We have developed new proteomic approaches for tracking changes in histone post-translational modifications (PTMs) at sites of UV damage repair in human cells (Aim 1). We have captured repair sites marked by EdU (Ethynyl deoxyUridine) and analyzed the associated proteins by mass spectrometry. Thus, we have uncovered a new function for DNAJC9 and MCM2 histone chaperones in coordinating old and new histone dynamics at repair sites (Plessier et al., BioRxiv 2024). We have also identified a number of histone modifiers and readers of histone PTMs and are now moving to their characterization. In addition, we have developed a complementary proteomics method, which relies on proximity biotinylation at repair sites, allowing us to monitor changes in histone PTMs associated with early repair steps. In parallel to these unbiased methods, we took a candidate approach using immunofluorescence to detect histone modifications of interest, mitotic phosphorylations in particular, at sites of local UV damage. Thus, we have uncovered local alterations in mitotic phosphorylation on histone H3 and deciphered a chromatin-marking pathway that controls the segregation of UV damage in mitosis (Ferrand*, Dabin* et al., Nat Commun, 2025).
We have also run studies to analyze DNA methylation maintenance at UV damage sites (Aim 2). Through imaging and proteomic approaches, we have identified key molecular players involved in DNA methylation dynamics at UV sites and their connection with UV damage repair factors (Aims 2&4). We have employed Nanopore sequencing to map both DNA methylation and the position of repair patches and we have investigated how DNA methylation maintenance impacts cell survival, genome integrity and transcription regulation post UV (Mori et al., manuscript in preparation).
Regarding the analysis of higher-order chromatin organization (Aim 3), we have set up an innovative approach to target UV damage to heterochromatin domains in mouse cells (Fortuny et al., Nature Commun 2021; Chansard*, Pobega* et al., Front Cell Dev Biol 2022). By imaging the response to UV damage in pericentric heterochromatin domains in real time, we have uncovered an interplay between histone modifying enzymes and histone variant deposition in safeguarding higher-order chromatin integrity (Aims 3&4). We have also combined cell imaging and genome-wide analyses of nascent transcripts to dissect the interplay between transcription and chromatin restoration after DNA damage (Aim 4) by focusing on the human histone chaperone complex HIRA (Bouvier et al., Nat Commun, 2021).
