RESUMEN
BACKGROUND: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation. RESULTS: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. CONCLUSIONS: We discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Cromatina/química , ADN/química , ADN/metabolismo , Genoma , Humanos , Células K562 , Masculino , Ratones , Neoplasias/genética , Motivos de Nucleótidos , Unión Proteica , Espermátides/metabolismo , Espermatozoides/metabolismo , Transcripción GenéticaRESUMEN
FOXP3 is a lineage-specific transcription factor that is required for regulatory T cell development and function. In this study, we determined the crystal structure of the FOXP3 forkhead domain bound to DNA. The structure reveals that FOXP3 can form a stable domain-swapped dimer to bridge DNA in the absence of cofactors, suggesting that FOXP3 may play a role in long-range gene interactions. To test this hypothesis, we used circular chromosome conformation capture coupled with high throughput sequencing (4C-seq) to analyze FOXP3-dependent genomic contacts around a known FOXP3-bound locus, Ptpn22. Our studies reveal that FOXP3 induces significant changes in the chromatin contacts between the Ptpn22 locus and other Foxp3-regulated genes, reflecting a mechanism by which FOXP3 reorganizes the genome architecture to coordinate the expression of its target genes. Our results suggest that FOXP3 mediates long-range chromatin interactions as part of its mechanisms to regulate specific gene expression in regulatory T cells.
Asunto(s)
Cromosomas/química , ADN/química , Factores de Transcripción Forkhead/química , Animales , ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Modelos Moleculares , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genéticaRESUMEN
In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A-containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. Whereas the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail does not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling but nevertheless elevates chromosome loss. N-tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN.
Asunto(s)
Centrómero/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Electroforesis en Gel de Campo Pulsado , Fluorescencia , Histonas/metabolismo , Immunoblotting , Mutación/genética , Reacción en Cadena de la Polimerasa , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
A large number of cis-regulatory sequences have been annotated in the human genome, but defining their target genes remains a challenge. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C)-based techniques. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C). We determined over one million long-range chromatin interactions at 5-10-kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts after TNF-α signalling in these cells. Unexpectedly, we found that TNF-α-responsive enhancers are already in contact with their target promoters before signalling. Such pre-existing chromatin looping, which also exists in other cell types with different extracellular signalling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is relatively stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.
Asunto(s)
Cromatina/metabolismo , Mapeo Cromosómico , Genoma Humano , Línea Celular , Cromatina/química , Cromatina/genética , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Humanos , Imagenología Tridimensional , Regiones Promotoras Genéticas/fisiología , Unión Proteica , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Cromatina/genética , Cromatina/metabolismo , Genes Reguladores , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Cromatina/química , Regulación de la Expresión Génica , Ratones , Regiones Promotoras Genéticas/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genéticaRESUMEN
It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.
Asunto(s)
Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Redes Reguladoras de Genes , Células Precursoras de Linfocitos B/metabolismo , Factores de Transcripción TCF/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Células Cultivadas , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Linfopoyesis/genética , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/patología , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción TCF/genética , Transactivadores/genética , Transactivadores/metabolismo , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.
Asunto(s)
Linaje de la Célula/genética , Epigénesis Genética , Fibroblastos/citología , Fibroblastos/metabolismo , Genoma Humano/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Celular , Cromatina/genética , Islas de CpG/genética , Metilación de ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genes del Desarrollo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Noncoding RNAs (ncRNAs) have recently been discovered to regulate mRNA transcription in trans, a role traditionally reserved for proteins. The breadth of ncRNAs as transacting transcriptional regulators and the diversity of signals to which they respond are only now becoming recognized. Here we show that human Alu RNA, transcribed from short interspersed elements (SINEs), is a transacting transcriptional repressor during the cellular heat shock response. Alu RNA blocks transcription by binding RNA polymerase II (Pol II) and entering complexes at promoters in vitro and in human cells. Transcriptional repression by Alu RNA involves two loosely structured domains that are modular, a property reminiscent of classical protein transcriptional regulators. Two other SINE RNAs, human scAlu RNA and mouse B1 RNA, also bind Pol II but do not repress transcription in vitro. These studies provide an explanation for why mouse cells harbor two major classes of SINEs, whereas human cells contain only one.
Asunto(s)
Elementos Alu/genética , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , ARN Mensajero/metabolismo , ARN no Traducido , Elementos de Nucleótido Esparcido Corto , Transcripción Genética , Animales , Línea Celular , Humanos , Ratones , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
We previously found that the SINE-encoded mouse B2 RNA binds RNA polymerase II and represses mRNA transcription during the cellular heat-shock response. In vitro B2 RNA assembles into preinitiation complexes on promoter DNA via its interaction with the polymerase, thus rendering the complexes inactive. With the goal of understanding which regions of B2 RNA are important for high-affinity binding to RNA polymerase II and repression of transcription, we performed a structural and deletion analysis of a 178 nucleotide (nt) B2 RNA. We describe an experimentally derived secondary structure model for B2 RNA, and show that RNA polymerase II protects a specific region from RNase digestion. Deletion studies revealed that a 51-nt region of B2 RNA is sufficient for high-affinity binding to RNA polymerase II, association with preinitiation complexes, and repression of transcription in vitro, the latter of which involves a large predominately single-stranded region within the RNA. In addition, this piece of B2 RNA blocked the polymerase from properly associating with template DNA during the assembly of elongation complexes. Further deletion revealed that a 33-nt piece of B2 RNA binds RNA polymerase II, associates with preinitiation complexes, but cannot repress transcription. These results support a model in which RNA polymerase II contains a high-affinity ncRNA docking site to which a distinct region of B2 RNA binds, thereby allowing another region of the RNA to repress transcription. Moreover, the mechanism of transcriptional repression by B2 RNA likely involves disrupting critical contacts between RNA polymerase II and the promoter DNA.
Asunto(s)
ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN no Traducido/química , ARN no Traducido/metabolismo , Transcripción Genética , Secuencia de Bases , Huella de ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , ARN no Traducido/genética , Eliminación de Secuencia , Moldes GenéticosRESUMEN
B2 RNA is a small noncoding RNA polymerase III transcript that represses mRNA transcription in response to heat shock in mouse cells. Here we define the mechanism by which B2 RNA inhibits RNA polymerase II (Pol II) transcription. Using a purified Pol II transcription system, we found that B2 RNA potently inhibits transcription by binding to core Pol II with high affinity and specificity. Through this interaction, B2 RNA assembles into preinitiation complexes at the promoter and blocks RNA synthesis. Once B2 RNA is removed from preinitiation complexes, transcriptional activity is restored. Our studies describe a previously unobserved mechanism of transcriptional repression by a small RNA and suggest that B2 RNA associates with Pol II at promoters in heat shocked cells to actively inhibit transcription.