Ricerc@Sapienza

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 aect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a
tissue-specific manner.