Gene expression regulation (III)

The role of phase separation in RNA polymerase II transition from initiation to elongation

Project summary

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 explain 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 hypothesize 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.

References

• Boehning, M., Dugast-Darzacq, C., Rankovic, M., Hansen, A.S., Yu, T., Marie-Nelly, H., McSwiggen, D.T., Kokic, G., Dailey, G.M., Cramer, P., et al. (2018). RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat Struct Mol Biol 25, 833-840.
• Boija, A., Klein, I.A., Sabari, B.R., Dall'Agnese, A., Coffey, E.L., Zamudio, A.V., Li, C.H., Shrinivas, K., Manteiga, J.C., Hannett, N.M., et al. (2018). Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842-1855 e1816.
• Cho, W.K., Spille, J.H., Hecht, M., Lee, C., Li, C., Grube, V., and Cisse, II (2018). Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412-415.
• Churchman, L.S., and Weissman, J.S. (2011). Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368-373.
• 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.
• Guo, Y.E., Manteiga, J.C., Henninger, J.E., Sabari, B.R., Dall'Agnese, A., Hannett, N.M., Spille, J.H., Afeyan, L.K., Zamudio, A.V., Shrinivas, K., et al. (2019). Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543-548.
• Jeronimo, C., Bataille, A.R., and Robert, F. (2013). The writers, readers, and functions of the RNA polymerase II C-terminal domain code. Chem Rev 113, 8491-8522.
• Lu, H., Yu, D., Hansen, A.S., Ganguly, S., Liu, R., Heckert, A., Darzacq, X., and Zhou, Q. (2018). Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558, 318-323.
• Sabari, B.R., Dall'Agnese, A., Boija, A., Klein, I.A., Coffey, E.L., Shrinivas, K., Abraham, B.J., Hannett, N.M., Zamudio, A.V., Manteiga, J.C., et al. (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science 361.
• Wang, J., Choi, J.M., Holehouse, A.S., Lee, H.O., Zhang, X., Jahnel, M., Maharana, S., Lemaitre, R., Pozniakovsky, A., Drechsel, D., et al. (2018). A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 174, 688-699 e616.

Summary of responsibilities

This project combines the use of state-of-the-art functional genomics, genome editing, and biochemistry. The student/postdoc will be in charge of executing and analyzing the vast majority of the experiments for the project and will be assisted during his/her training by Dr. Robert and senior members of the lab. The IRCM has several core laboratories with expertise that will enhance the training experience of the candidate.

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.
• Drouin, S., Laramee, L., Jacques, P.E., Forest, A., Bergeron, M., and Robert, F. (2010). DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes. PLoS Genet 6, e1001173.
• Jeronimo, C., Bataille, A.R., and Robert, F. (2013). The writers, readers, and functions of the RNA polymerase II C-terminal domain code. Chem Rev 113, 8491-8522.
• Jeronimo, C., Collin, P., and Robert, F. (2016a). The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain. J Mol Biol 428, 2607-2622.
• 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.
• Uwimana, N., Collin, P., Jeronimo, C., Haibe-Kains, B., and Robert, F. (2017). Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighboring genes. Nucleic Acids Res 45, 6417-6426.

Job requirements

We seek a highly motivated individual with a genuine interest in understanding the mechanistic aspects of molecular processes. A background in molecular biology and/or biochemistry is mandatory. Knowledge in bioinformatics, computer programming or statistics are assets but not mandatory. Only candidates with very good academic track records will be considered.

Starting date: As soon as possible.

How to apply

Send your candidacy by e-mail to:

Francois Robert

francois.robert@ircm.qc.ca