UC Berkeley

Cell identity is maintained by the selective activation and silencing of distinct gene programs that give rise to distinct cellular functions. Epigenetic regulation occurs often independent of DNA sequence and relies on integrating regulatory information encoded by chemical modifications made directly to chromatin. These modifications can establish and maintain distinct chromatin states, where compact chromatin is transcriptionally silent and open chromatin is transcriptionally permissive. These states can be propagated through cell division, and their establishment and maintenance are essential for embryonic development and the maintenance of cell identity throughout life.The Polycomb group proteins are evolutionarily conserved chromatin regulators that play a central role in gene silencing during development. The mammalian system functions through two multi-subunit complexes, Polycomb Repressive Complex 1 and 2 (PRC1/2). PRC1 compacts chromatin and ubiquitylates histone H2A at lysine 119 (H2AK119Ub), while PRC2 catalyzes the mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27me1/2/3). H3K27me3 is the defining chromatin mark of Polycomb-repressed domains and serves as a recruitment site for chromobox-containing PRC1 subunits. Additionally, PRC2 accessory subunits harbor ubiquitin-interaction motifs that interact with PRC1-mediated H2AK119Ub, creating two self-reinforcing feedback loops for gene silencing. The coordinated activities of both Polycomb complexes are essential, and their dysregulation drives developmental disorders and cancers.PRC2 is a multi-subunit histone methyltransferase composed of four core subunits: EZH2, SUZ12, EED, and RBAP48. EZH2 harbors the catalytic SET domain responsible for lysine methyltransferase activity and contains multiple regulatory elements including the CXC domain and bridge helix, which contact nucleosomal DNA, and the stimulatory response motif (SRM), which communicates allosteric signals to the active site. EED, a WD40 repeat protein, recognizes H3K27me3 through an aromatic cage and relays this signal to the EZH2 SRM to allosterically stimulate further methylation. This read-write mechanism helps spread the silencing mark across repressive domains. SUZ12 serves as the structural scaffold of the complex, bridging the catalytic lobe (EZH2, EED, and the SUZ12 VEFS domain) and the targeting lobe (RBAP48 and the SUZ12 N-terminal zinc finger and C2 domains) through flexible hinges that allow large conformational rearrangements. RBAP48 and SUZ12 together form the primary interaction platform for accessory cofactors.The PRC2 core associates with a variety of accessory proteins that influence its catalytic activity, chromatin binding affinity, and genomic targeting. These associations define two major PRC2 subtypes with distinct biological functions. PRC2.1 incorporates one of three Polycomb-like (PCL) proteins (PHF1, MTF2, or PHF19) and either EPOP or PALI1, whereas PRC2.2 incorporates AEBP2 and JARID2. The two subtypes regulate largely overlapping but non-identical sets of target genes, and maintaining the proper balance between subtypes is essential for normal development. Mutations or expression changes to PRC2 core and accessory subunits that affect this balance are frequently observed in cancer. Despite their importance, structural characterization of PRC2 has been largely limited to PRC2.2 complexes and partial assemblies, leaving the architectural basis for PRC2.1 subtype-specific functions unresolved.PRC2 activity is also tightly regulated through its integration of chromatin signals in the surrounding environment. In addition to its interaction with the catalytic products of both Polycomb complexes, histone modifications associated with active transcription directly inhibit PRC2 catalytic function. This antagonism ensures that PRC2 is excluded from actively transcribed genomic regions and confines its activity to appropriate repressive domains. Understanding how PRC2 reads and integrates these competing chromatin inputs at the molecular level is essential to explain how robust boundaries between active and silent chromatin states are established and maintained.The chapters that follow leverage advanced cryo-EM sample preparation approaches, together with biochemical and functional characterization, to address outstanding questions about how PRC2 catalytic and targeting activities are regulated through its interactions with regulatory accessory subunits and the surrounding chromatin environment.The first chapter describes the optimization and application of streptavidin affinity grids for cryo-electron microscopy (cryo-EM) sample preparation. A persistent challenge in structural studies of large, dynamic macromolecular complexes such as PRC2 is the tendency for particles to adopt preferential orientations or become damaged at the air–water interface during grid preparation. Streptavidin affinity grids, which capture biotinylated samples onto a two-dimensional crystalline streptavidin lattice, circumvent these issues by enriching intact complexes away from the interface. The streptavidin affinity grid technology developed and refined here was instrumental to all structural studies described in this dissertation and enabled the visualization of flexible, fragile PRC2–chromatin complexes that had been refractory to conventional cryo-EM approaches.The second chapter presents cryo-EM structures that reveal the structural basis by which active transcription-associated histone post-translational modifications (PTMs) inhibit PRC2 activity. Using PRC2.2 complexes containing AEBP2 and JARID2 bound to modified nucleosome substrates, we show that H3K36me3 reduces engagement of PRC2 with the histone H3 tail and alters the complex’s interaction with chromatin, while H3K4me3 acts as an allosteric antagonist that engages the EED regulatory site and competes with PRC2-activating peptides. Together, these results illuminate the complex interplay between PRC2, its accessory subunits, and the chromatin environment in regulating its function.The third chapter presents the first cryo-EM structures of intact PRC2.1 subtype complexes engaged with chromatin substrates. These structures reveal that while PRC2 subtypes share conserved principles of nucleosome engagement and allosteric activation through the EZH2–EED axis, distinct accessory factors exploit overlapping interaction surfaces within the PRC2 core to drive large rearrangements of the targeting lobe, creating architecturally distinct complexes. This work further identifies novel nucleic-acid interaction surfaces contributed by both the PRC2 core subunit SUZ12 and the accessory factor PALI1, supporting a model in which the targeting and catalytic activities of PRC2 are partially decoupled. Together, these findings provide a structural framework for understanding how the balance between PRC2 subtypes is achieved and how its disruption may contribute to developmental defects and disease.