RESUMEN
Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.
Asunto(s)
Cromosomas/química , Clonación Molecular/métodos , Epigénesis Genética/fisiología , Regulación de la Expresión Génica/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Factor de Unión a CCCTC , Cromatina/química , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias , Impresión Genómica/fisiología , Hígado/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Largo no Codificante , ARN no Traducido/genética , Proteínas Represoras/metabolismo , TransactivadoresRESUMEN
The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.
Asunto(s)
Proteínas de Unión al ADN/genética , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , Mutación , ARN no Traducido/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Animales , Factor de Unión a CCCTC , Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Metilación , Ratones , Unión Proteica , ARN Largo no Codificante , Proteínas Represoras/metabolismoRESUMEN
The repression of the maternally inherited Igf2 allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at the H19 imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenous H19 ICR lead to loss of CTCF binding and Igf2 imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derived H19 ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/metabolismo , Proteínas Represoras , Factores de Transcripción/genética , Animales , Factor de Unión a CCCTC , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Masculino , Ratones , ARN Largo no Codificante , ARN no Traducido/genética , Factores de Transcripción/metabolismoRESUMEN
The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.
