RESUMEN
Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales.
Asunto(s)
Linaje de la Célula , Cromatina/metabolismo , Genoma , Proteínas Nucleares/análisis , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/química , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , Células-Madre Neurales/química , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , CohesinasRESUMEN
Two mutant alleles of the same gene, each located in one of the two homologous chromosomes, may in some instances restore the wild-type function of the gene. This is the case with certain combinations of mutant alleles in the mod(mdg4) gene. This gene encodes several different proteins, including Mod(mdg4)2.2, a component of the gypsy insulator. This protein is encoded by two separate transcription units that can be combined in a trans-splicing reaction to form the mature Mod(mdg4)2.2-encoding RNA. Molecular characterization of complementing alleles shows that they affect the two different transcription units. Flies homozygous for each allele are missing the Mod(mdg4)2.2 protein, whereas wild-type trans-heterozygotes are able to synthesize almost normal levels of the Mod(mdg4)2.2 product. This protein is functional as judged by its ability to form a functional insulator complex. The results suggest that the interallelic complementation in the mod(mdg4) gene is a consequence of trans-splicing between two different mutant transcripts. A conclusion from this observation is that the trans-splicing reaction that takes place between transcripts produced on two different mutant chromosomes ensures wild-type levels of functional protein.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Trans-Empalme , Factores de Transcripción/genética , Animales , Western Blotting , Prueba de Complementación Genética , Inmunohistoquímica , Análisis de Secuencia de ADN , Alas de AnimalesRESUMEN
CTCF plays a central role in vertebrate insulators and forms part of the Fab-8 insulator in Drosophila. dCTCF is present at hundreds of sites in the Drosophila genome, where it is located at the boundaries between bands and interbands in polytene chromosomes. dCTCF colocalizes with CP190, which is required for proper binding of dCTCF to chromatin, but not with the other gypsy insulator proteins Su(Hw) or Mod(mdg4)2.2. Mutations in the CP190 gene affect Fab-8 insulator activity, suggesting that CP190 is an essential component of both gypsy and dCTCF insulators. dCTCF is present at specific nuclear locations, forming large insulator bodies that overlap with those formed by Su(Hw), Mod(mdg4)2.2, and CP190. The results suggest that Su(Hw) and dCTCF may be the DNA-binding components of two different subsets of insulators that share CP190 and cooperate in the formation of insulator bodies to regulate the organization of the chromatin fiber in the nucleus.