RESUMEN
Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks" of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Regulación de la Expresión Génica , Elementos Aisladores/fisiología , ARN Polimerasa II/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Ojo/metabolismo , Redes Reguladoras de Genes , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de Proteínas , Interferencia de ARN , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Elementos Aisladores/genética , Modelos Biológicos , Nucleosomas/fisiología , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Análisis por Micromatrices , Datos de Secuencia Molecular , Análisis de Componente Principal , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.