panoramaProteomicsDelphine PfliegerMus Musculushttps://panoramaweb.org/WFyfDU.urldelphine.pflieger@cea.frUniversity Grenoble Alpes, CEA, INSERM, UA13 BGE, CNRS, CEA, UAR 2048, Grenoble, FrancePanoramaPublicDynamic histone post-translational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L- over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and post-meiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25-35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.Both L-lactyl and D-lactyl enantiomers modify histones in mouse testis.Manessier Julie J, Hijazi Hassan H, Vizzini Lisa L, Brugière Sabine S, Courçon Marie M, Masselon Christophe C, de la Iglesia Alberto A, Cocquet Julie J, Pflieger Delphine DfalseBoth L-lactyl and D-lactyl enantiomers modify histones in mouse testisDynamic histone post-translational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the first one may more likely modify histones due to abundant L-lactate produced by glycolysis. Here we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, while several lysines of H3 and H4 exhibit a balanced amount of both enantiomers, residues 18 and 79 from histone H3 (H3K18 and H3K79) appear to be significantly more D-lactylated, while H3K23 is more L-lactylated. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing 0.01 to 0.5%, which contrasts with acetylation whose abundance substantially varies between lysines. Interestingly, lactylation is more abundant than acetylation on the C-terminal half of H3 and H4. These results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, and supporting a site-selective addition of the enantiomers.Tue Jun 23 00:00:00 GMT+01:00 2026PXD0702741009042285509