Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference

Diverse Roles for the TFIIH Complex in Transcription and mRNA Processing

TFIIH is a general transcription factor involved in the regulation of RNA Polymerase II (Pol II) transcription. It is a large, multi-subunit complex that contains 10 subunits, but can dissociate into a 7-subunit core and a 3-subunit CDK-activating kinase (CAK). The 7-subunit core is known to be important for DNA repair, and the CAK is known to be involved in cell cycle regulation but has no known transcriptional roles. The full 10-subunit TFIIH complex is recruited to transcription start sites and is essential for Pol II initiation. The translocase activity of TFIIH core subunit XPB acts to &ldquo;melt&rdquo; the dsDNA, allowing Pol II to begin transcribing. The TFIIH kinase, CDK7, is known to phosphorylate the C-terminal domain (CTD) of Pol II, but few other targets have been identified due to a lack of reliable CDK7 inhibitors. This phosphorylation of the Pol II CTD facilitates the transition from initiation to elongation and is also important for proper recruitment of mRNA processing factors such as capping enzyme, elongation factors, splicing factors, and termination factors; however, no direct interactions have been identified between CDK7 and these factors. Furthermore, CDK7 has recently emerged as a potential therapeutic target for many aggressive, metastatic cancers, pushing the development of selective, next-generation inhibitors.Here, I describe my recent work in collaboration with others to evaluate the role of TFIIH in Pol II transcription and mRNA processing, with an emphasis on CDK7 kinase activity. We utilized a next-generation, covalent CDK7 inhibitor with unmatched selectivity in SILAC-MS experiments in the first large-scale identification of CDK7 kinase substrates in human cells, using the HL60 leukemia cell line. These experiments found novel, high-confidence targets including the splicing factors SF3B1 and U2AF2, elongation factors SPT5 (DSIF) and SUPT16H (FACT), and the T-loop sites of CDK12 and CDK13 among other targets. These results revealed direct interactions between CDK7 and mRNA processing factors and implicated CDK7 in Pol II transcription more broadly than previously thought. Additionally, we found inhibition of CDK7 to be correlated with reduced gene expression and diverse splicing defects, further implicating these interactions with identified factors. Validation of these identified targets with biochemical assays led to the discovery that TFIIH itself regulates CDK7 kinase activity and controls substrate specificity. The TFIIH core was found to restrict kinase activity to the Pol II CTD and dissociation of the CAK important for permitting CDK7 phosphorylation of these novel targets; this suggested that CAK dissociation may be a key regulatory step to control CDK7 kinase activity. Furthermore, while the CAK complex had previously been found to activate the cell cycle kinases CDK1, CDK2, and CDK4/6 through phosphorylation of their T-loops, we discovered that it also acts as a &ldquo;master regulator&rdquo; of the transcriptional kinases CDK9, CDK12, and CDK13. Taken together, these results suggested new models for CDK7 function in Pol II transcription and mRNA processing and implicated CAK dissociation from TFIIH as essential for kinase activation, ensuring CDK7 activation can be spatially and temporally linked to transcription.Ongoing work utilizing next-generation, non-covalent CDK7 inhibitors in combination with RNA-seq experiments in the OV90 ovarian cancer cell line has found defects in gene expression and diverse splicing changes, similar to our previous work. Major advancements will be made through future work involving nascent transcriptomics experiments, PRO-seq. Inhibition of CDK7 in these experiments will elucidate the role of CDK7 in promoter-proximal pausing, elongation, and transcription factor activity (eRNA production). Taken together, we have discovered new mechanisms for how CDK7 kinase activity is regulated, how CDK7 regulates the activity of other transcription-associated kinases, and how CDK7-dependent phosphorylation may govern a myriad of stages in Pol II transcription. These results have broad therapeutic implications given the prominent role for CDK7 in development and disease and future work will continue to clarify these roles.</p

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning

Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6.

TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II preinitiation complex, TFIIH regulates pol II enzyme activity in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit, but the functional significance of specific residues is unclear. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and 12 conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Our biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to preinitiation complexes in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH and for pol II transcription

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference