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    Chromatin and the Regulation of DNA Double-Strand Breaks

    Scientific Abstract:
    Double-strand breaks (DSBs) are a threat to genome integrity, thereby contributing to the development of cancer. Women inheriting mutations affecting the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to early onset breast cancer; BRCA1 has been implicated in the repair of DSBs by homologous recombination, while BRCA2 has been proposed to regulate DNA recombination reactions mediated by the DNA strand-exchange factor RAD51. Organisms spend considerable effort to avoid and repair DSBs. However, some important biological processes, including meiosis, require the deliberate formation of DSBs. Successful meiosis requires programmed DSBs to be formed and repaired using much of the same recombinational machinery utilized to repair DSBs in somatic cells. Given the predictable nature of meiotic breaks, meiosis provides an excellent opportunity to study and understand break formation and repair. The germline of C. elegans is an especially valuable model system. As cells progress through meiosis they move through the germline, thereby allowing the temporal order of events to be elucidated. Additionally, homologs of BRCA1, BRCA2 and RAD51 are encoded by the C. elegans genome, indicating a high degree of conservation of break repair machinery. Recently, links have been elucidated between DSBs and chromatin; the C. elegans protein HIM-17 is required for proper competence for DSB formation as well as chromatin methylation. Additionally, the partial phenotypes of him-17 intermediate alleles are altered by loss of LIN-35/Rb, the only C. elegans homolog of the Retinoblastoma (Rb) protein. As Rb proteins are known to function in chromatin modulation, it was hypothesized that chromatin is a critical component of controlling and regulating DSBs. Our specific aims are to investigate the role of the chromatin-associated protein HIM-17 in promoting DSB formation and histone methylation, the relationship of methylation to DSB competence and the role of LIN-35/Rb and Rb-related pathways in modulating DSB competence. We will identify factors that functionally interact with HIM-17 and LIN-35 using biochemistry and genetic screens and with molecular tools, microscopy, and DNA microarrays determine how these proteins impact DSB formation and chromatin methylation. The knowledge gained from these studies will help us to elucidate the mechanisms of DSB initiation and the roles played by proteins such as BRCA1 and BRCA2. This understanding will increase our knowledge of how DSBs and loss of DSB repair proteins contribute to tumorigenesis.

    Lay Abstract:
    DNA double-strand breaks (DSBs) are a threat to the human genome and health since DSBs contribute to the development of cancer. Women who inherit defects in a gene that functions in DSB repair, including BRCA1 and BRCA2, are predisposed to develop early onset breast cancer. Humans and other organisms spend considerable efforts to avoid and repair breaks that form spontaneously. However, some biological processes, including meiosis, the process by which sperm and eggs are produced, require the formation and repair of programmed DSBs. The programmed breaks that form during meiosis utilize the same repair machinery as DSBs that contribute to cancers. Therefore, the predictable nature of meiosis makes it a valuable model in which to study DNA break formation and repair. The meiosis of the worm C. elegans is particularly amenable to scientific study as it allows biologists to utilize powerful techniques, including high-resolution microscopy, genome-wide DNA microarrays, biochemistry and genetics. Additionally, the worm genome encodes BRCA1- and BRCA2-like proteins. Recently, links have been elucidated between DSBs and the chromosomal protein structure, called chromatin; the C. elegans protein HIM-17 is required for the proper formation of meiotic DSBs, as well as for the proper modification of chromatin. Without HIM-17, chromatin does not become methylated, which alters the nature of the chromatin structure. Additionally, partially functional versions of the HIM-17 protein have different effects on meiosis when the LIN-35/Rb protein is also lost; LIN-35/Rb is the C. elegans equivalent of the Retinoblastoma (Rb) protein. As Rb proteins have also been shown to alter chromatin structure, it was hypothesized that chromatin is a critical component of controlling and regulating DSBs. Our specific aims are to investigate the role of HIM-17 in promoting DSB formation, the relationship of chromatin methylation to DSB formation and the role of LIN-35/Rb and Rb-related pathways in DSB formation. We will identify proteins that function with HIM-17 and LIN-35 using biochemistry and genetic screens and with microscopy, molecular tools and genomics tools we will determine how these proteins impact DSB formation and chromatin methylation. These studies will improve our knowledge of how DSBs occur and therefore increase our understanding of how DSBs and the loss of DSB repair proteins, including BRCA1 and BRCA2, contribute to tumorigenesis and cancer.