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1.
Mol Cell ; 81(10): 2216-2230.e10, 2021 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-33848455

RESUMEN

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Secuencia de Bases , Reparación del ADN por Unión de Extremidades , Eucromatina/metabolismo , Reordenamiento Génico , Genoma Humano , Heterocromatina/metabolismo , Humanos , Mutación INDEL/genética , Células K562 , Cinética , Unión Proteica , Reproducibilidad de los Resultados
2.
EMBO J ; 39(6): e103159, 2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32080885

RESUMEN

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes.


Asunto(s)
Cromosomas/genética , Replicación del ADN/genética , Genoma/genética , Lámina Nuclear/genética , Activación Transcripcional/genética , Animales , Línea Celular , Núcleo Celular/genética , Cromatina/genética , Células Madre Embrionarias , Femenino , Humanos , Interfase , Ratones , Neuropilina-1/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción SOXD/genética , Transgenes
3.
Cell ; 132(6): 925-8, 2008 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-18358805

RESUMEN

During cell division the cohesin complex mediates the pairing of sister chromatids. Emerging evidence shows that cohesin also has roles in interphase cells. New studies, including that of Gullerova and Proudfoot (2008) in this issue, reveal how cohesin is targeted to specific sites on chromosomes and implicate cohesin in the regulation of gene expression.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Animales , Ciclo Celular , Cromosomas/metabolismo , Evolución Molecular , Cohesinas
4.
Genome Res ; 27(10): 1634-1644, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28916540

RESUMEN

Cellular senescence is a mechanism that virtually irreversibly suppresses the proliferative capacity of cells in response to various stress signals. This includes the expression of activated oncogenes, which causes Oncogene-Induced Senescence (OIS). A body of evidence points to the involvement in OIS of chromatin reorganization, including the formation of senescence-associated heterochromatic foci (SAHF). The nuclear lamina (NL) is an important contributor to genome organization and has been implicated in cellular senescence and organismal aging. It interacts with multiple regions of the genome called lamina-associated domains (LADs). Some LADs are cell-type specific, whereas others are conserved between cell types and are referred to as constitutive LADs (cLADs). Here, we used DamID to investigate the changes in genome-NL interactions in a model of OIS triggered by the expression of the common BRAFV600E oncogene. We found that OIS cells lose most of their cLADS, suggesting the loss of a specific mechanism that targets cLADs to the NL. In addition, multiple genes relocated to the NL. Unexpectedly, they were not repressed, implying the abrogation of the repressive activity of the NL during OIS. Finally, OIS cells displayed an increased association of telomeres with the NL. Our study reveals that senescent cells acquire a new type of LAD organization and suggests the existence of as yet unknown mechanisms that tether cLADs to the NL and repress gene expression at the NL.


Asunto(s)
Senescencia Celular , Regulación de la Expresión Génica , Genoma Humano , Mutación Missense , Lámina Nuclear , Proteínas Proto-Oncogénicas B-raf , Sustitución de Aminoácidos , Línea Celular , Humanos , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Proteínas Proto-Oncogénicas B-raf/biosíntesis , Proteínas Proto-Oncogénicas B-raf/genética
5.
Cell Rep ; 42(10): 113124, 2023 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-37733591

RESUMEN

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 cells can acquire resistance to Taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here, we investigate how the ABCB1 gene is derepressed. ABCB1 activation is associated with reduced H3K9 trimethylation, increased H3K27 acetylation, and ABCB1 displacement from the nuclear lamina. While altering DNA methylation and H3K27 methylation had no major impact on ABCB1 expression, nor did it promote resistance, disrupting the nuclear lamina component Lamin B Receptor did promote the acquisition of a Taxol-resistant phenotype in a subset of cells. CRISPRa-mediated gene activation supported the notion that lamina dissociation influences ABCB1 derepression. We propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.


Asunto(s)
Resistencia a Antineoplásicos , Neoplasias , Humanos , Resistencia a Antineoplásicos/genética , Paclitaxel/farmacología , Resistencia a Múltiples Medicamentos/genética , Neoplasias/genética , Metilación de ADN/genética , Línea Celular Tumoral
6.
Genome Biol ; 22(1): 36, 2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33446254

RESUMEN

We report SPIN, an integrative computational method to reveal genome-wide intranuclear chromosome positioning and nuclear compartmentalization relative to multiple nuclear structures, which are pivotal for modulating genome function. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and putative associations with nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to other 3D genome features and genome function (transcription and replication timing). SPIN provides critical insights into nuclear spatial and functional compartmentalization.


Asunto(s)
Núcleo Celular/genética , Genoma Humano , Compartimento Celular , Cromatina , Mapeo Cromosómico , Cromosomas , Replicación del ADN , Histonas , Humanos , Células K562 , Modelos Genéticos
7.
Cancer Res ; 62(11): 3233-43, 2002 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-12036939

RESUMEN

The ordered expression of genes after growth factor stimulation in G(1) supportsthe onset of DNA replication. To characterize regulatory events during S-phase when cell cycle progression has become growth factor independent, we have profiled the expression of over 7,000 human genes using GeneChip DNA microarray analysis. HeLa cells were synchronized at the beginning of S-phase by thymidine/aphidicolin block, and RNA populations were analyzed throughout the S and G(2) phases. Expression of genes involved in DNA replication is maximal during early S-phase, whereas histone mRNAs peak at mid S-phase. Genes related to cell proliferation, including those encoding cyclins, oncoproteins, growth factors, proteins involved in signal transduction, and DNA repair proteins, follow distinct temporal patterns of expression that are functionally linked to initiation of DNA replication and progression through S-phase. The timing of expression for many genes in tumor-derived HeLa cells is highly conserved when compared with normal cells. In contrast, a number of genes show growth phenotype-related expression patterns that may directly reflect loss of stringent growth control in tumor cells. Our data reveal there is a core subset of cell growth-related genes that is fundamental to cycling cells irrespective of cell growth phenotype.


Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/genética , Replicación del ADN/genética , Proteínas de Unión al ADN , Regulación Leucémica de la Expresión Génica , Nucleosomas/genética , División Celular/genética , ADN/biosíntesis , ADN/genética , Reparación del ADN/genética , Factores de Transcripción E2F , Fase G1/genética , Perfilación de la Expresión Génica , Células HeLa , Histonas/genética , Humanos , Mitosis/genética , Nucleosomas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/genética , ARN/metabolismo , Fase S/genética , Factores de Transcripción/genética
8.
Nat Protoc ; 2(6): 1467-78, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17545983

RESUMEN

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein-DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.


Asunto(s)
Metilación de ADN , ADN/metabolismo , Genómica/métodos , Proteínas/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Animales , Sitios de Unión , Línea Celular , ADN/química , Humanos , Ratones , Unión Proteica
9.
Genome Res ; 16(12): 1493-504, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17038565

RESUMEN

Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1beta) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1-4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3' ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family.


Asunto(s)
Proteínas de Unión al ADN/genética , Heterocromatina/química , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Línea Celular , Línea Celular Tumoral , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Mapeo Cromosómico , Cromosomas Humanos Par 19 , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Elementos de Nucleótido Esparcido Largo , Metiltransferasas/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Protaminas/química , Unión Proteica , Proteínas Represoras/química
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