Two breakpoint clusters at fragile site FRA3B form phased nucleosomes.
ABSTRACT: Fragile sites are gaps and breaks in metaphase chromosomes generated by specific culture conditions. Fragile site FRA3B is the most unstable site and is directly involved in the breakpoints of deletion and translocation in a wide spectrum of cancers. To learn about the general characteristics of common fragile sites, we investigated the chromatin structure of the FRA3B site. Because FRA3B spans several hundred kilobases, we focused our study on two breakpoint clusters found in FRA3B. Using various nucleases, we demonstrated that these two regions contain phased nucleosomes, regardless of treatment with aphidicolin. Because these regions are located in intron 4 of the FHIT gene, it is very interesting to observe phased nucleosomes over these regions, which are several hundred kilobases downstream from the promoter. Further, by using nucleosome assembly assays, we demonstrate that these two regions do not contain strong nucleosome positioning elements. These results suggest that other factors appear to cooperate with the DNA sequence of these regions to impart nucleosome phasing. This study provides the first information on the chromatin structure of breakpoint regions in a common fragile site. The observation of phased nucleosomes over these breakpoint regions could offer a foundation to understand the expression of fragile sites.
Project description:We have sequenced 870 kilobases of the FHIT/FRA3B locus, from FHIT intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6. 2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between FHIT exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are (i) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; (ii) that 9 of the 10 FHIT allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and (iii) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.
Project description:Common fragile sites (CFSs) are loci that preferentially exhibit metaphase chromosome gaps and breaks after partial inhibition of DNA synthesis. The fragile site FRA3B, which lies within the FHIT tumor-suppressor gene, is a site of frequent heterozygous and homozygous deletions in many cancer cells and precancerous lesions. The great majority of FHIT and other CFS-associated gene rearrangements in tumors are submicroscopic, intralocus deletions of hundreds of kilobases that often result in inactivation of associated genes. Although CFS instability leads to chromosome gaps and breaks and translocations, there has been no direct evidence showing that CFS instability or replication stress can generate large submicroscopic deletions of the type seen in cancer cells. Here, we have produced FHIT/FRA3B deletions closely resembling those in tumors by exposing human-mouse chromosome 3 somatic hybrid cells to aphidicolin-mediated replication stress. Clonal cell populations were analyzed for deletions by using PCR, array comparative genomic hybridization (aCGH), and FISH. Thirteen percent to 23% of clones exhibited submicroscopic FHIT deletions spanning approximately 200-600 kb within FRA3B. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus, with a 2- to 12-fold reduction in metaphase gaps and breaks compared with controls. Sequence analysis showed no regions of homology at breakpoints and suggests involvement of NHEJ in generating the deletions. Our results demonstrate that replication stress induces a remarkably high frequency of tumor-like microdeletions that reduce fragility at a CFS in cultured cells and suggests that similar conditions during tumor formation lead to intralocus deletion and inactivation of genes at CFSs and perhaps elsewhere in the genome.
Project description:Common fragile sites are regions that show elevated susceptibility to DNA damage, leading to alterations that can contribute to cancer development. FRA3B, located at chromosome region 3p14.2, is the most frequently expressed human common fragile site, and allelic losses at FRA3B have been observed in many types of cancer. The FHIT gene, encompassing the FRA3B region, is a tumor-suppressor gene. To identify the features of FHIT/FRA3B that might contribute to fragility, sequences of the human FHIT and the flanking PTPRG gene were compared with those of murine Fhit and Ptprg. Human and mouse orthologous genes, FHIT and Fhit, are more highly conserved through evolution than PTPRG/Ptprg and yet contain more sequence elements that are exquisitely sensitive to genomic rearrangements, such as high-flexibility regions and long interspersed nuclear element 1s, suggesting that common fragile sites serve a function. The conserved AT-rich high-flexibility regions are the most characteristic of common fragile sites.
Project description:The instability of common fragile sites (CFSs) contributes to the development of a variety of cancers. The ATR-dependent DNA damage checkpoint pathway has been implicated in maintaining CFS stability, but the mechanism is incompletely understood. The goal of our study was to elucidate the action of the ATR protein in the CFS-specific ATR-dependent checkpoint response. Using a chromatin immunoprecipitation assay, we demonstrated that ATR protein preferentially binds (directly or through complexes) to fragile site FRA3B as compared to non-fragile site regions, under conditions of mild replication stress. Interestingly, the amount of ATR protein that bound to three regions of FRA3B peaked at 0.4microM aphidicolin (APH) treatment and decreased again at higher concentrations of APH. The total amounts of cellular ATR and several ATR-interacting proteins remained unchanged, suggesting that ATR binding to the fragile site is guided initially by the level of replication stress signals generated at FRA3B due to APH treatment and then sequestered from FRA3B regions by successive signals from other non-fragile site regions, which are produced at the higher concentrations of APH. This decrease in ATR binding to fragile site FRA3B at the higher concentrations of APH may account for the increasing number of chromosome gaps and breaks observed under the same conditions. Furthermore, inhibition of ATR kinase activity by treatment with 2-aminopurine (2-AP) or by over-expression of a kinase-dead ATR mutant showed that the kinase activity is required for the binding of ATR to fragile DNAs in response to APH treatment. Our results provide novel insight into the mechanism for the regulation of fragile site stability by ATR.
