Chromosomal common fragile sites (CFSs) are specific loci that show non-random gaps, breaks, or rearrangements in metaphase chromosomes when cells are cultured under conditions that inhibit or impair DNA replication, such as in the presence of aphidicolin (APH) (1
). CFSs are highly unstable regions of the genome, and molecular deletions and rearrangements within CFSs have been identified in a variety of human tumors. Moreover, a high frequency of loss of heterozygosity at known CFSs, likely mediated by replication stress, occurs during the pre-malignant and pre-invasive stages of many types of human tumors (2
). These features have led us and others to hypothesize that CFSs play a mechanistic role in the recurring chromosomal rearrangements, deletions and somatic recombination observed in tumor cells.
Although extensive effort has been invested in cloning CFSs and characterizing genetic rearrangements of CFSs in cancer cells, little progress has been made in elucidating the mechanism(s) of fragile site induction. To date, 89 CFSs have been identified in humans, among which 13 have been cloned and characterized at the molecular level (1
). On the basis of sequence analysis of the cloned CFSs, a number of molecular features have been identified, including high A/T content, low gene content, high-flexibility, and high content of Long Interspersed Nuclear Elements (LINE) and Medium Reiterated (MER) repeats. Several lines of evidence suggest that DNA replication is involved in the induction of fragile sites (3
). We and other investigators have shown that CFSs replicate in mid-late S phase, and that exposure to APH further delays the timing of replication (3
). Furthermore, expression of CFSs is induced by conditions that impair replication, such as culturing cells in the presence of the DNA polymerase inhibitor, APH, and expression is enhanced by G2
/M checkpoint inhibitors, such as caffeine. By examining the location of APH-induced breaks, CFSs have been demonstrated to lie at the interface of R- and G-bands, suggesting that CFSs are regions of unusual chromatin conformation, that replicate late in S phase (6
Chromatin conformation influences DNA replication in at least two ways. First, as demonstrated in the Xenopus
egg extract system, binding of the origin recognition complex to DNA is negatively regulated by DNA methylation (7
). Second, replication origin activity, including origin assembly and origin activation timing, can be positively regulated by histone acetylation in a variety of systems (8
). However, whether histone acetylation is required for origin selection or replication-timing specification remains an open question (10
On the basis of these observations, we hypothesized that CFSs represent sequences that are inherently difficult to replicate. Moreover, perturbation of DNA replication within CFSs by APH treatment may be mediated, in part, by specific epigenetic patterns at CFSs, resulting in incomplete DNA replication and, ultimately, leading to the formation of gaps, breaks, or rearrangements in metaphase chromosomes (referred to as ‘CFS expression’ in this report). To test these hypotheses, we examined the chromatin modification pattern within six of the most highly expressed human CFSs in a human lymphoblastoid cell line, using a Chromatin immunoprecipitation (ChIP)-on-chip assay described previously (11
). Here, we show that the majority of these CFSs are characterized by less acetylation, a feature typical of a more condensed chromatin structure, than their flanking non-fragile sequences (NCFSs). By using FRA3B as an example, we demonstrate that chromatin within the fragile site sequences is relatively more compact than that of the flanking NCFSs. Furthermore, we demonstrate that modifying chromatin structure at CFSs by treatment with trichostatin A (TSA) and/or 5-azadeoxycytidine (5-Aza) increases histone acetylation, decreases CpG methylation at CFSs and, notably, reduces fragile site expression. Taken together, our results demonstrate that histone hypoacetylation is a feature of the chromatin at CFSs, and that there is a link between histone acetylation, chromatin compaction, and the level of chromosome breakage observed at CFSs.