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1.
Cell ; 175(1): 224-238.e15, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30173918

RESUMO

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.


Assuntos
Cromatina/genética , Repetições de Microssatélites/fisiologia , Expansão das Repetições de Trinucleotídeos/fisiologia , Adulto , Encéfalo/citologia , Encéfalo/patologia , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/fisiologia , Linhagem Celular , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Ilhas de CpG/genética , Ilhas de CpG/fisiologia , DNA/genética , Doença/etiologia , Doença/genética , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína do X Frágil da Deficiência Intelectual/fisiologia , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Genoma Humano/genética , Humanos , Masculino , Repetições de Microssatélites/genética , Expansão das Repetições de Trinucleotídeos/genética
2.
Nature ; 576(7785): 158-162, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31776509

RESUMO

Features of higher-order chromatin organization-such as A/B compartments, topologically associating domains and chromatin loops-are temporarily disrupted during mitosis1,2. Because these structures are thought to influence gene regulation, it is important to understand how they are re-established after mitosis. Here we examine the dynamics of chromosome reorganization by Hi-C after mitosis in highly purified, synchronous mouse erythroid cell populations. We observed rapid establishment of A/B compartments, followed by their gradual intensification and expansion. Contact domains form from the 'bottom up'-smaller subTADs are formed initially, followed by convergence into multi-domain TAD structures. CTCF is partially retained on mitotic chromosomes and immediately resumes full binding in ana/telophase. By contrast, cohesin is completely evicted from mitotic chromosomes and regains focal binding at a slower rate. The formation of CTCF/cohesin co-anchored structural loops follows the kinetics of cohesin positioning. Stripe-shaped contact patterns-anchored by CTCF-grow in length, which is consistent with a loop-extrusion process after mitosis. Interactions between cis-regulatory elements can form rapidly, with rates exceeding those of CTCF/cohesin-anchored contacts. Notably, we identified a group of rapidly emerging transient contacts between cis-regulatory elements in ana/telophase that are dissolved upon G1 entry, co-incident with the establishment of inner boundaries or nearby interfering chromatin loops. We also describe the relationship between transcription reactivation and architectural features. Our findings indicate that distinct but mutually influential forces drive post-mitotic chromatin reconfiguration.


Assuntos
Cromatina , Fase G1 , Mitose , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Camundongos , Coesinas
3.
Mol Cell ; 66(1): 102-116.e7, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388437

RESUMO

Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Sistemas CRISPR-Cas , Linhagem Celular , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Edição de Genes/métodos , Hibridização in Situ Fluorescente , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Imagem Individual de Molécula/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
4.
Nat Methods ; 16(7): 633-639, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235883

RESUMO

Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.


Assuntos
Regulação da Expressão Gênica , Engenharia de Proteínas , Animais , Proteínas de Transporte/genética , Células Cultivadas , Proteínas de Ligação a DNA , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Luz , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética
5.
Cell Genom ; 3(8): 100356, 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37601975

RESUMO

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)-present in some but not all cells-remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e-4), with recurrent somatic deletions of exons 1-5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5' deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk.

6.
Nat Neurosci ; 25(4): 474-483, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35332326

RESUMO

Chromosomal organization, scaling from the 147-base pair (bp) nucleosome to megabase-ranging domains encompassing multiple transcriptional units, including heritability loci for psychiatric traits, remains largely unexplored in the human brain. In this study, we constructed promoter- and enhancer-enriched nucleosomal histone modification landscapes for adult prefrontal cortex from H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from 388 controls and 351 individuals diagnosed with schizophrenia (SCZ) or bipolar disorder (BD) (n = 739). We mapped thousands of cis-regulatory domains (CRDs), revealing fine-grained, 104-106-bp chromosomal organization, firmly integrated into Hi-C topologically associating domain stratification by open/repressive chromosomal environments and nuclear topography. Large clusters of hyper-acetylated CRDs were enriched for SCZ heritability, with prominent representation of regulatory sequences governing fetal development and glutamatergic neuron signaling. Therefore, SCZ and BD brains show coordinated dysregulation of risk-associated regulatory sequences assembled into kilobase- to megabase-scaling chromosomal domains.


