Investigating phase separation mechanisms for transcriptional control

Abstract

Transcription of protein-coding genes by RNA polymerase (Pol) II is a highly coordinated process. In metazoan cells, transcription is regulated both at the initiation step by recruitment of the Pol II machinery as well as during early elongation by promoter-proximal pausing. Prior to transcription initiation, Pol II forms short-lived clusters near active gene promoters, but the underlying molecular basis has remained unknown. Pol II possesses a disordered C-terminal heptad repeat domain (CTD) that is essential for factor recruitment during the transcription cycle. CTD length is organism-specific with 52 repeats in human and 26 repeats in yeast. In this work, we report that the human and yeast CTD can undergo concentration-dependent liquid-liquid phase separation in vitro, based on weak multivalent repeat-repeat interactions. We show that this behavior strongly correlates with the repeat number, as the shorter yeast CTD forms less-stable droplets. Shortening of the CTD in human cells to the length of the yeast CTD reduces Pol II clustering and chromatin-association, while artificial extension has the contrary effect. Repeat-repeat interactions are sensitive to CTD phosphorylation by the transcription factor IIH kinase CDK7, which dissolves CTD droplets in vitro. Together these results imply a model for gene activation that involves CTD-mediated clustering of initiation-competent Pol II and release through CTD phosphorylation upon transcription initiation. Heat shock causes the accumulation of the negative elongation factor (NELF) at chromatin, which stabilizes paused Pol II within the promoter-proximal region of downregulated target genes. In this work, we show that NELF clusters in nuclear puncta upon heat shock, which possess properties consistent with phase-separated condensates. In vitro, purified NELF complex self-interacts to form phase-separated droplets with liquid-like properties. We show that multivalent interactions between the disordered NELF tentacles are essential for NELF phase separation in vitro and stress-induced condensation in vivo. Phosphorylation by positive elongation factor b (P-TEFb) counteracts NELF phase separation in vitro and is prevented through the inactivation of P-TEFb upon heat shock in vivo. Sumoylation is further required for stress-induced NELF condensation, as NELF itself can be sumoylated in vitro and interacts with SUMO2/3 in a chain length-dependent manner. Together with published data, our results suggest a model that involves stress-induced sequestration of promoter-proximal paused Pol II by NELF near downregulated gene promoters. Taken together, the findings presented in this work indicate that phase separation mechanisms regulate key steps of eukaryotic gene transcription and provide a basis to further analyze the role of phase separation within the Pol II transcription cycle, as well as investigate its modulation in the future.