Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes

Abstract

Nucleosomes carry extensive post-translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysine 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27-specific lysine methyltransferase (KMT) polycomb repressive complex 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3-mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.

The Chd1 chromatin remodeler shifts hexasomes unidirectionally

“…Since the nucleosome is pseudo-symmetric with two copies of each core histone (H2A, H2B, H3 and H4), asymmetry occurs when each copy possesses distinct epigenetic modifications. Recent advances have revealed asymmetry at the single nucleosome level (Rhee et al, 2014; Voigt et al, 2012), yet with challenges in synthesizing uniform populations of asymmetrically modified nucleosomes (Lechner et al, 2016; Liokatis et al, 2016), the biological significance of the vast majority of asymmetric marks remains unclear.…”

“…Trimethylation of H3K27 is carried out by PRC2, and while H3K4me3 blocks modification of K27 on the same H3 tail, PRC2 can deposit a H3K27me3 mark on the opposing H3 tail of the same nucleosome (Lechner et al, 2016; Voigt et al, 2012). In addition to generating nucleosomes with asymmetric H3K4me3/H3K27me3, PRC2 is also activated by the mark it deposits, with substrate preference for asymmetric nucleosomes containing one H3K27me3 (Lechner et al, 2016; Margueron et al, 2009).…”

Differentially Isotope‐Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time‐Resolved NMR Spectroscopy

“…At the same time, new chemical tools are emerging for reconstitution of asymmetrically modified nucleosomes to be used for functional studies. [20] Into this highly growing field, our approach aims to uncover mechanistic facets on the establishment and propagation of asymmetrically modified nucleosomes.…”

Controlling the supramolecular assembly of nucleosomes asymmetrically modified on H4

“…This allowed us to synthesize asymmetrically modified nucleosomes, carrying H3K4me3 and H3K27me3 on different H3 copies. 12 After the formation of nucleosomes, the crosslink was reversed and the lnc-tag was removed using tobacco etch virus (TEV) protease (Scheme 1A-C).Based on these studies, we thus wondered if our synthetic method could be adapted to synthesize crosslinked versions of differentially modified H4 ( xlnc H4). In cells, H4 is however acetylated at its N-terminus, 13 precluding the use of the original lnc-tag strategy.…”

“…4,11 We recently developed a chemical method to address this problem and control supramolecular nucleosome assembly. 12 Our synthetic approach was based on the inclusion of a link-and-cut (lnc)-tag that enabled transient crosslinking (by a disulfide bond) of H3 species during nucleosome reconstitution. This allowed us to synthesize asymmetrically modified nucleosomes, carrying H3K4me3 and H3K27me3 on different H3 copies.…”