Acetyl-methyllysine marks chromatin at active transcription start sites

Lysine residues in histones and other proteins can be modified by post-translational modifications (PTMs) that encode regulatory information. Acetylation and methylation of histone lysine residues are especially important for regulating chromatin and gene expression. Pathways involving these PTMs are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here, we report the discovery of cellular lysine residues that are both methylated and acetylated on the same sidechain to form acetyl-methyllysine (Kacme). We show that Kacme is found on histone H4 across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin and is regulated in response to biological signals. Analysis of nascent transcription and promoter-proximal pausing in human cells demonstrates that higher levels of Kacme are associated with higher levels of initiation and transcription. H4Kacme can be installed by enzymatic acetylation of monomethylated lysine peptides and is resistant to deacetylation by some HDACs in vitro. Further, Kacme can be bound by chromatin proteins that recognize modified lysine residues as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a new cellular PTM with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a PTM with fundamental importance to chromatin biology. ChIP-seq with Kacme and H4Kac antibodies in S2 cells +/- heatshock, ChIP-seq with Kacme and H4Kac antibodies in K562 and HEK 293T cells, ChIP-seq with H4Kacme antibodies in HEK 293T cells, and CUT&RUN with Kacme and H4Kac antibodies in K562 cells. TT-TL-seq and STL-seq performed in HEK 293T cells. TT-TL-seq performed in HEK 293T cells treated with TSA (400 nM) or DMSO for 15 hours. All experiments performed in duplicate. Inputs included.