Periodic Reporting for period 3 - REMIND (Epigenome maintenance in response to DNA damage)
Berichtszeitraum: 2022-03-01 bis 2023-08-31
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 aims to fill this important gap by profiling the epigenome of repair patches following
UVC damage in human cells and by characterizing the molecular players contributing to chromatin
restoration/plasticity. We propose an integrated approach that tackles this question at different levels of
chromatin organization, from histone and DNA modifications up to higher-order chromatin folding.
Building on our unique expertise and through the development of powerful novel methodologies, combining
cutting-edge imaging, proteomics and epigenomic technologies, we will elucidate mechanisms for (1)
histone modification re-establishment and maintenance and (2) DNA methylation inheritance at repair sites.
We will also investigate how repair-associated changes in DNA and histone modifications reflect at the level
of (3) higher-order chromatin organization in the tridimensional nuclear space, and dissect (4) functional
crosstalks between the epigenetic changes that arise in damaged chromatin.
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 aims to fill this important gap by profiling the epigenome of repair patches following
UVC damage in human cells and by characterizing the molecular players contributing to chromatin
restoration/plasticity. We propose an integrated approach that tackles this question at different levels of
chromatin organization, from histone and DNA modifications up to higher-order chromatin folding.
Building on our unique expertise and through the development of powerful novel methodologies, combining
cutting-edge imaging, proteomics and epigenomic technologies, we will elucidate mechanisms for (1)
histone modification re-establishment and maintenance and (2) DNA methylation inheritance at repair sites.
We will also investigate how repair-associated changes in DNA and histone modifications reflect at the level
of (3) higher-order chromatin organization in the tridimensional nuclear space, and dissect (4) functional
crosstalks between the epigenetic changes that arise in damaged chromatin.
Since the beginning of the project, we have made significant progress on all Aims of the proposed work and implemented minor deviations from the original work plan.
We have developed proteomic approaches for tracking changes in histone post-translational modifications (PTMs) at sites of UV damage repair in human cells (Aim 1). We have improved the originally proposed protocol and we have validated this method by western blot at different time points after UV irradiation. We obtained mass spectrometry results at 1h and 24h post UV damage and we validated them by orthogonal approaches including immunofluorescence after local UV irradiation. Thus, we uncovered a novel histone chaperone involved in chromatin repair, DNAJC9, which contributes both to new histone deposition and old histone recycling (manuscript in preparation). We have also identified a number of histone modifiers and readers of histone PTMs and are now moving to their characterization. In parallel, we are developing 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 (manuscript under review).
We have also run studies to analyze DNA methylation maintenance at UV damage sites (Aim 2). 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 decided to employ Nanopore sequencing to map both DNA methylation and the position of repair patches and are now setting up the methodology.
Regarding the analysis of higher-order chromatin organization (Aim 3), we revised our priorities regarding the originally proposed chromatin conformation capture approach because of recent works on a similar topic. Instead, we set up an innovative approach to target UV damage to heterochromatin domains in mouse cells. This imaging-based method revealed core principles of higher-order chromatin maintenance following UV damage (Fortuny et al., Nature Commun 2021), including heterochromatin folding and the interplay with the maintenance of heterochromatin-specific histone marks (Aims 3&4). We are now investigating the response to DNA damage in another type of heterochromatin domain, the inactive X chromosome in female mammalian cells.
We have developed proteomic approaches for tracking changes in histone post-translational modifications (PTMs) at sites of UV damage repair in human cells (Aim 1). We have improved the originally proposed protocol and we have validated this method by western blot at different time points after UV irradiation. We obtained mass spectrometry results at 1h and 24h post UV damage and we validated them by orthogonal approaches including immunofluorescence after local UV irradiation. Thus, we uncovered a novel histone chaperone involved in chromatin repair, DNAJC9, which contributes both to new histone deposition and old histone recycling (manuscript in preparation). We have also identified a number of histone modifiers and readers of histone PTMs and are now moving to their characterization. In parallel, we are developing 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 (manuscript under review).
We have also run studies to analyze DNA methylation maintenance at UV damage sites (Aim 2). 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 decided to employ Nanopore sequencing to map both DNA methylation and the position of repair patches and are now setting up the methodology.
Regarding the analysis of higher-order chromatin organization (Aim 3), we revised our priorities regarding the originally proposed chromatin conformation capture approach because of recent works on a similar topic. Instead, we set up an innovative approach to target UV damage to heterochromatin domains in mouse cells. This imaging-based method revealed core principles of higher-order chromatin maintenance following UV damage (Fortuny et al., Nature Commun 2021), including heterochromatin folding and the interplay with the maintenance of heterochromatin-specific histone marks (Aims 3&4). We are now investigating the response to DNA damage in another type of heterochromatin domain, the inactive X chromosome in female mammalian cells.
Future developments include an in-depth analysis of the proteome at UV damage repair patches by mass spectrometry in order to to profile alterations in histone modifications. We will also analyze the dependency of histone mark changes on UV damage repair and examine the temporal and functional coordination between histone PTM changes and transcription regulation at repair sites.
In parallel, we will develop imaging and sequencing approaches to study the maintenance of DNA methylation at UV damage sites. This should reveal the molecular pathways involved in DNA methylation alteration/re-establishment coupled to UV damage repair. We will also investigate potential cross-talks between DNA methylation factors and damage-associated histone dynamics and the functional relevance of DNA methylation maintenance post UV damage for transcriptional silencing and genome stability.
Regarding higher-order chromatin structures, we will dissect the impact of heterochromatin organization on DNA repair fidelity and the molecular mechanisms that maintain heterochromatin marks following DNA damage.
In parallel, we will develop imaging and sequencing approaches to study the maintenance of DNA methylation at UV damage sites. This should reveal the molecular pathways involved in DNA methylation alteration/re-establishment coupled to UV damage repair. We will also investigate potential cross-talks between DNA methylation factors and damage-associated histone dynamics and the functional relevance of DNA methylation maintenance post UV damage for transcriptional silencing and genome stability.
Regarding higher-order chromatin structures, we will dissect the impact of heterochromatin organization on DNA repair fidelity and the molecular mechanisms that maintain heterochromatin marks following DNA damage.