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The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here, we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones, followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nano-LC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression 1 . These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues 2-7 . One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation 3,8 . As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen 9-11 , we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Protein acetylation plays a critical role in biological processes by regulating the functions and properties of proteins. Thus, the study of protein acetylation dynamics is critical for understanding of how this modification influences protein stability, localization, and function. Here we performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry (MS) based proteomics through utilization of 13 C-glucose or D 3 -acetate, which are metabolized into acetyl-coA, labeling acetyl groups through subsequent incorporation into proteins. Samples were collected at eight time points to monitor rates and trends of heavy acetyl incorporation. Through this platform, we characterized around 1,000 sites with significantly increasing acetylation trends, which we clustered based on their rates of acetylation. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. Among others, we identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop, which could explain self-acetylation as an important feedback mechanism to regulate acetyltransferases. Overall, our studies highlight the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation.