A Modular Ligation Strategy for Asymmetric Bivalent Nucleosomes Trimethylated at K36 and K27

Abstract

In nature, individual histones in the same nucleosome can carry identical (symmetric) or different (asymmetric) post‐translational modification (PTM) patterns, increasing the combinatorial complexity. Embryonic stem cells exhibit “bivalent” nucleosomes, some of which are marked by an asymmetric arrangement of H3K36me3 (an activating PTM) and H3K27me3 (a repressive PTM). Here we describe a modular synthetic method to access such asymmetrically modified nucleosomes and show that H3K36me3 inhibits the activity of the methyltransferase PRC2 locally while still prolonging its chromatin binding time.

Structural basis for PRC2 decoding of active histone methylation marks H3K36me2/3

“…On isolated H3 N-terminal peptides, H3K36me3 did not inhibit the formation of H3K27me1 ( Figure 3D ), consistent with earlier findings that on peptide substrates H3K36me3 only has a minor effect on the k cat of H3K27 methylation ( Schmitges et al, 2011 ; Jani et al, 2019 ). Also, H3K36me3 does not diminish the affinity of PRC2 for binding to mononucleosomes ( Figure 3A,B ) and does not reduce the residence time of PRC2 on nucleosome arrays ( Guidotti et al, 2019 ). Taken together, a possible scenario would therefore be that within the time frame of the PRC2-nucleosome binding and reaction cycle, docking of the H3K36 side chain in the EZH2 CXC -DNA interface is critical for rapid alignment of the H3 N-terminus on the EZH2 surface into a catalytically competent state.…”

“…While recent structural studies have uncovered the allosteric activation mechanism for PRC2 ( Jiao and Liu, 2015 ; Justin et al, 2016 ), the molecular basis of PRC2 inhibition by active chromatin marks has remained enigmatic. In particular, in nucleosome-binding assays, PRC2–DNA interactions make the largest contribution to the nucleosome-binding affinity of PRC2 ( Wang et al, 2017 ; Choi et al, 2017 ) and H3K4me3, H3K36me2 and H3K36me3 do not seem to have a major effect on this binding affinity ( Schmitges et al, 2011 ; Guidotti et al, 2019 ; Jani et al, 2019 ). Instead, these three modifications were found to reduce the k cat of PRC2 for H3K27 methylation ( Schmitges et al, 2011 ; Jani et al, 2019 ).…”

Structural basis for PRC2 decoding of active histone methylation marks H3K36me2/3

“…On isolated H3 N-terminal peptides, H3K36me3 did not inhibit the formation of H3K27me1 ( Figure 3D ), consistent with earlier findings that on peptide substrates H3K36me3 only has a minor effect on the kcat of H3K27 methylation (Schmitges et al, 2011) (Jani et al, 2019). Also, H3K36me3 does not diminish the affinity of PRC2 for binding to mononucleosomes ( Figure 3A, B ) and does not reduce the residence time of PRC2 on nucleosome arrays (Guidotti et al, 2019). Taken together, a possible scenario would therefore be that within the time frame of the PRC2 nucleosome binding and reaction cycle, docking of the H3K36 side chain in the EZH2 CXC -DNA interface is critical for rapid alignment of the H3 N-terminus on the EZH2 surface into a catalytically competent state.…”

“…While recent structural studies have uncovered the allosteric activation mechanism for PRC2 (Jiao and Liu, 2015; Justin et al, 2016), the molecular basis of PRC2 inhibition by active chromatin marks has remained enigmatic. In particular, in nucleosome binding assays, PRC2-DNA interactions make the largest contribution to the nucleosome-binding affinity of PRC2 (Choi et al, 2017; Wang et al, 2017) and H3K4me3, H3K36me2 and H3K36me3 do not seem to have a major effect on this binding affinity (Guidotti et al, 2019; Jani et al, 2019; Schmitges et al, 2011). Instead, these three modification were found to reduce the k cat of PRC2 for H3K27 methylation (Jani et al, 2019; Schmitges et al, 2011).…”

Applying Peptide and Protein Synthesis to Study Post-translational Modifications in Epigenetics and Beyond

“…[20] Using this library we could show that PRC2 methyltransferase activity is activated by pre-existing H3K27me3 in asymmetric nucleosomes across the nucleosome surface, allowing the enzyme to propagate this PTM along the chromatin fiber. In contrast, PRC2 activity was found to be locally inhibited by pre-existing H3K4me3 or H3K36me3, limiting its encroachment into euchromatin [21,23] (Fig. 3c).…”