AND OBJECTIVE: We found previously that the SIRT1 deacetylase provides a critical link between DNA repair, aging and genomic instability. Specifically, we found that DNA damage can cause the redistribution of SIRT1 on chromatin, resulting in its recruitment to DNA breaks and concomitant deregulation of otherwise SIRT1 regulated genomic loci. We were further able to demonstrate the direct involvement of SIRT1 in DNA break repair and propose SIRT1 redistribution as a molecular mechanism linking DNA damage to large-scale epigenetic deregulation, even of undamaged loci. This work further led to the more general hypothesis that an evolutionarily conserved, DNA damage-induced redistribution of chromatin modifiers (or 'RCM response') may underlie the alterations in gene expression and genomic stability that characterize eukaryotic aging. Elucidating the impact of the DNA damage response on chromatin organization is, thus, vital to improve our understanding of often detrimental, age-related (epi-)genomic changes and the associated organismal decline, and may eventually help to promote genomic stability and maintain a more youthful epigenetic profile. RESULTS AND FUTURE DIRECTIONS: To test if DNA damage can indeed result in a genome-wide remodeling of chromatin beyond the redistribution of SIRT1, we used an RNA interference based screening approach to identify additional DNA-repair linked chromatin modifiers from a comprehensive list of Gene Ontology annotated chromatin remodelers. Our preliminary results suggested that a large number of chromatin-modifying enzymes are involved in DNA break repair. Interestingly, many of these proteins are transcriptional repressors and associated with the formation of silent chromatin. While it has been postulated that chromatin surrounding DNA breaks is made accessible for efficient repair factor access, our data suggest large-scale chromatin compaction at sites of DNA damage, possibly a mechanism to help confine the site of damage and to aid the repair process. We are currently investigating the hypothesis that DNA breaks can induce break-associated chromatin compaction using a range of molecular biology and cell-based imaging techniques. Initial fluorescence-based in situ hybridization (FISH) analyses support our model and provide the basis for an in depth analysis of the role of DNA-repair related chromatin modifiers during this process. We have further established several independent cell-based systems to induce defined DNA breaks in mammalian cells, thereby enabling us to investigate direct association of said chromatin modifiers with DNA breaks. Using these systems, we validated the recruitment of two repressive histone variants that were amongst the top 5 hits in our screen, to sites of DSBs. This recruitment is downstream of early DNA damage signaling through H2AX phosphorylation and correlates with the acquisition of the repressive histone H3 K9 methyl-mark at the DSB site. Importantly, our screen identified an H3K9 methyltransferase with no known function in DNA break repair. Using RNAi based knock-down, we are currently investigating if this enzyme is responsible for H3K9 methylation of DSB proximal histones. We are further interested in understanding the role of both the histone variants and the H3K9 histone methyltransferase with regard to chromatin reorganization at DSBs and how these proteins may affect the proposed chromatin compaction. Together, these data point to a novel DNA repair module that may critically modulate the chromatin microenvironment surrounding a DSB. To study the latter we are currently performing genome wide circular chromatin configuration capture assays (4C), characterizing the three-dimensional chromatin microenvironment around at the DSB site before and after break induction. In combination with FISH validation, this approach will further allow us to address the impact of the DNA repair relevant repressive chromatin components on break-proximal structural changes. Consistent with far-reaching DSB-induced chromatin remodeling, we found that genes within up to 4 Mb from the break site can be transiently repressed upon induction of a single DSB. This finding implies that previously reported break-proximal interference with RNA polymerase II transcription can occur over megabase regions of the genome, highlighting the far-reaching impact of DSB-mediated chromatin remodeling. IMPLICATIONS: The implications of chromatin modifiers as critical mediators of DNA repair are two-fold: (1) Only a small number of chromatin modifiers has been conclusively linked to the DNA repair process. A comprehensive analysis will significantly improve our understanding of mammalian DNA repair and its impact on genomic instability and cancer. (2) DNA damage induced reorganization of chromatin may explain epigenetic changes observed with age and/or during malignant transformation. A global disturbance of nuclear integrity has been tightly linked to aging, cancer and degenerative diseases and this project is expected to shed light on the contribution of DNA repair associated chromatin reorganization to these processes. Interestingly, the histone variants identified in our screen are associated with heterochromatin alterations in senescent cells and have further been shown to protect from skin metastasis through epigenetic silencing of the tumor promoter CDK8. Together, these observations corroborate the link between DNA damage, age-related (epi)genomic reorganization and ultimatley cancer progression. Unlike mutations in DNA, epigenetic changes are, at least in theory, reversible and may prove to serve as a target for directed therapeutic intervention, aiming at the restoration of tissue-appropriate gene regulation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011282-02
Application #
8349443
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2011
Total Cost
$458,761
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Shi, Lei; Oberdoerffer, Philipp (2012) Chromatin dynamics in DNA double-strand break repair. Biochim Biophys Acta 1819:811-9