Periodic Reporting for period 4 - REMIND (Epigenome maintenance in response to DNA damage)
Berichtszeitraum: 2023-09-01 bis 2025-02-28
Cell viability and homeostasis rely on the stable maintenance of the epigenetic information conveyed by chromatin, which associates DNA and histone proteins in the cell nucleus and governs gene expression programs. Yet, epigenome integrity is challenged during all DNA transactions, including DNA damage
repair. While much effort has been devoted to characterizing chromatin alterations in response to DNA damage and how they contribute to the repair response, our knowledge of this fundamental process is largely incomplete, and whether and how epigenetic features are re-established following a genotoxic stress challenge is still unexplored. Thus, a comprehensive framework of the mechanisms underlying the maintenance of epigenome integrity in response to DNA damage is lacking. The present project fills this important gap by profiling the epigenome of repair patches following UV damage in human cells and by characterizing the molecular players contributing to chromatin restoration/plasticity. We took an integrated approach that tackles this question at different levels of chromatin organization, from histone and DNA modifications up to higher-order chromatin folding.
We have developed powerful novel methodologies, combining cutting-edge imaging, proteomics and epigenomic technologies to elucidate mechanisms for (1) histone modification re-establishment and maintenance and (2) DNA methylation inheritance at repair sites. We have also investigated how repair-associated changes in DNA and histone modifications reflect at the level of (3) higher-order chromatin organization, and dissected (4) functional crosstalks between the epigenetic changes that arise in damaged chromatin.
repair. While much effort has been devoted to characterizing chromatin alterations in response to DNA damage and how they contribute to the repair response, our knowledge of this fundamental process is largely incomplete, and whether and how epigenetic features are re-established following a genotoxic stress challenge is still unexplored. Thus, a comprehensive framework of the mechanisms underlying the maintenance of epigenome integrity in response to DNA damage is lacking. The present project fills this important gap by profiling the epigenome of repair patches following UV damage in human cells and by characterizing the molecular players contributing to chromatin restoration/plasticity. We took an integrated approach that tackles this question at different levels of chromatin organization, from histone and DNA modifications up to higher-order chromatin folding.
We have developed powerful novel methodologies, combining cutting-edge imaging, proteomics and epigenomic technologies to elucidate mechanisms for (1) histone modification re-establishment and maintenance and (2) DNA methylation inheritance at repair sites. We have also investigated how repair-associated changes in DNA and histone modifications reflect at the level of (3) higher-order chromatin organization, and dissected (4) functional crosstalks between the epigenetic changes that arise in damaged chromatin.
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).
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).
Setting up the capture and proteomic analysis of repair patches was a technical breakthrough and revealed new functions for histone chaperones in coordinating old and new histone dynamics at repair sites, thus expanding the histone chaperone network involved in repair-coupled chromatin restoration. This new methodology will be instrumental for further characterization of epigenome alterations during the repair process.
Our investigations of mitotic histone modifications at UV damage sites unveiled an unexpected contribution of damaged chromatin marks to controlling DNA damage segregation through mitosis. The discovery that chromatin alterations impinge on genome stability not only by regulating DNA repair but also by controlling DNA damage segregation in mitosis broadens the scope of genome maintenance mechanisms.
By dissecting the regulatory mechanisms for transcription recovery following UV damage, we discovered a non-canonical function of the histone chaperone HIRA, that operates independently of H3.3 histone deposition.
Together, these discoveries constitute major steps forward in our mechanistic understanding of genome and epigenome maintenance.
Our investigations of mitotic histone modifications at UV damage sites unveiled an unexpected contribution of damaged chromatin marks to controlling DNA damage segregation through mitosis. The discovery that chromatin alterations impinge on genome stability not only by regulating DNA repair but also by controlling DNA damage segregation in mitosis broadens the scope of genome maintenance mechanisms.
By dissecting the regulatory mechanisms for transcription recovery following UV damage, we discovered a non-canonical function of the histone chaperone HIRA, that operates independently of H3.3 histone deposition.
Together, these discoveries constitute major steps forward in our mechanistic understanding of genome and epigenome maintenance.