Controlling the supramolecular assembly of nucleosomes asymmetrically modified on H4

Abstract

In stem cells, H4 proteins carrying different modifications coexist within single nucleosomes. For functional studies, we report the synthesis of such asymmetric nucleosomes. Asymmetry is achieved by transiently crosslinking H4 by a traceless, protease-removable tag introduced via an isopeptide linkage. These nucleosomes are used to study Set8 activity, a key methyltransferase.Nucleosomes, the basic unit of chromatin, organize 147 bp of DNA wrapped around two of each core histone H3, H4, H2A and H2B.1 The histone proteins carry combinations of post-translational modifications (PTMs, or marks), which are implicated in regulating chromatin function. 2 In particular, methylation of lysine residues on H3 and H4 has clearly defined roles in gene activation and repression, with implication for cell differentiation, development and disease. 3Detailed MS studies found that key methyl-marks in embryonic stem cells (ESCs) exist in asymmetric nucleosomes, i.e. nucleosomes carrying two differently modified copies of H3 or H4. 4 These PTMs include H4 monomethylated at lysine 20 (H4K20me1), as well as H3K4me3, H3K36me3 and H3K27me3. 5 H4K20 methylation is a critical modification involved in heterochromatin silencing, and also in DNA replication and the DNA damage response. 6 The methyltransferase Set8 (also known as PR-Set7 or KMT5A) is responsible for H4K20 monomethylation, whereas two further methyltransferases, Suv4-20h1 and Suv4-20h2, catalyze di-and trimethylation of this residue. 6 Together, H4K20 methylation is involved in chromatin structure regulation, 7 and serves as a binding site for chromatin regulators, including 53BP1 8 and L3MBTL1. The establishment, maintenance and function of nucleosomes asymmetrically modified on H4 is not well understood. This is mainly due to the fact that such nucleosomes are not easily available for detailed in vitro mechanistic studies. Here, we thus developed a synthetic strategy enabling the traceless synthesis of nucleosomes containing differentially modified H4 proteins, with a focus on H4K20me1. While expressed protein ligation (EPL) methods readily enable the installation of combinations of marks on nucleosomes, 10 the reconstitution of asymmetric chromatin is not straightforward and is largely based on the attachment of affinity tags, followed by multi-step purification schemes. 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. 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 mo...

A Genetically Encoded Approach for Breaking Chromatin Symmetry

“…We began by exploring options for the chemoselective and traceless tethering of defined H3 monomers such that their association would be maintained throughout the nucleosome reconstitution process. 25 − 27 In particular, we wanted these tethers to be genetically encoded, biorthogonal, and irreversible. The so-called SpyCatcher/SpyTag method presented itself as an optimal method for efficiently generating obligate protein heterodimers, as it results in an autocatalyzed isopeptide bond between two fragments of a split protein domain, covalently fusing the two polypeptide chains.…”

“… 28 , 29 Moreover, chemical tethering approaches rely on sophisticated synthetic chemistry manipulations that are not accessible to every laboratory. 25 − 27 …”

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

“…To complete our library, we also synthesized and reconstituted nucleosomes containing asH3K27me3 ( 8C , following published protocols) as well as nucleosomes symmetrically modified with H3K36me3 ( 8D , Supporting Information). Together with our previously developed syntheses, we have now established synthetic access to all known asymmetric PTM combinations found in stem cells, which sets the stage for mechanistic studies of stem cell specific PTM regulation.…”

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

“…Finally, the lnc-tag is removed using TEV protease, leaving no trace of the whole procedure. Using this approach [21] (and variants thereof targeting H4, [22] Fig. 3c) we synthesized the whole ensemble of nucleosomes carrying asymmetric modifications on H3 and H4 which were found in stem cells.…”