Visualizing stimulated chromatin regulatory proteins bound to substrate
EMSL Project ID
50702
Abstract
The regulation of gene activity is a key regulatory process during cellular differentiation and is a crucial requirement for development in multicellular organisms. A large number of mechanisms have evolved to maintain developmentally important genes in either the "on" or "off" state in the appropriate cellular environments. One of the most important complexes involved in maintaining genes in the repressed state during development is the evolutionarily conserved Polycomb group (PcG) of protein complexes. PcG complexes exert silencing function by interacting with chromatin, which is comprised of DNA, histones, and other regulatory proteins, and are frequently misregulated in human malignancies. Polycomb repressive complex 2 (PRC2) is a multi-subunit protein complex that methylates histone H3 at lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) is strongly associated with silenced genes. However, the role of H3K27me3 in silencing is not fully understood. H3K27me3 directly interacts with several repressive complexes, and can allosterically activate PRC2, suggesting leading to models of recruitment and positive feedback culminating in the stabilization of silenced states. Understanding the molecular details underlying the interactions of PRC2 modules with chromatin is a prerequisite for targeting key protein-protein interactions (PPIs) with small molecule inhibitors for therapeutic purposes. As an important first step in understanding the structural role of PRC2-chromatin interactions in maintaining gene repression, we have generated several core PRC2 complexes and have developed purification and grid freezing protocols that allowed us to generate a 3.9 Å cryo-EM map of human PRC2 containing EZH1. EZH1 and EZH2 are the two primary catalytic subunits found in PRC2 and dictate several biochemical differences between PRC2 containing one or the other. Our preliminary structure has allowed us to map some of the biochemical differences to different structural features, such as understanding how several domains underlie the differences in substrate binding and catalysis. However, several questions remain that can be addressed by further structural investigations with cryo-EM at PNCC. First, what is the role of the stimulatory response motif in allosteric activation of PRC2 containing EZH1? Secondly, what is the mechanism for inhibitor resistance by PRC2-EZH1? Project Details
Start Date
2019-03-15
End Date
2019-12-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Grau D, Zhang Y, Lee CH, Valencia-Sánchez M, Zhang J, Wang M, Holder M, Svetlov V, Tan D, Nudler E, Reinberg D, Walz T, Armache KJ. Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction. Nat Commun. 2021 Jan 29;12(1):714. doi: 10.1038/s41467-020-20775-z. PMID: 33514705.