Instability of chromosomal fragile sites is related directly to many cancers. Three types of fragile sites generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA 16B, a distamycin-A-inducible site, are involved in the formation of cancers. The CCG repeating sequence found in the FRA11B site along with four other folate-sensitive sites, has been shown to resist assembly into nucleosomes and the degree of CpG methylation further excludes nucleosome assembly proportionally. These results provide an intriguing model for the nature of fragile sites in which nucleosome exclusion at these sites would create unusual chromatin structure to allow chromosomal rearrangement and viral integration, resulting in fragile site- specific tumorigenesis. Three goals are proposed : (1) The chromatin structure of DNA sequences derived from fragile sites will be investigated in vitro. We showed that the CCG repeating DNA of the FRA11B site excludes nucleosome. We will employ the same techniques for two other different groups of fragile sites, the FRA3B and FRA16B. Further, nucleosome arrays formed over large DNAs of three fragile sites will be characterized by nuclease probing and electron microscopy to determine the stability and physical structure of nucleosomes formed over these sites. (2) The in vivo chromatin structure of these three fragile sites will be examined. Nucleosome position will be mapped over fragile site DNAs in cell lines from fragile site-expressing individuals or an EBV episomal system. The effect of the fragile site-inducing chemicals on the nucleosome pattern will also be examined. (3) The location of fragile sites in higher-order chromatin domains will be explored. Various cell lines will be used to map the location of each fragile site and its adjacent region relative to MARs/SARs. This association will be further investigated for its cell-cycle dependency, the nature of the interaction, and the involvement of topoisomerase II cleavage sites. These experiments will contribute information about the nature of these fragile sites and their role in the formation of cancer, and also address some fundamental biological questions, such as determinants of chromatin structure.
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