The projects in our laboratory focus on how regulatory complexes alter chromatin structure at scales that include short clusters of nucleosomes, kilobase-sized domains, and, within phase separated puncta. The focus is on complexes and regulatory RNAs that can alter these interactions. We use prior genetic studies on development, primarily those on the Polycomb-Group (PcG) and trithorax-group (trxG), to define complexes that we study. These complexes regulate the classic epigenetic paradigm of stable memory of cell type across numerous generations of cell divisions. Understanding their mechanism should lead to general insights on how transcriptional memory is maintained across mitosis and meiosis in mammals.
Polycomb Repressive Complex 1 (PRC1)
PRC1 family complexes function as the major ‘engines’ of repression in the PcG. Canonical PRC1 complexes in mammals contain one of five CBX proteins, CBX2, CBX4, CBX6, CBX7 or CBX8. They also include a RING protein, a PCGF protein and a PH protein. One major focus of the group is to understand the function of each of the CBX proteins. At least three of these proteins (CBX2,4,8) contain a central domain that compacts chromatin structure and generates phase separation. We use mutational analysis to understand the relationship between phase separation and compaction and cell culture studies to understand how the CBX proteins function on chromatin and in gene regulation during differentiation. We are also exploring various gain-of-function strategies to determine how these domains might alter regulation when they are placed in proteins that do not normally contain them, or when their function is induced at abnormal times.
We are applying similar approaches to proteins in the PCGF family and in the PH family. Studies on PCGF family proteins will allow us to understand the interface between canonical PRC1 complexes and the non-canonical PRC1 complexes. These latter complexes are primarily responsible for ubiquitylation of histone H2A, a second activity of the PRC1 family, and we are interested in how that activity coordinates with chromatin compaction. The PH proteins, on the other hand, form networks that have been implicated in generating long range interactions. Studying these proteins will help us understand their function in formation of higher order domains.
Polycomb Repressive Complex 2 (PRC2)
PRC2 is primarily known for its ability to methylate histone H3 on lysine 27 (H3K27me3). We are interested in understanding how this function integrates with PRC1 function to generate heritably repressed domains. However, we are also interested in exploring distinct functions for this complex, including alternative substrates for methylation and alternative functions that exist outside of this well-studied enzymatic activity.
The SWI/SNF (aka BAF) family of complexes in mammals has been implicated in enhancer function, which in turn involves long range interactions in chromatin. We are interested in understanding how specific subunits of the SWI/SNF family influence long range interactions using the same technologies that we use to understand these functions with the PcG complexes. Using structural analysis and studies of functional interactions, we are further interested in determining the interactions between SWI/SNF subunits within their complex and between those subunits and outside proteins.
In addition to characterizing the mechanisms of epigenetic regulatory complexes using biochemistry and molecular approaches in cell culture, we examine how these mechanisms might function during mammalian development and differentiation. One major area of study is in how the different PcG complexes function during specific stages of mouse development. We examine gene regulation during the development of specific lineages in mouse lines with specific PcG protein and domain. Currently, we are characterizing PRC1 function during spermatogenesis. We are additionally developing projects that examine PRC1 function and interactions during neurogenesis, and in the future are interested in understanding the interplay between PcG complexes and SWI/SNF complexes in these systems.