PRC2 bridges non-adjacent nucleosomes to establish heterochromatin

Abstract

Polycomb repressive complex 2 (PRC2) maintains transcriptionally silent heterochromatin by installing and spreading repressive histone methylation marks along nucleosome arrays. Despite extensive research, the mechanism by which PRC2 engages with chromatin remains incompletely understood. Here we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the interactions of PRC2 with polynucleosome substrates. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides interacting with bare DNA, mononucleosomes, and neighboring nucleosome pairs, PRC2 is also found to bridge non-adjacent nucleosomes, an activity associated with chromatin compaction. Furthermore, the distribution and stability of these PRC2-chromatin interaction modes are differentially modulated by accessory PRC2 cofactors, oncogenic histone mutations, and the methylation state of chromatin. Overall, this work provides a paradigm for understanding the physical basis of epigenetic maintenance mediated by Polycomb group proteins. Eukaryotic chromatin is modified by a plethora of epigenetic machineries that add or erase specific histone posttranslational modifications, thereby exerting transcriptional control (1). Some of these complexes are recruited to and stimulated by their own enzymatic products, which leads to the modification of neighboring nucleosomes and eventually the formation of large-scale chromatin domains. One preeminent example for such a positive feedback mechanism is mediated by the Polycomb repressive complex 2 (PRC2), which catalyzes methylation of the lysine 27 residue on histone H3 (H3K27), resulting in the final enzymatic product, trimethylated H3K27 (H3K27me3), a hallmark of facultative heterochromatin (2). PRC2 maintains transcriptional silencing of a large number of genes involved in development and cancer (3). The PRC2 core complex comprises four subunits: EED, EZH2, RBBP4, and SUZ12. In the proposed "read-and-write" model, EED recognizes an existing H3K27me3 mark and allosterically activates the methyltransferase EZH2, which in turn modifies a neighboring nucleosome (4,5). This model is supported by a recent cryo-electron microscopy (EM) structure of PRC2 engaging a dinucleosome, in which the EED subunit interacts with an H3K27me3-containing nucleosome and the EZH2 subunit of the same PRC2 complex engages an adjacent unmodified nucleosome (6). However, due to the prohibitive conformational heterogeneity of polynucleosome arrays, the binding configuration of PRC2 on its natural chromatin substrate has thus far been refractory to structural interrogation. Moreover, the activity of the PRC2 core complex is influenced by diverse regulatory factors, including accessory subunits, histone modifications, DNA methylation, and RNA [reviewed in (7,8)]. How PRC2 integrates signals from these regulatory factors to achieve chromatin targeting and H3K27me3 spreading remains incompletely understood. To address these knowledge gaps, in this work we developed experi...