Mediator is a universal transcriptional coactivator. It is recruited to enhancers by sequence-specific transcription factors and allows communication between the enhancers and their target promoters. How this communication is established in not fully understood but a generally accepted view is that the enhancer-bound Mediator physically reaches the promoter (probably via a looping mechanism) and stabilizes the pre-initiation complex (PIC) formed by RNA polymerase II (RNAPII) and other general transcription factors. This model, however, raises a conundrum: stabilizing the PIC would lead to transcription activation only if PIC formation was rate-limiting; yet, PIC formation is not rate-limiting at most genes in metazoans. In this project, we hypothesize that Mediator regulates transcription by controlling transcriptional bursting.
Single-molecule RNA fluorescence in situ hybridization and live-imaging have revealed that transcription occurs in bursts. During a burst (“on” state), several initiation events lead to convoys of RNAPII actively transcribing the gene whereas between bursts (“off” state) the gene is kept silent. Recent work showed that cellular stimuli led to increased burst initiation but not to increased polymerase recruitment rates within bursts. This suggests that transcription is mainly regulated by modulating the time a gene is in the “on” state rather than by regulating the different transcription steps (PIC formation, initiation, pausing, elongation) within a burst. Moreover, the use of allele-specific RNA-seq from single cells recently showed that cell-type-specific gene expression is primarily regulated by burst frequency and that this is mediated by enhancers. Based on these data, we hypothesize that Mediator (which mediates its effect from enhancers) regulates gene expression in part by regulating burst frequency. In other words, Mediator favors the transition from the “off” state into the “on” state, therefore, providing more opportunities for the target gene to make transcripts, rather than impinging directly on the different transcription steps.
To test how Mediator regulates burst kinetics, we will perform allele-specific single-cell RNA-seq in mouse fibroblasts derived from CAST/EiJ x C56BL/6J crosses, in WT and Mediator depletion conditions. The polymorphisms between the two mice genetic backgrounds provide a way to monitor the expression of each allele independently in single cells. By modeling the expression distribution of the two alleles using the two-state model of transcription, bursting parameters (burst frequency and burst size) can be inferred from these measurements. Mediator depletion will be performed using a powerful induced-degron strategy enabled by the use of CRISPR/Cas9 genome editing. Combined with other genomic assays, these experiments will allow us to establish a role for Mediator in transcriptional bursting and dissect the mechanisms involved.
Summary of responsibilities
This project combines the use of state-of-the-art functional genomic, genome editing, and protein depletion technologies. 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.
Recent selected publications
· Jeronimo C, Robert F. The Mediator Complex: At the Nexus of RNA Polymerase II Transcription. Trends Cell Biol. 2017 Oct;27(10):765-783.
· Jeronimo C, Langelier M-F, Bataille AR, Pascal JM, Pugh BF, Robert F. (2016) Tail and Kinase modules differently regulate core Mediator recruitment and function in vivo. Mol Cell. 2016 Nov 3;64(3):455-466. doi: 10.1016/j.molcel.2016.09.002. Epub 2016 Oct 20.
· Jeronimo C, Robert F. Kin28 regulates the transient association of Mediator with core promoters. Nat Struct Mol Biol. 2014 May;21(5):449-55.
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.
Tagged as: Life Sciences