Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin

Abstract

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the “1–3” bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2–chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Cooperative DNA looping by PRC2 complexes

“…PRC2 mediated looping observed here could play important roles for histone mark spreading inside the nucleus ( 32 , 70 ). As shown by Reinberg and coworkers ( 71 ), chromatin loops may serve as nucleation centers from which PRC2 can spread histone marks to both neighboring and distal nucleosomes via 3D diffusion.…”

“…Electrostatic interactions between protein and DNA were treated with the Debye–Hückel theory. These near-atomistic models have been widely used to study a wide range of protein-DNA complexes with great success ( 32 , 52–57 ).…”

“…Single-molecule pulling experiments were performed at room temperature on a LUMICKS C-Trap instrument equipped with dual-trap optical tweezers as previously described ( 32 ). Briefly, a piece of 10-kb biotinylated-DNA was tethered between two 3.23-micron streptavidin-coated and optically trapped polystyrene beads in PBS buffer.…”

“…A recent cryo-EM study captured PRC2 with a di-nucleosome, resolving a structure in which the protein complex simultaneously contacts two nucleosomes ( 20 ). We further demonstrated that such contacts could extend beyond nearest neighbors, using a combination of single-molecular force spectroscopy and computational modeling ( 32 ). Importantly, we found that PRC2 chromatin interactions are significantly weakened with the presence of AEBP2, supporting the role of accessory proteins and PRC2 conformation on chromatin binding.…”

Chromatin dynamics controls epigenetic domain formation

“…The combination of 1D and 3D models, which integrates the rates of spreading and chromatin dynamics created here, could be a step towards fulfilling this goal. Indeed, there is experimental evidence pointing to the importance of both linear (nearest-neighbor) [45] spreading of H3K9me3 marks, as well as spatial spreading [46] of H3K27 marks, showing that chromatin silencing could proceed by both mechanisms, as accounted for in Scheme II of our model.…”