RESUMEN
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Regiones Promotoras Genéticas , Sistemas CRISPR-Cas , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Regulación de la Expresión Génica , Fosforilación , Unión Proteica , Respuesta al Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
Femto-seq is a novel nanoscale optical method that can be used to obtain DNA sequence information from targeted regions around a specific locus or other nuclear regions of interest. Two-photon excitation is used to photobiotinylate femtoliter volumes of chromatin within the nucleus, allowing for subsequent isolation and sequencing of DNA, and bioinformatic mapping of any nuclear region of interest in a select set of cells from a heterogenous population.