Assuntos
Transtorno Bipolar , Esquizofrenia , Adulto , Transtorno Bipolar/genética , Encéfalo , Cromatina , Humanos , Lisina/genética , Esquizofrenia/genética
7.
Genome Biol ; 21(1): 219, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859248

RESUMO

An important unanswered question in chromatin biology is the extent to which long-range looping interactions change across developmental models, genetic perturbations, drug treatments, and disease states. Computational tools for rigorous assessment of cell type-specific loops across multiple biological conditions are needed. We present 3DeFDR, a simple and effective statistical tool for classifying dynamic loops across biological conditions from Chromosome-Conformation-Capture-Carbon-Copy (5C) and Hi-C data. Our work provides a statistical framework and open-source coding libraries for sensitive detection of cell type-specific loops in high-resolution 5C and Hi-C data from multiple cellular conditions.


Assuntos
Cromatina/química , Cromossomos/química , Modelos Genéticos , Benchmarking , Epigenômica , Humanos , Conformação Molecular
8.
Nat Genet ; 52(10): 1076-1087, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32868908

RESUMO

Animal chromosomes are partitioned into contact domains. Pathogenic domain disruptions can result from chromosomal rearrangements or perturbation of architectural factors. However, such broad-scale alterations are insufficient to define the minimal requirements for domain formation. Moreover, to what extent domains can be engineered is just beginning to be explored. In an attempt to create contact domains, we inserted a 2-kb DNA sequence underlying a tissue-invariant domain boundary-containing a CTCF-binding site (CBS) and a transcription start site (TSS)-into 16 ectopic loci across 11 chromosomes, and characterized its architectural impact. Depending on local constraints, this fragment variably formed new domains, partitioned existing ones, altered compartmentalization and initiated contacts reflecting chromatin loop extrusion. Deletions of the CBS or the TSS individually or in combination within inserts revealed its distinct contributions to genome folding. Altogether, short DNA insertions can suffice to shape the spatial genome in a manner influenced by chromatin context.


Assuntos
Fator de Ligação a CCCTC/genética , Cromatina/genética , Cromossomos/genética , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Genoma Humano/genética , Humanos , Ligação Proteica/genética , Domínios Proteicos/genética
9.
Cell Syst ; 8(3): 197-211.e13, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30904376

RESUMO

Chromosome-Conformation-Capture-Carbon-Copy (5C) is a molecular technology based on proximity ligation that enables high-resolution and high-coverage inquiry of long-range looping interactions. Computational pipelines for analyzing 5C data involve a series of interdependent normalization procedures and statistical methods that markedly influence downstream biological results. A detailed analysis of the trade-offs inherent to all stages of 5C data analysis has not been reported. Here, we provide a comparative assessment of method performance at each step in the 5C analysis pipeline, including sequencing depth and library complexity correction, bias mitigation, spatial noise reduction, distance-dependent expected and variance estimation, statistical modeling, and loop detection. We discuss methodological advantages and disadvantages at each step and provide a full suite of algorithms, lib5C, to allow investigators to test the range of approaches on their own 5C data. Principles learned from our comparative analyses can be applied to protein-independent proximity ligation-based data, including Hi-C, 4C, and Capture-C.


Assuntos
Núcleo Celular , Cromatina/metabolismo , Modelos Biológicos , Conformação Molecular , Animais , Camundongos
10.
Cell Stem Cell ; 18(5): 611-24, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27152443

RESUMO

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.


Assuntos
Reprogramação Celular/genética , Genoma , Conformação de Ácido Nucleico , Animais , Fator de Ligação a CCCTC , Linhagem da Célula/genética , Cromatina/química , Células Clonais , Elementos Facilitadores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo
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