Thesis defended on April 30, 2025 to obtain the degree of Doctor of the Université Grenoble Alpes - Speciality: Chemistry Biology.​

Abstract:
Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the HTT gene, leading to an extended polyglutamine (polyQ) stretch (>35) in the huntingtin protein. Although the mutant gene is inherited at birth, motor symptoms typically emerge in midlife. Early hallmarks of HD include transcriptional dysregulation and metabolic disturbances. Histone post-translational modifications (hPTMs) play a critical role in gene regulation and are increasingly recognized as a bridge between metabolism and epigenetics. Notably, acetylation of lysine 27 of histone H3 (H3K27ac) is decreased at enhancers and gene bodies of neuronal identity genes, correlating with their downregulation. This project employed mass spectrometry to explore the role of hPTMs beyond acetylation in HD, focusing on novel acylations like lactylation and crotonylation. 

I established a comprehensive workflow for histone sample preparation and proteomics data analysis. The bioinformatics pipeline was compiled into an R package, histonePTM (https://github.com/HijaziHassan/histonePTM/), enabling automated and reproducible hPTM analysis. 

While only the H3.3 variant exists in humans, mice possess additional mouse-specific H3 variants (H3mm). Two variants, H3mm7 and H3mm13, showed high transcript levels in mouse brain and testes. These variants differ from H3.3 by a single amino acid substitution within the H3K27-R40 stretch, which contains three lysine (K) residues. A rigorous manual validation approach was developed to analyze 40 sequence-PTM combinations. While canonical H3 and H3.3 variants were identified and quantified, only the unmodified form of H3K27R40 was detected for H3mm7 at low abundance, and no peptides were identified for H3mm13. Consequently, these mouse-specific variants were excluded from future experiments. 

Using the established workflow, hPTMs on histones H3 and H4 were screened in the striata of wild-type and R6/1 HD mice. No significant differences in hPTM abundance were observed, consistent with prior findings on localized acetylation dysregulation. Notably, several lysines were confidently identified as lactylated, though their quantification remains challenging. Given the cellular heterogeneity of the striatum, Fluorescence-Activated Nuclei Sorting (FANS) was used to try to isolate nuclei from neurons versus glial cells for histone extraction. Although the initial attempt was unsuccessful due to limited sample amounts, recommendations for methodological improvements were provided for future studies. 

A major challenge in histone proteomics is the co-elution and co-fragmentation of positional isomers, complicating accurate identification and quantification. While standard derivatization of endogenously non-modified lysines using propionic anhydride increases peptide hydrophobicity, it is insufficient for resolving certain isobaric peptides, particularly from the H4 N-terminus. Trimethylacetic anhydride (TMA), a recently introduced derivatization agent, has shown promise in improving peptide separation. In this project, we optimized the TMA derivatization protocol and characterized its impact on hPTM identification and quantification. Finally, we demonstrated its utility in stable isotope tracing experiments, which consists of following the kinetics of incorporation of heavy labeled acetyl into histones after injecting 13C-glucose into mice.

In my PhD, I established a robust protocol for histone derivatization for their proteomic analysis and a bioinformatics tool for hPTM analysis. I implemented them to study hPTMs in the context of HD. Future work will focus on improving lactylation quantification and enhancing cell-type-specific histone analyses to further elucidate epigenetic alterations in HD.​

Key words :
Huntington disease, Quantitative proteomics, Mass spectrometry, Histone lysine acylations