Impaired Replication Timing Promotes Tissue-Specific Expression of Common Fragile Sites

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.

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response

“…Furthermore, the effect was independent of fragile site coincidence even though fragile sites are renowned harbours of long genes ( 56 , 78 ) and the mean length of established fusions also significantly exceeded that of a simulated dataset produced using the same mapping pipeline (Figure 3E ). Operationally, the replication of long genes is limited by their transcription that may be incomplete over more than one round of cell division ( 79 ). Where long genes are also poor in initiation sites, delayed replication in concert with the extended distance of fork travel renders these sites vulnerable to instability ( 78 ).…”

Common Fragile Sites Are Characterized by Faulty Condensin Loading after Replication Stress

“…Unlike constitutively fragile locations, such as fragile X, CFSs form in a cell-type-specific manner, leading to suggestions that an epigenetic component has a role in their fragility ( Letessier et al., 2011 ; Le Tallec et al., 2011 ). A number of factors have been identified, including late replication timing, transcription of long genes, and features of the underlying DNA sequence ( Blin et al., 2019 ; Brison et al., 2019 ; Fungtammasan et al., 2012 ; Helmrich et al., 2011 ; Maccaroni et al., 2020 ; Okamoto et al., 2018 ; Le Tallec et al., 2014 ; Wei et al., 2016 ; Wilson et al., 2015 ). CFSs also require FANCD2 for efficient replication ( Madireddy et al., 2016 ; Pentzold et al., 2018 ) and have been identified as regions in which active DNA synthesis is apparent on mitotic chromosomes in a process dependent on POLD3 and the Mus81 nuclease ( Minocherhomji et al., 2015 ).…”

Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms

“…As cells differentiate from a highly proliferative pluripotent stem cell, they alter replication timing in a cell-type-dependent manner as well as origin usage [ 206 , 207 , 208 ]. Furthermore, common fragile sites show lineage specificity, indicating that different regions of the genome are sensitive to perturbation of the replication program depending on the cell type [ 209 ]. It is therefore conceivable that differences in the replication program sensitize specific cell types to depletion of origin licensing or origin firing factors [ 209 , 210 , 211 ].…”

“…Furthermore, common fragile sites show lineage specificity, indicating that different regions of the genome are sensitive to perturbation of the replication program depending on the cell type [ 209 ]. It is therefore conceivable that differences in the replication program sensitize specific cell types to depletion of origin licensing or origin firing factors [ 209 , 210 , 211 ]. For example, we recently proposed that NK cells are sensitive to MCM10 depletion due to enhanced telomere erosion [ 85 ].…”