PROJECTS
The laboratory is currently organized around four different research axes:
Dissecting the role of the Mediator Kinase module in transcriptional regulation in vivo
Mediator is a highly conserved transcriptional co-activator. It is composed of 25 (yeast) to 30 (human) polypeptides organized into four modules named Head, Middle, Tail and Kinase. Mediator interacts with both the transcription factors bound to enhancers and with RNA polymerase II and many general transcription factors at core promoters, therefore relaying regulatory signals from enhancers to promoters. In the recent years, the Robert lab has made significant contributions to our understanding of Mediator function, notably by dissecting the different Mediator modules dynamically interacting with chromatin during transcription. This work revealed the very dynamic nature of the Kinase module. The Kinase module is recruited to enhancers together with the rest of Mediator but is ejected from Mediator upon the transient interaction of Mediator with the core promoter. Interestingly, the Kinase module acts as a negative regulator of Mediator by impairing Mediator-enhancer interactions. In this project, we are now further dissecting the mechanism and function of the Kinase module. This is of primary importance since subunits of the Kinase module are frequently mutated in human diseases including many cancers. Reaching a high mechanistic-level of understanding of the function of the Kinase module will be key to grasp its role in human diseases.
The role of Mediator in the regulation of promoter-proximal pausing
In addition to its well-known role in transcription initiation, Mediator also regulates other aspects of transcription, notably promoter-proximal pausing. Shortly after initiation, RNA polymerase II makes a pause and this constitutes a major regulatory step for the expression of several genes. While Mediator has been shown to regulate pause release, the mechanism remains ill defined. In this project, we use high-resolution mapping technologies such as ChIP-exo to dissect the role of Mediator in pausing.
The role of the RNA polymerase II CTD in the regulation of chromatin at active genes
Chromatin was long known to impact on transcription. More recently, we and others have shown that transcription can also influence chromatin in many ways. As a postdoc, Dr. Robert and his colleagues were among the first to show that RNA polymerase II (RNAPII) can actively regulate chromatin, notably by recruiting the histone methyltransferase (HMT) Set1. More recently, the Robert lab and others have shown that RNAPII can recruit more chromatin regulators (e.g. the Rpd3S and Set3C HDACs and the Set2 HMT). Quite interestingly, in all cases, the phosphorylation of the C-terminal domain (CTD) of RNAPII is mediating these interactions. This is reminiscent with the well described role of the CTD in the recruitment of mRNA processing enzymes. There is therefore an emerging theme in the literature where the RNAPII CTD is viewed as a landing pad for the recruitment of various factors, including chromatin regulators, in order to couple transcription with related processes. In this project, we use combinations of proteomic, genomic and genetic approaches to identify new CTD-recruited factors and characterize their function.
The role of histone chaperones FACT and Spt6 in the regulation of chromatin structure
Over the years we have done significant contributions to the understanding of the role of the variant histone H2A.Z, a non-allelic variant of H2A that is involved in transcription, anti-silencing, centromeres and perhaps also in heterochromatin functions. How H2A.Z can fulfill so many different functions remains enigmatic but it has clear implications in cell differentiation and cancer. As a postdoc, Dr. Robert, in collaboration with the Gaudreau lab (Université de Sherbrooke), was among the firsts to show that H2A.Z is involved in transcription. Later, we and many others have shown that it occupies one or two nucleosomes in most promoters and we showed that it also localizes to facultative heterochromatin in human cells. It is well known that H2A.Z is deposited in chromatin by the Swr1 complex, but we have recently shown that other activities, namely the histone chaperones FACT and Spt6, are involved in determining the localization of H2A.Z along the genome. We are currently extending our study of FACT and Spt6 to the regulation of other epigenetic marks.