Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

Abstract

Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments.

A Chemical Biology Approach to Reveal Sirt6-targeted Histone H3 Sites in Nucleosomes

“…However, this has been hampered due to the formidable difficulty of their synthesis using traditional methods. 13, 14 Despite the remaining doubts of Sirt6 biochemistry, mounting evidence has proven the critical functions of Sirt6 in metabolism, inflammation, and genome stability. 15 Transgenic mice overexpressing Sirt6 have low LDL cholesterol and triglyceride levels, reduced body fat, improved glucose tolerance, and a long lifespan.…”

Emerging Chemistry Strategies for Engineering Native Chromatin

“…Protein ligation methods, by contrast, offer a great deal more flexibility in terms of the type and number of PTMs that can be installed into a histone. 26 Both total synthesis and semisynthesis schemes can be used, in each case exploiting traceless versions of native chemical ligation to assemble the target histone from recombinant and/or synthetic peptide segments (Figure 2B). 26 These strategies are especially well suited to installing PTMs into the flexible N- or C-terminal tails of histones, where in fact the majority of marks reside, but are somewhat more cumbersome when it comes to the modifying residues in the middle of histones (although this is still possible).…”

“…26 Both total synthesis and semisynthesis schemes can be used, in each case exploiting traceless versions of native chemical ligation to assemble the target histone from recombinant and/or synthetic peptide segments (Figure 2B). 26 These strategies are especially well suited to installing PTMs into the flexible N- or C-terminal tails of histones, where in fact the majority of marks reside, but are somewhat more cumbersome when it comes to the modifying residues in the middle of histones (although this is still possible). Unnatural amino acid (UAA) mutagenesis (Figure 2C), which in its most commonly used incarnation relies on the amber suppression strategy, 27 does not suffer from any sequence restrictions and, like cysteine-based bioconjugation, is operationally straightforward to perform, at least once the necessary orthogonal tRNA/tRNA synthetase pair for the UAA of interest has been developed.…”

2016 EMBO Chemical Biology Conference

“…Sophisticated semisynthesis strategies have been developed for the preparation of selectively modified chromatin, forming the basis of a powerful toolkit for addressing the histone code hypothesis . However, the nucleosome building blocks of chromatin consist of dimers of the four core histone proteins.…”