Final Report Summary - CHROMATINDAMAGE (Regulation of chromatin compaction in response to DNA damage in mammalian cells)
Project objectives:
Understanding how to recover a fully functional chromatin when its integrity has been challenged by genotoxic stress is a critical issue. In this project, we proposed to pursue two complementary approaches in order to uncover the regulatory mechanisms modulating chromatin organisation in response to DNA damage in mammalian cells. First, we wanted to quantify changes in chromatin compaction in cells exposed to genotoxic stress (task 1). Second, we planned to screen for factors involved in chromatin dynamics, in particular remodelling factors that are recruited to sites of DNA damage (task 2).
Progress on task 1: FLIM-FRET based analysis of chromatin compaction changes in mammalian cells exposed to genotoxic stress
As planned in the research proposal, we have established stable human U2OS and murine NIH3T3 cell lines expressing H2B-EGFP and mcherry-H2B for FLIM-FRET based analysis of chromatin compaction in vivo. We have also tested two different FLIM-FRET systems available at the imaging facility of the Institut Curie. Having finally set up the experiment on a Leica SP5 confocal microscope, we have assessed the impact of genotoxic agents including H2O2 and radiomimetic drugs on chromatin compaction in human and murine cells. However, due to the complexity of the analysis, we could not reach clear conclusions and therefore, we could not yet examine the effect of inhibition or siRNA-mediated depletion of DNA damage response factors as initially planned.
Corrective action:
To get assistance in these technically challenging studies, Dr Sophie Polo has established a collaboration with Dr N. Audugé in M. Coppey's laboratory (Institut Jacques Monod, Paris, France), who is highly experienced in the technique and has recently developed a fast acquisition FLIM-FRET system for high-resolution studies in live cells.
Thus, for the time being, this part of the project has been put on hold, allowing us to take task 2 to completion.
Progress on task 2: Screen for factors involved in chromatin dynamics that are recruited to sites of DNA damage
Due to the availability of reagents in the host laboratory, we have decided to focus our studies on histone chaperones rather than chromatin remodelling factors. Using local UVC irradiation through micropore filters and laser micro-irradiation, we have screened known human histone H3 chaperones for their ability to accumulate at DNA damage sites. Among them, we have found that subunits of the H3.1-specific chaperone complex chromatin assembly factor-1 (CAF-1) and the H3.3-specific chaperone complex histone regulator A (HIRA) are recruited to damage sites.
By combining SNAP-labelling of newly synthesised histones with localised irradiation of cells, we have shown that H3.3 histones are deposited de novo at DNA damage sites by the HIRA complex.
Furthermore, our data demonstrate that HIRA recruitment to damaged chromatin is an early event in the DNA damage response coupled to DNA damage detection by Cullin4-containing E3 ubiquitin ligase complexes. In line with these results, our molecular dissection of the mechanism reveals that HIRA is targeted to damage sites in a ubiquitin-dependent manner.
Most importantly, our findings highlight the functional relevance of this pathway for the cellular response to genotoxic stress in priming chromatin for transcription recovery after repair of DNA damage. Whether HIRA-dependent transcription recovery after damage is mediated by the deposition of H3.3 and the presence of specific histone modifications associated with transcription activation deserves further investigation.
Scientific impact:
Together, our study defines a novel pathway for restoring transcriptionally active chromatin upon DNA damage, linking restoration of chromatin structure and function during the response to genotoxic stress. Our work represents the first study to put forward the role of a histone chaperone in transcription recovery following DNA damage. These findings are presented in a manuscript submitted to Science.
Our new understanding of the contribution of chromatin dynamics to transcriptional control after DNA damage opens up avenues to unravel how seemingly minor defects in resetting chromatin in response to genotoxic stress could influence transcriptional programs and thus have a profound impact on cell fate.
Understanding how to recover a fully functional chromatin when its integrity has been challenged by genotoxic stress is a critical issue. In this project, we proposed to pursue two complementary approaches in order to uncover the regulatory mechanisms modulating chromatin organisation in response to DNA damage in mammalian cells. First, we wanted to quantify changes in chromatin compaction in cells exposed to genotoxic stress (task 1). Second, we planned to screen for factors involved in chromatin dynamics, in particular remodelling factors that are recruited to sites of DNA damage (task 2).
Progress on task 1: FLIM-FRET based analysis of chromatin compaction changes in mammalian cells exposed to genotoxic stress
As planned in the research proposal, we have established stable human U2OS and murine NIH3T3 cell lines expressing H2B-EGFP and mcherry-H2B for FLIM-FRET based analysis of chromatin compaction in vivo. We have also tested two different FLIM-FRET systems available at the imaging facility of the Institut Curie. Having finally set up the experiment on a Leica SP5 confocal microscope, we have assessed the impact of genotoxic agents including H2O2 and radiomimetic drugs on chromatin compaction in human and murine cells. However, due to the complexity of the analysis, we could not reach clear conclusions and therefore, we could not yet examine the effect of inhibition or siRNA-mediated depletion of DNA damage response factors as initially planned.
Corrective action:
To get assistance in these technically challenging studies, Dr Sophie Polo has established a collaboration with Dr N. Audugé in M. Coppey's laboratory (Institut Jacques Monod, Paris, France), who is highly experienced in the technique and has recently developed a fast acquisition FLIM-FRET system for high-resolution studies in live cells.
Thus, for the time being, this part of the project has been put on hold, allowing us to take task 2 to completion.
Progress on task 2: Screen for factors involved in chromatin dynamics that are recruited to sites of DNA damage
Due to the availability of reagents in the host laboratory, we have decided to focus our studies on histone chaperones rather than chromatin remodelling factors. Using local UVC irradiation through micropore filters and laser micro-irradiation, we have screened known human histone H3 chaperones for their ability to accumulate at DNA damage sites. Among them, we have found that subunits of the H3.1-specific chaperone complex chromatin assembly factor-1 (CAF-1) and the H3.3-specific chaperone complex histone regulator A (HIRA) are recruited to damage sites.
By combining SNAP-labelling of newly synthesised histones with localised irradiation of cells, we have shown that H3.3 histones are deposited de novo at DNA damage sites by the HIRA complex.
Furthermore, our data demonstrate that HIRA recruitment to damaged chromatin is an early event in the DNA damage response coupled to DNA damage detection by Cullin4-containing E3 ubiquitin ligase complexes. In line with these results, our molecular dissection of the mechanism reveals that HIRA is targeted to damage sites in a ubiquitin-dependent manner.
Most importantly, our findings highlight the functional relevance of this pathway for the cellular response to genotoxic stress in priming chromatin for transcription recovery after repair of DNA damage. Whether HIRA-dependent transcription recovery after damage is mediated by the deposition of H3.3 and the presence of specific histone modifications associated with transcription activation deserves further investigation.
Scientific impact:
Together, our study defines a novel pathway for restoring transcriptionally active chromatin upon DNA damage, linking restoration of chromatin structure and function during the response to genotoxic stress. Our work represents the first study to put forward the role of a histone chaperone in transcription recovery following DNA damage. These findings are presented in a manuscript submitted to Science.
Our new understanding of the contribution of chromatin dynamics to transcriptional control after DNA damage opens up avenues to unravel how seemingly minor defects in resetting chromatin in response to genotoxic stress could influence transcriptional programs and thus have a profound impact on cell fate.