In the nuclei of all eukaryotic cells, highly conserved histone proteins (H2A, H2B, H3, and H4) function as a scaffolding core to package genomic DNA into repeating nucleosomal units that are further folded into higher-order chromatin fibers. While genomic DNA is the ultimate template of our heredity, chemical modifications of DNA and histones define epigenome, which regulates a variety of chromatin-templated processes, including transcription, DNA replication, and DNA damage repair. As the available antibodies to histone PTMs and mass spectrometrybased proteomics are expanding, the number and variety of histone PTMs have turned out to be much greater than previously expected. Therefore, the combination of histone PTMs can create myriad "histone codes", each of which may have a distinct impact on chromatin structures and gene expression. To understand how histone codes regulate biological processes, it is also important to examine the specific consequences of each histone PTM. Toward this objective, several methods to regioselectively introduce histone PTMs entirely through artificial means, i.e., independent of histone-modifying enzymes, have emerged. They include genetic code expansion, histone semisynthesis using protein trans-splicing, posttranslational chemical mutagenesis, and chemical catalyst-promoted incorporation of histone PTMs. Since the former three methods need genetic manipulation, modifying endogenous histones is challenging. In contrast, Lys-acylating chemical catalysts do not require genetic manipulation, and thus have unique utilities in epigenetic studies as well as therapeutic purposes. We previously developed a PEG-LANA-DSSMe catalyst, conjugating a prodrug disulfide form (DSSMe) of a thiol-tethered 4-dimethylaminopyridine (DSH) catalyst moiety with the N-terminus of a histone-binding LANA (latency associated nuclear antigen, residue 5-15) peptide and a poly(ethylene glycol) (PEG) moiety (Figure ). DSSMe generated DSH under reducing conditions in cells or in the presence of tris(2carboxyethyl)phosphine (TCEP) in test tubes. DSH activated thioesters, such as acyl-CoAs, through the dynamic thiol-thioester exchange and intramolecular acyl group transfer, forming a reactive acyl pyridinium intermediate (Figure ). The catalyst bound to the acidic patch of nucleosomes via LANA, and efficiently promoted H2BK120 (the 120th Lys of H2B)-selective acylation in living cells in the presence of cell permeable acetyl thioesters (NAC-Ac) as acetyl donors. Quantification of acetylation yield of recombinant nucleosomes acetylated by PEG-LANA-DSSMe showed that H2BK120 was acetylated in ~80%, and a proximate H2BK116 residue was also acetylated in ~10% yield. Other Lys residues in a nucleosome were rarely acetylated (<5% yield).