Class I Histone Deacetylases (HDAC1–3) are Histone Lysine Delactylases

Abstract Lysine l -lactylation [K( l -la)] is a newly discovered histone mark that can be stimulated under conditions of high glycolysis, such as the Warburg effect. K( l -la) is associated with functions that are different from the widely studied histone acetylation. While K( l -la) can be introduced by the acetyltransferase p300, histone delactylase enzymes remain unknown. Here, we report the systematic evaluation of zinc- and NAD + -dependent HDACs for their ability to cleave ε- N - l -lactyllysine marks. Our screens identified HDACs 1–3 and SIRT1–3 as delactylases in vitro. HDACs 1–3 show robust activity toward not only K( l -la) but also K( d -la) and diverse short-chain acyl modifications. We further confirmed the de- l -lactylase activity of HDACs 1 and 3 in cells. Identification of p300 and HDAC3 as regulatory enzymes suggests that histone lactylation is installed and removed by enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.