The repair of DNA damage is critical to protect the integrity of the genome and prevent genotoxic events which can lead to cancer. However, mammalian cells contain a diverse array of functional and structural chromatin domains, which differ in the pattern of histone modifications, chromatin binding proteins and in the density of nucleosome packing. Consequently, remodeling of the chromatin structure at sites of damage is critical for the detection and repair of DNA damage. However, the underlying mechanism driving changes in nucleosome structure at DSBs remains poorly defined. Our preliminary data demonstrates that a histone variant, histone H2A.Z, is rapidly exchanged onto nucleosomes at DNA double-strand breaks (DSBs). The exchange of H2A.Z at DSBs alters nucleosomes dynamics, driving the formation of open, flexible chromatin domains at the break. Further, this H2A.Z exchange promotes specific patterns of histone modification and is critical for controlling resection and processing of the DNA at the break site. The central hypothesis is that H2A.Z exchange drives remodeling of the chromatin at DSBs and controls both histone modification and end processing of the DNA. A library of Zinc Finger Nucleases (ZFNs) will be used to create sequence-specific DSBs in actively transcribed genes and compact, intergenic regions. We will determine how H2A.Z exchange remodels chromatin structure in genes and intergenic regions and identify key differences in the mechanism of repair between these distinct chromatin domains. We will determine how H2A.Z exchange impacts the positioning of nucleosomes at DSBs and determine how nucleosome positioning at DSBs impacts the subsequent modification of histones and the extent of end resection. Further, we will identify key domains on H2A.Z which alter nucleosome dynamics and promote the formation of open chromatin structures at DSBs. In addition, we will determine how the presence of H2A.Z-nucleosomes at DSBs impacts the mechanism and fidelity of DSB repair. By using Zinc Finger Nucleases to create DSBs in genes and intergenic regions, we can determine how DSB repair proceeds in distinct functional domains and unravel the importance of H2A.Z in altering the local chromatin architecture at DSBs. Further, because many tumor cells have both altered chromatin organization and increased levels of histone H2A.Z, this work will provide new insights into how chromatin structure and H2A.Z impacts processes related to carcinogenesis, tumor progression and the sensitivity of tumors to both radiation therapy and chemotherapy.
Chromatin organization and histone modifications are frequently altered in cancer, and may contribute to the etiology and progression of tumors. By exploring how chromatin and histones contribute to DNA repair and genome stability, we can identify new targets, including inhibitors of H2A.Z exchange, as clinical radiation sensitizers. Further, understanding how increased H2A.Z expression in tumor cells impacts sensitivity to radiation and chemotherapy can be used to predict choice of therapy for specific tumor types.
|Ayrapetov, Marina K; Gursoy-Yuzugullu, Ozge; Xu, Chang et al. (2014) DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin. Proc Natl Acad Sci U S A 111:9169-74|