Session: Session 5

What do antibodies directed against rare modifications of histones actually bind?

Marie COURCON¹, Julie MANESSIER¹, Hassan HIZAJI¹, Sabine BRUGIERE¹, Alberto DE LA IGLESIA³, Julie COQUET³, Jean-Baptiste REISER², Delphine PFLIEGER¹

¹Exploring the Dynamics of Proteomes (EDyP), University Grenoble Alpes, CEA, Inserm, IRIG-BGE UA13, CNRS/CEA FR2048, GRENOBLE, France
²Institut de Biologie Structurale, UMR 5075, Univ. Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France , GRENOBLE, France
³Group "Epigenetics & Spermatogenesis", Lab “From Gametes to Birth: Genomics, Epigenetics and Physiopathology of Reproduction”, Institut Cochin -Inserm U1016-CNRS UMR8104-Université Paris Cité, Paris, France

In the nucleus of eukaryotes, DNA wraps around octamers of histones to form a structure called chromatin. Post-translational modifications (PTMs) of histones play a crucial role in regulating gene expression. Since its discovery in 2019, histone lysine lactylation has been studied in many contexts for its role in the metabolic regulation of gene expression. More precisely, the genome-wide distribution of a specific lactylated histone lysine is established using antibodies directed against this mark. Then, this large-scale information is integrated with gene expression data collected by RNA-seq. However, whether these antibodies are really specific for the desired histone marks remains a scarsely addressed question.

We performed immuno-precipitation (IP) of histones with an antibody raised against lactylated lysine 18 from histone H3 (H3K18lac). The enriched histone material was digested with trypsin and analyzed by LC-MS/MS to quantify the extent of enrichment of peptides containing the targeted PTM compared to their abundance in the original histone sample. Besides, we also assessed the specificity of the antibody by testing its affinity for variably modified peptides centered on H3K18 (e.g. non-modified, acetylated, etc., peptides) using Surface Plasmon Resonance (SPR).

Both IP followed by LC-MS analysis and SPR experiments provide coherent results. They show that the antibody raised against H3K18lac exhibits very strong affinity for histone H3 bearing lactylation on Lys18 or Lys23. In addition, the antibody binds significantly the non-modified and acetylated peptide forms.

Given the relative stoichiometries of acetylated versus lactylated H3K18 (ratio >100), it is unlikely that genome-wide distributions established for lactylated histone residues can be fully trusted. We recommend establishing in parallel the distributions of the lactylated and acetylated forms of a given histone lysine of interest, to search for specific signals detected for the lactyl form.