RESUMEN
Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3' UTRs.
Asunto(s)
Proteínas Bacterianas/genética , Cromatina/aislamiento & purificación , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Endonucleasas/genética , Genes Reguladores/genética , Histonas/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3' , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Cromatina/genética , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endonucleasas/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Paused RNA polymerase (Pol II) is a pervasive feature of Drosophila embryos and mammalian stem cells, but its role in development is uncertain. Here, we demonstrate that a spectrum of paused Pol II determines the "time to synchrony"-the time required to achieve coordinated gene expression across the cells of a tissue. To determine whether synchronous patterns of gene activation are significant in development, we manipulated the timing of snail expression, which controls the coordinated invagination of â¼1,000 mesoderm cells during gastrulation. Replacement of the strongly paused snail promoter with moderately paused or nonpaused promoters causes stochastic activation of snail expression and increased variability of mesoderm invagination. Computational modeling of the dorsal-ventral patterning network recapitulates these variable and bistable gastrulation profiles and emphasizes the importance of timing of gene activation in development. We conclude that paused Pol II and transcriptional synchrony are essential for coordinating cell behavior during morphogenesis.
Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Datos de Secuencia Molecular , Morfogénesis , Regiones Promotoras GenéticasRESUMEN
Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.