High resolution mapping of elongating RNA polymerase II (RNAPII) in yeast using Native Elongating Transcript sequencing (NET-seq) revealed frequent pausing events in the 5’end of genes (Churchman and Weissman, 2011). More recently, we found that these early elongation pausing events are reduced dramatically when the RNAPII C-terminal domain (CTD) is mutated to replace its tyrosines with phenylalanines (Collin et al., 2019). Because the CTD is well known to recruit different factors to the elongating RNAPII, we then performed a proteomic analysis of this RNAPII-Y1F mutant but found no tyrosine-dependent RNAPII interactor likely to provide an explanation to the loss of pausing phenotype. Mounting evidence suggests that the CTD can form protein condensates in cells via liquid-liquid phase separation (Boehning et al., 2018; Guo et al., 2019; Lu et al., 2018). Protein condensates formed by transcription factors, co-activators and RNAPII (via the CTD) are major players in gene expression regulation, notably by providing local environments favorable for the assembly of macromolecular assemblies such as super-enhancers (Boija et al., 2018; Cho et al., 2018; Sabari et al., 2018). Interestingly, CTD phosphorylation, which is a very dynamic process during transcription elongation (Jeronimo et al., 2013), was shown to affect the ability of the CTD to form condensates (Boehning et al., 2018) and to drive RNAPII from one type of condensate to another (Guo et al., 2019; Lu et al., 2018). Based on these data, and because tyrosine residues are often determinant for the ability of a protein to form condensates (Wang et al., 2018), we hypothesise that 1) the transition from initiation to elongation involves a redistribution of RNAPII from initiation-type condensates into elongation-type condensates; 2) this transition is a rate limiting step resulting in pausing and 3) the phosphorylation of the CTD provides the driving force for this transition. This hypothesis will be addressed using a combination of in vitro and in vivo phase separation assays, combined with genomic technologies such as NET-seq and others.
Boehning, M. et al. (2018). Nat Struct Mol Biol 25, 833-840.
Boija, A. et al. (2018). Cell 175, 1842-1855 e1816.
Cho, W.K. et al. (2018). Science 361, 412-415.
Churchman, L.S., and Weissman, J.S. (2011). Nature 469, 368-373.
Collin, P. et al. (2019). Mol Cell 73, 655-669 e657.
Guo, Y.E. et al. (2019). Nature 572, 543-548.
Jeronimo, C., Bataille, A.R., and Robert, F. (2013). Chem Rev 113, 8491-8522.
Lu, H. et al. (2018). Nature 558, 318-323.
Sabari, B.R. et al. (2018). Science 361.
Wang, J. et al. (2018). Cell 174, 688-699 e616.
Selected publications from the Robert lab
Bataille, A.R., Jeronimo, C., Jacques, P.E., Laramee, L., Fortin, M.E., Forest, A., Bergeron, M., Hanes, S.D., and Robert, F. (2012). A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes. Mol Cell 45, 158-170.
Collin, P., Jeronimo, C., Poitras, C., and Robert, F. (2019). RNA Polymerase II CTD Tyrosine 1 Is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway. Mol Cell 73, 655-669 e657.
Jeronimo, C., Langelier, M.F., Bataille, A.R., Pascal, J.M., Pugh, B.F., and Robert, F. (2016b). Tail and Kinase Modules Differently Regulate Core Mediator Recruitment and Function In Vivo. Mol Cell 64, 455-466.
Jeronimo, C., Poitras, C., and Robert, F. (2019). Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications. Cell Rep 28, 1206-1218 e1208.
Jeronimo, C., and Robert, F. (2014). Kin28 regulates the transient association of Mediator with core promoters. Nat Struct Mol Biol 21, 449-455.
Jeronimo, C., and Robert, F. (2017). The Mediator Complex: At the Nexus of RNA Polymerase II Transcription. Trends Cell Biol 27, 765-783.
Jeronimo, C., Watanabe, S., Kaplan, C.D., Peterson, C.L., and Robert, F. (2015). The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations. Mol Cell 58, 1113-1123.