AND OBJECTIVEDNA damage-induced chromatin reorganization is emerging to be a key aspect of eukaryotic DNA repair. Much has been learned about the role of histone modifications as landing pads for repair effectors, thereby modulating or directing their recruitment to sites of damage. Recent work suggests that structural changes in the break-surrounding chromatin environment may be equally important in directing the repair process, with implications ranging from repair factor accessibility to break-proximal transcriptional silencing. In light of this complexity, we decided to take an unbiased approach to dissect the role of chromatin in DNA repair, identify its most critical components and explore their functional relevance.RESULTS AND FUTURE DIRECTIONS: To gain insight into the role of chromatin in DSB repair, we performed RNAi-based high-throughput screening of a comprehensive list of 400 Gene Ontology-annotated chromatin modifiers. Repair efficiency was determined using the previously established, U2OS cell-based DR-GFP reporter system, in which DNA repair by homologous recombination (HR) results in restoration of a functional GFP gene and HR efficiency can, thus, be measured as the fraction of GFP+ cells. We found a large number of chromatin-modifying enzymes to be involved in DNA break repair, and in contrast to previous reports suggesting DSB-induced chromatin relaxation, many of these proteins were transcriptional repressors and/or associated with the formation of silent chromatin. Specifically, we identified a DNA repair module consisting of two macro-histone variants and a histone 3 lysine 9 methyltransferase (H3K9MT) with no previously known function in DNA break repair, which promotes a biphasic change in the DSB-proximal chromatin micro-environment. In agreement with DSB-induced chromatin decondensation reported previously, we observe initial depletion of macroH2A along with repressive chromatin marks from the break site, and initiation of the DNA damage response is not affected by either the H3K9MT or the macro-histone variants. However, within minutes after DSB induction, we detect DNA damage signaling-dependent, DSB-proximal enrichment of macro-histone variants as well as the silent chromatin mark H3-dimethyl-K9 and the responsible HMT. We show that macroH2A is responsible for H3K9MT recruitment to breaks. Importantly, macroH2A depletion causes a significant reduction in H3K9 dimethylation and concomitant break-associated chromatin reorganization. Further investigation revealed that both macroH2A and the H3K9MT are essential for efficient DSB repair by homologous recombination, but have little to no effect on non-homologous end-joining, suggesting that repressive chromatin may set the stage for downstream repair factor assembly and, specifically repair pathway choice. Fittingly, we found that loss of macroH2A or the H3K9MT significantly reduced DSB recruitment of the HR mediator and tumor suppressor BRCA1 but not the NHEJ-associated repair protein 53BP1. Our data thus implicate the formation of repressive chromatin as a key initiating step in directing repair outcome, which is expected to have significant consequences for genomic integrity in a chromatin context-dependent manner.To gain mechanistic insight into how repressive chromatin affects BRCA1 recruitment, we are currently investigating the impact of chromatin modifications and H3K9me2 in particular with regard to their ability to selectively interact with downstream repair factors. We are further investigating the consequences of forced changes in chromatin structure on repair factor retention at sites of DNA damage.Building on the identification of repressive chromatin as a key component of DSB repair, we seek to investigate the impact of DNA damage on chromatin integrity beyond the site of damage. To do so, we performed 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 allow us to address the impact of the DNA repair relevant repressive chromatin components on break-proximal structural changes both in cis and in trans to the break. DSB-induced epigenomic remodeling may further provide a molecular basis for DSB-associated transcriptional silencing, which has recently been suggested to protect sites of damage from potentially hazardous RNA polymerase encounters, a process thought to ensure unperturbed DNA repair.IMPLICATIONS: The implications of this work are two-fold: (1) Determining the factors that control the balance between BRCA1 and 53BP1 at DNA breaks is critical for our understanding of DSB repair in general and BRCA1-associated tumorigenesis in particular. Defects in BRCA1 function have been linked to tumor initiation and genomic instability. More recently, it has been suggested that a key role for BRCA1 may be to prohibit aberrant 53BP1 recruitment to sites of DNA damage, which accounts for many of the detrimental genomic effects of BRCA1 loss. The identification of selective, chromatin-based modulators of BRCA1/53BP1 recruitment to sites of damage, therefore, has implications for the targeted manipulation of repair outcome and possibly tumor initiation. (2) Global DNA damage induced reorganization of chromatin may explain epigenetic changes observed with age and/or during malignant transformation. A disturbance of nuclear integrity has been tightly linked to aging, cancer and degenerative diseases and our work sheds light on the molecular drivers of DNA repair associated chromatin reorganization. Interestingly, the histone variants identified in our screen are associated with heterochromatin alterations in senescent cells and have further been shown to protect from metastasis through epigenetic silencing of the tumor promoter CDK8. Together, this work is, thus, expected to reveal the functional interplay between DNA damage, age-related (epi)genomic reorganization and ultimately cancer development and/or progression.
|Shi, Lei; Oberdoerffer, Philipp (2012) Chromatin dynamics in DNA double-strand break repair. Biochim Biophys Acta 1819:811-9|