Thesis defended on October 28, 2025 to obtain the degree of Doctor of the Université Grenoble Alpes
Speciality: Chemistry Biology
Abstract
Gene expression is regulated by the dynamics of histone post-translational modifications. Over the past 15 years, a wide range of new structures have been discovered, collectively known as acylations. These structures finely regulate transcription through the metabolic state of the cell. However, their roles in the regulation of gene expression remain poorly characterized. My thesis project focused on this question/issue , in the context of murine spermatogenesis.
The first part of my project is the exploration of the following histone lysines modifications, crotonyl, butyryl and beta-hydroxybutyryl, by mass spectrometry and immunoprecipitation coupled with mass spectrometry (IP-MS) in an attempt to validate their presence in histones extracted from mouse testes. Experiments with these marks, mainly on lysine 27 of histone H3 (H3K27), as well as on other residues, did not lead to their identification. In addition to this search for acylations, I contributed to a study on the existence of mouse-specific variants. Particularly, I interpreted middle-down data which enabled to confidently validate the presence of the canonical histone H3.1 and the H3.3 and Testis-Specific H3 variants in mouse testes, but not the presence of mouse-specific variants, H3mm7 and H3mm13.
The second part of this project lies in the identification and characterization of lactylation, recently discovered. This work represents the most innovative and successful aspect of my thesis project. Indeed, I have established a mapping of lactylation and acetylation on numerous lysines of histone H3 and H4, by exploratory proteomic analysis (DDA). The near-impossibility of quantifying lactylation by DDA, due to oxidation events, prompted us to develop a targeted proteomic analysis method, using synthetic peptides labeled with stable isotopes and modified with the L-lactyl and D-lactyl enantiomers. The results showed that peptides carrying L-lactyl or D-lactyl were in similar abundance on most H3 and H4 lysines. The stoichiometry of lactylation was finally estimated: this modification is very low and stable (about 0.1%) on all lysines studied.
Finally, I focused on the characterization of an antibody directed against the H3K18(L-lactyl) mark. Using Surface Plasmon Resonance (SPR) and IP-MS, I showed that this antibody is not perfectly specific to its target. It also has a strong affinity for the lactyl mark on lysine H3K23. In addition, it recognizes forms close to lactyl such as hydroxy-iso-butyrylation, and to a lesser extent acetylation, which is relatively abundant on this residue. The mass spectrometry results showed a very strong enrichment of the lactylated marks on H3K18, H3K23, the acetylated mark on H3K18 and the di-acetylated form on H3K18/K23, both on H3.1 and on the TSH3 variant. Interestingly, this antibody shows similar recognition of the two enantiomers L-lactyl and D-lactyl.
My thesis work shows the complexity of the study of new acylations on histones. My project on lactylation highlights the presence of the two enantiomers L-lactyl and D-lactyl on a large number of lysines of histones H3 and H4, on mouse testis. The identification, validation and quantification of L- and D-lactylated histone peptides are the result of meticulous work. The study of lactylation has been expanding rapidly since its discovery in 2019, but the characterization of the two marks L-lactyl and D-lactyl is simultaneously less explored. My work is therefore part of an original and innovative approach.