Gene activation by transcription factors (TFs) is one of the most fundamental processes in a cell. In order to maintain cell homeostasis, properly respond to stress signals, or differentiate into a new cell type, DNA-binding transcription factors must find and bind to their cognate sequences in the genome. However, once bound, this TF binding must be faithfully relayed to the target promoter, sometimes located tens of kilobases away from the TF-binding site and elicit a change in gene expression. The relay and integration of gene activation signals is coordinated by large co-activator complexes that bind to both the TFs themselves and the general transcription machinery (GTFs). In humans, these complexes include TFIID, SAGA, and the Mediator complex. Each of these complexes have unique TF-binding, DNA-binding, and/or chromatin remodeling activities and genome-wide studies have found both overlapping and gene- and context-specific roles for each complex. Our goal is to better understand how these co-activators and GTFs regulate gene expression and activation through their interactions with Pol II. We are using state-of-the-art imaging techniques to visualize the dynamics of such complexes in living cells. Coupled with specific perturbations and biochemical and genomic approaches, we can gain mechanistic insight into fundamental co-activator mechanisms and dynamics.
Since the first characterization of the core components of the pre-initiation complex, many techniques to study the roles of these GTFs and co-activators have relied on static pictures of the transcriptional process and are only able to capture stable, long-lived interactions. By incorporating innovative, live-cell imaging techniques including single-particle tracking in human cells, we aim to uncover previously invisible phenomena underlying these fundamental transcriptional mechanisms.
– How stable or dynamic are co-activator and PIC components, and do they cluster within the nucleus (like Pol II)?
– How does each co-activator/GTF contribute both shared and specific roles in gene activation, such as their involvement in TBP recruitment or PIC stability?
– How do co-activators/GTFs interact with the pre-initiation complex and Pol II during each stage of transcription – including initiation, re-initiation, and elongation?
– How does chromatin remodeling by co-activators/GTFs affect transcriptional outcomes?
– How do shared protein subunits between some co-activators and GTFs (like TFIID and SAGA) regulate complex assembly and dynamics?