Synthesis of libraries of lysine-acetylated nucleosomes and their usage for probing bromodomains

Abstract

ε-N-acetylation of lysine residues is one of the most prevalent post-translation modifications of histones, often associated with chromatin remodeling and gene transcription. Bromodomains (BRDs) are conserved protein motifs that can recognize and bind acetylated lysine residues. Commonly, the interactions between BRDs and histone acetylation sites are investigated with peptides, omitting any impact of the nucleosome structure.

In this thesis, libraries of acetylated nucleosomes were generated by protein semi-synthesis and probed with a set of recombinant BRDs. Resorting to sortase-mediated ligation established for semi-synthesis of histone H3, 64 depsipeptides permutating the known acetylation site at K4, K9, K14, K18, K23 and K27 were synthesized and ligated with truncate nucleosomal H3. Protein trans-splicing was used to generate 16 nucleosomes with permuted acetylation sites at K5, K9, K13 and K15 of histone H2A.

The nucleosomal libraries were probed with recombinant BRDs derived from BAZ2B, CREBBP, BRD3(2) and the tandem BRD of BRD4(1/2). In general, mono-acetylated nucleosomes showed similar interaction profiles with these BRDs as reported with peptides. In the case of acetylated H3, additive acetylation marks enhanced nucleosomal recruitment of BRDs, and this effect appeared to result from enhanced direct binding rather than charge neutralization modulating tail accessibility. Nucleosomes with multiple acetylation marks on H3 and H2A showed enhanced recruitment of the tandem bromodomain BRD4(1/2), which likely resulted from independent binding events rather than simultaneous binding of the acetylated H3 and H2A tails.

Die Dissertation ist gesperrt bis zum 23. Januar 2026 !