , Deleção de Genes , Genoma Humano/genética , Repetições de Microssatélites/genética , Inativação do Cromossomo X , Animais , Sítios de Ligação/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Feminino , Humanos , Macaca mulatta , Camundongos , Ligação Proteica
11.
Proc Natl Acad Sci U S A ; 112(47): E6456-65, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26499245

RESUMO

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.


Assuntos
Cromatina/química , Cromatina/genética , Engenharia Genética , Genoma/genética , Conformação de Ácido Nucleico , Anisotropia , Pareamento de Bases , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Simulação por Computador , Difusão , Fractais , Humanos , Hibridização in Situ Fluorescente , Modelos Moleculares , Motivos de Nucleotídeos/genética , Polímeros/química , Probabilidade , Proteínas Repressoras/metabolismo
12.
Cell ; 159(7): 1665-80, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25497547

RESUMO

We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.


Assuntos
Núcleo Celular/genética , Cromatina/química , Genoma Humano , Animais , Fator de Ligação a CCCTC , Linhagem Celular , Núcleo Celular/química , Regulação da Expressão Gênica , Código das Histonas , Humanos , Camundongos , Conformação Molecular , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismo
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