Studies of biochemical crosstalk in chromatin with semisynthetic histones

Abstract

Reversible post-translational modifications of histone proteins in eukaryotic chromatin are closely tied to gene function and cellular development. Specific combinations of histone modifications, or marks, are implicated in distinct DNA-templated processes mediated by a range of chromatin-associated enzymes that install, erase and interpret the histone code. Mechanistic studies of the precise biochemical relationship between sets of marks and their effects on chromatin function are significantly complicated by the dynamic nature and heterogeneity of marks in cellular chromatin. Protein semisynthesis is a chemical technique that enables the piecewise assembly of uniformly and site-specifically modified histones in quantities sufficient for biophysical and biochemical analyses. Recent pioneering efforts in semisynthesis have yielded access to histones site-specifically modified by entire proteins, such as ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO). Herein, we highlight key studies of biochemical crosstalk involving Ub and SUMO in chromatin that were enabled by histone semisynthesis.

Chemoenzymatic Semisynthesis of Proteins

“…The disordered N- and C-terminal tails of histones that protrude from the nucleosome core have long been known to serve as hubs for a plethora of PTMs (Figure ), which have been correlated with the transcriptional state of the underlying genomic locus . Accordingly, unraveling the complex biochemical and biophysical implications of histone modifications (colloquially referred to as “marks”) has become a dynamic and cross-disciplinary area of inquiry, in which peptide and protein synthesis have played pivotal roles (for detailed accounts that emphasize the important role of chemistry in the area, we direct the reader to refs − ).…”

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

“…14,[20][21][22][23][24][25][26] Protein chemistry also enables the flexible alteration of protein at the histone and/or modifications for comprehensive mechanistic investigation. [27][28][29][30][31][32][33][34][35][36][37][38][39] Moreover, mechanism-based chemical probes can be used for trapping specific reaction states of the PTM enzymes for structural determination. These efforts have greatly expanded our understanding of the underlying molecular mechanisms of histone PTM crosstalks.…”

“…For instance, homogeneously modified histones prepared by chemical protein synthesis allow the examination of direct or indirect PTM interplays 14,20–26 . Protein chemistry also enables the flexible alteration of protein at the histone and/or modifications for comprehensive mechanistic investigation 27–39 . Moreover, mechanism‐based chemical probes can be used for trapping specific reaction states of the PTM enzymes for structural determination.…”

Examining histone modification crosstalk using immobilized libraries established from ligation-ready nucleosomes

“…Designer nucleosomes with dened PTM patterns are commonly generated by protein semisynthesis of synthetic histones tail peptides that are fused to distinct recombinant globular domains of the core histones. [23][24][25][26] The individually reconstituted semisynthetic histones are then assembled with the remaining core histones into nucleosomes and used for biochemical studies. In tour de force work, Muir and coworkers have extended this approach to assembling libraries of 64 and 115 modied nucleosomes where the DNA used for individual reconstitution was barcoded.…”