PROJECTS
The laboratory’s research program is organized around three complementary axes:
Dissecting the role of the Mediator Kinase module in transcriptional regulation in vivo
Mediator is a highly conserved transcriptional coactivator composed of 25 polypeptides in yeast and 30 in humans, organized into four modules: Head, Middle, Tail, and Kinase. By interacting with transcription factors bound to enhancers, as well as with RNA polymerase II and numerous general transcription factors at core promoters, Mediator relays regulatory signals from enhancers to promoters. In recent years, the Robert lab has made significant contributions to our understanding of Mediator function, notably by characterizing how its different modules interact dynamically with chromatin during transcription. This work revealed the highly dynamic nature of the Kinase module: it is recruited to enhancers with the rest of Mediator but is released when Mediator transiently interacts with the core promoter. The Kinase module also acts as a negative regulator by impairing Mediator–enhancer as well as Mediator–RNA polymerase II interactions. We are now further investigating the mechanisms and functions of this module. This work is particularly important because Kinase module subunits are frequently mutated in human diseases, including many cancers. Achieving a detailed mechanistic understanding of the Kinase module will therefore be essential for elucidating its role in disease.
The role of Mediator in the regulation of promoter-proximal pausing
In addition to its well-established role in transcription initiation, Mediator has been proposed to regulate other stages of transcription, notably promoter-proximal pausing. Shortly after initiation, RNA polymerase II pauses in a major regulatory step that may serve as a checkpoint before elongation. The paused polymerase can either enter the elongation phase or terminate prematurely, allowing a new round of initiation. Although Mediator has been implicated in pause release, the underlying mechanism remains poorly defined. In this project, we combine omics approaches to model transcription kinetics in Drosophila cells, allowing us to distinguish Mediator’s effects on pausing from its effects on initiation.
Roles of the RNA polymerase II CTD
The C-terminal domain (CTD) of RNA polymerase II has traditionally been viewed as a platform that recruits factors—including RNA-processing proteins and chromatin regulators—to couple transcription with related processes. More recently, the CTD has also been proposed to engage in weak, multivalent interactions with itself and with other proteins. These interactions may confine the polymerase to subnuclear regions or promote the formation of protein clusters, hubs, or condensates, particularly near enhancers. In the lab, we combine proteomic, genomic, imaging, and genetic approaches to characterize the organization of the CTD interactome and determine how the weak and multivalent interactions fine-tune gene expression.