Abstract
Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes’ CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.
Highlights
Common fragile sites (CFSs) are regions in which the DNA is prone to gaps, breakage, or constriction that can be visualized on metaphase chromosomes when cells are under replicative stress [1]; they represent about 1% of the whole genome [2], spanning from hundreds to thousands of kilobases in size
In our fibroblast cell lines, we found the frequency of their expression under our experimental conditions to be 18.5% for 1p31.1 located on chromosome 1 in Medical Research Council cell strain 5 (MRC-5) and a 5.3% frequency of expression in Institute for Medical Research-90 cells (IMR-90); 3q13.3 located on chromosome 3 had a 7.5% frequency of expression in MRC-5 and 26.8% frequency of expression in IMR-90 (Figure 1C)
Our work confirmed the tissue specificity associated with CFSs for 1p31.1 and 3q13.3 that are expressed in MRC-5 and, as we found, in IMR-90 fibroblast cells
Summary
Common fragile sites (CFSs) are regions in which the DNA is prone to gaps, breakage, or constriction that can be visualized on metaphase chromosomes when cells are under replicative stress [1]; they represent about 1% of the whole genome [2], spanning from hundreds to thousands of kilobases in size. The frequency of sister chromatid exchange is higher at CFSs compared to non-fragile regions. They represent sites of exogenous viral DNA integration. These features make CFSs extremely recombinogenic and genetically unstable regions of the human genome [3,4]. Despite these destabilizing characteristics, CFSs can be found in almost every organism, from prokaryotes to eukaryotes, suggesting a positive evolutionary selection of these loci [5]. FRAgile site 3B (FRA3B) and FRA16D CFSs co-localize with two tumor suppressor genes, the fragile histidine triad (FHIT) gene and the WW domain-containing oxidoreductase (WWOX), respectively [7,8], two genes known for their involvement in chromosomal aberrations and found mutated in different type of tumors [9,10]
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