Project description:It has been suggested that delayed DNA replication underlies fragility at common human fragile sites, but specific sequences responsible for expression of these inducible fragile sites have not been identified. One approach to identify such cis-acting sequences within the large nonexonic regions of fragile sites would be to identify conserved functional elements within orthologous fragile sites by interspecies sequence comparison. This study describes a comparison of orthologous fragile regions, the human FRA3B/FHIT and the murine Fra14A2/Fhit locus. We sequenced over 600 kbp of the mouse Fra14A2, covering the region orthologous to the fragile epicenter of FRA3B, and determined the Fhit deletion break points in a mouse kidney cancer cell line (RENCA). The murine Fra14A2 locus, like the human FRA3B, was characterized by a high AT content. Alignment of the two sequences showed that this fragile region was stable in evolution despite its susceptibility to mitotic recombination on inhibition of DNA replication. There were also several unusual highly conserved regions (HCRs). The positions of predicted matrix attachment regions (MARs), possibly related to replication origins, were not conserved. Of known fragile region landmarks, five cancer cell break points, one viral integration site, and one aphidicolin break cluster were located within or near HCRs. Thus, comparison of orthologous fragile regions has identified highly conserved sequences with possible functional roles in maintenance of fragility.
Project description:The hypothesis that chromosomal fragile sites may be "weak links" that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B. Sequence analysis of 276 kb of the FRA3B/FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.
Project description:Chromosomal common fragile sites (CFSs) are genetically unstable regions of the genome that are induced by conditions that impair DNA replication. In this report, we show that treatment with the DNA polymerase inhibitor, aphidicolin (APH), slows the replication rate throughout S phase. To investigate the unusual sensitivity of CFSs to APH-induced replication stress, we examined replication dynamics within a 50 kb region of the most frequently expressed CFS, FRA3B. We mapped four origins of replication, ori 1-4, using two independent methods. In untreated cells, we detected significantly less newly replicated DNA at FRA3B ori 1-3, as compared with three control origins located within non-fragile regions (NCFSs). In APH-treated cells, all FRA3B and control origins tested were active; however, there was a significant increase of nascent strand DNA at the control origins and, to a lesser extent, at the FRA3B ori 1-3. On the basis of these observations and the theoretical modeling of the nascent strand abundance assay developed in this study, we hypothesize that CFS origins may be less efficient, and that APH treatment slows replication fork movement near these origins to a greater extent, resulting in impaired DNA replication and, ultimately, leading to the genetic instability characteristic of CFSs.
Project description:Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome.
Project description:Chromosomal positions of common fragile sites differ in lymphoblasts and fibroblasts, with positions dependent on the epigenetically determined density of replication origins at these loci. Because rearrangement of fragile loci and associated loss of fragile gene products are hallmarks of cancers, we aimed to map common fragile sites in epithelial cells, from which most cancers derive. Among the five most frequently activated sites in human epithelial cells were chromosome bands 2q33 and Xq22.1, which are not among top fragile sites identified in lymphoblasts or fibroblasts. FRA16D at 16q23 was among the top three fragile sites in the human epithelial cells examined, as it is in lymphoblasts and fibroblasts, while FRA3B at 3p14.2, the top fragile locus in lymphoblasts, was not fragile in most epithelial cell lines tested. Epithelial cells exhibited varying hierarchies of fragile sites; some frequent epithelial cell fragile sites are apparently not frequently altered in epithelial cancers and sites that are frequently deleted in epithelial cancers are not necessarily among the most fragile. Since we have reported that loss of expression of the FRA3B-encoded FHIT protein causes increased replication stress-induced DNA damage, we also examined the effect of FHIT-deficiency on markers of genome instability in epithelial cells. FHIT-deficient cells exhibited increases in fragile breaks and in ?H2AX and 53BP1 foci in G1 phase cells, confirming in epithelial cells that the FHIT gene and encompassing FRA3B, is a "caretaker gene" necessary for maintenance of genome stability.
Project description:Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ?147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ?190 bp, corresponding to a ?45 bp linker. However, cortical neurons have a shorter average spacing of ?165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (?165 bp), whereas OPCs (?182 bp) and astrocytes (?183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (?182 bp) is longer than expected from the genomic average for DRG neurons, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average for these cells (?183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.