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Understanding the molecular mechanisms underlying chromatin regulation, the complexity of which seems to deepen with each passing year, requires a multidisciplinary approach. While many different tools have been brought to bear in this area, here we focus on those that have emerged from the field of chemical biology. We discuss methods that allow the generation of what is now commonly referred to as "designer chromatin," a term that was coined by the late C. David (Dave) Allis. Among Dave's many talents was a remarkable ability to "brand" a nascent area (or concept) such that it was immediately relatable to the broader field. This also had the entirely intentional effect of drawing more people into the area, something that as this brief review attempts to convey has certainly happened when it comes to getting chemists involved in chromatin research., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

It is well established that chromatin is a destination for signal transduction, affecting many DNA-templated processes. Histone proteins in particular are extensively post-translationally modified. We are interested in how the complex repertoire of histone modifications is coordinately regulated to generate meaningful combinations of "marks" at physiologically relevant genomic locations. One important mechanism is "cross-talk" between pre-existing histone post-translational modifications and enzymes that subsequently add or remove modifications on chromatin. Here, we use chemically defined "designer" nucleosomes to investigate novel enzymatic cross-talk relationships between the most abundant histone ubiquitylation sites, H2AK119ub and H2BK120ub, and two important histone methyltransferases, Dot1L and PRC2. Although the presence of H2Bub in nucleosomes greatly stimulated Dot1L methylation of H3K79, we found that H2Aub did not influence Dot1L activity. In contrast, we show that H2Aub inhibited PRC2 methylation of H3K27, but H2Bub did not influence PRC2 activity. Taken together, these results highlight how the position of nucleosome monoubiquitylation affects the specificity and direction of cross-talk with enzymatic activities on chromatin.

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

Activation of the agr system, a major regulator of staphylococcal virulence, is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor histidine protein kinase. There are four known agr specificity groups in Staphylococcus aureus, and we have previously localized the determinant of AIP receptor specificity to the C-terminal half of the AgrC sensor domain. We have now identified the specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the AgrC-I and AgrC-IV sequences revealed a set of five divergent residues in the region of the second extracellular loop of the receptor that could be responsible. Accordingly, we exchanged these residues between AgrC-I and AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of blaZ to the principal agr promoter, P3. Exchange of all five residues caused a complete switch in receptor specificity. Replacement of two of the AgrC-IV residues by the corresponding residues in AgrC-I caused the receptor to be activated by AIP-I nearly as well as the wild type AgrC-I receptor. Replacement of two different AgrC-I residues by the corresponding AgrC-IV residues broadened receptor recognition specificity to include both AIPs. Various types of intermediate activity were observed with other replacement mutations. Preliminary characterization of the AgrC-I-AIP-I interaction suggests that ligand specificity may be sterically determined.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.