Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation

• Published in: Molecular cell Vol. 84; no. 15; pp. 2856 - 2869.e9
• Main Authors: Versluis, Philip, Graham, Thomas G.W., Eng, Vincent, Ebenezer, Jonathan, Darzacq, Xavier, Zipfel, Warren R., Lis, John T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.08.2024
• Subjects: Animals; CRISPR-Cas Systems; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; DSIF; Gene Expression Regulation; heat shock; Heat-Shock Response - genetics; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; live-cell imaging; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleosomes - genetics; Nucleosomes - metabolism; P-TEFb; PAF1; Phosphorylation; Polytene Chromosomes - genetics; Polytene Chromosomes - metabolism; Positive Transcriptional Elongation Factor B - genetics; Positive Transcriptional Elongation Factor B - metabolism; Promoter Regions, Genetic; Protein Binding; RNA Polymerase II; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; Spt6; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional Elongation Factors - genetics; Transcriptional Elongation Factors - metabolism; live-cell imaging; Spt6; PAF1; heat shock; DSIF; RNA Polymerase II; P-TEFb
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2024.07.009

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. [Display omitted] •In early heat shock, RNA Pol II and DSIF are not recycled for rounds of transcription•P-TEFb and PAF1 transiently associate with RNA Pol II, and Spt6 interacts stably•PAF1 and Spt6 are not recruited to RNA Pol II without P-TEFb activity•PAF1 aids RNA Pol II elongation, particularly when nucleosomes are encountered Versluis et al. track transcription factor dynamics in live cells, observing that RNA Pol II and DSIF show similar kinetics at Hsp70, while P-TEFb and PAF1 rapidly exchange between neighboring Hsp70 chromatids. Elongation factors, PAF1 and Spt6, display different binding kinetics but require P-TEFb activity to be recruited to Hsp70.