Chromatin modification and DNA replication at common fragile sites, a source of chromosomal instability

Chromosomal fragile sites are specific loci that show non-random gaps, breaks, or rearrangements in metaphase chromosomes when cells are cultured under conditions that inhibit DNA replication. The common fragile sites (CFSs) exhibit several features characteristic of highly unstable or recombigenic regions of the genome, including sites for sister chromatid exchanges, chromosomal deletions and rearrangements, integration of transfected plasmid DNA or exogenous viral sequences, and the initiation of breakage-fusion-bridge cycles, leading to gene amplification. Loss of heterozygosity and amplification of fragile site regions have been observed in a variety of tumors, suggesting a potential role for fragile sites in chromosomal instability and tumorigenesis.; To date, the molecular basis for chromosome fragility at CFSs is unknown. Data from our laboratory and others suggest that fragile site expression is related to DNA replication. It has been known that histone modifications greatly impact DNA replication. Hence, I examined the chromatin modification pattern of several CFSs, and its role in fragile site expression and in origin firing at CFSs.; We developed a microarray platform, and combined it with either chromatin immunoprecipitation to examine histone modifications, or with nascent strand abundance assay to analyze origin firing at CFSs. The data from those studies demonstrated that chromatin at CFSs may be heterochromatic due to hypoacetylation of historic H3 and hypermethylation of CpG dinucleotides. Relaxing the chromatin conformation at CFSs by trichostatin A (increases histone acetylation) and/or 5-Azacytodine (decreases CpG methylation) can reduce fragile site expression, partially through the facilitation of origin firing via the increase of the origin density within CFSs. Our results also suggest that there may be subtypes of CFSs that are induced by somewhat different mechanisms that involve DNA replication.; Based on results from my studies and those of others, I think that the tight chromatin conformation at CFSs sensitizes them to replication stress, and impairs DNA replication, resulting in incomplete replication at CFSs, which could be the source of chromosomal instability and contribute to tumorigenesis.