Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.histone acetylation | chromatin dynamics | native chemical ligation
Post-translational modification (PTM) of histones plays a central role in genome regulation. Engineering histones with defined PTMs on one or multiple residues is crucial for understanding their function within nucleosomes and chromatin. We introduce a sequential native chemical ligation strategy suitable for the preparation of fully synthetic histone proteins, which allows for site-specific incorporation of varied PTMs throughout the sequence. We demonstrate this method with the generation of histone H3 acetylated at lysine 56 [H3(K56ac)]. H3(K56ac) is essential for transcription, replication, and repair. We examined the influence of H3(K56ac) on the targeting of a model DNA binding factor (LexA) to a site ∼30 bp within the nucleosome. We find that H3(K56ac) increases LexA binding to its DNA target site by 3-fold at physiological ionic strength. We then demonstrate that H3(K56ac) facilitates LexA binding by increasing DNA unwrapping and not by nucleosome repositioning. Furthermore, we find that H3(K56Q) quantitatively imitates H3(K56ac) function. Together these studies introduce powerful tools for the analysis of histone PTM functions.
Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.
C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully- or semi-synthetic proteins. However, efficient generation of C-terminal thioesters via Fmoc solid phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc-SPPS. Here, we demonstrate that this approach results in the formation of side products by over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which may be purified and converted in situ to thioester under ligation conditions. This method is compatible with automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.
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