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

Autor: Simona Giunta, Klizia Maccaroni, Elisa Balzano, Franca Pelliccia, Federica Mirimao
Jazyk: angličtina
Rok vydání: 2020
Předmět:
0301 basic medicine
Aphidicolin
DNA Replication
Male
lcsh:QH426-470
chromosomal instability
replicative stress
Nerve Tissue Proteins
tissue specificity
Biology
Origin of replication
Article
Cell Line
Dystrophin
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
DNA Replication Timing
Genetics
Humans
human fetal fibroblasts
Gene
Genetics (clinical)
Cells
Cultured
long genes
Replication timing
Neuronal growth regulator 1
Chromosomal fragile site
Chromosome Fragile Sites
common fragile sites
DNA replication timing
fluorescent in situ hybridization
gene length
DNA replication
Membrane Proteins
Cell biology
lcsh:Genetics
030104 developmental biology
chemistry
Chromosomes
Human
Pair 1
Organ Specificity
030220 oncology & carcinogenesis
Female
Chromosomes
Human
Pair 3
Zdroj: Genes
Genes, Vol 11, Iss 3, p 326 (2020)
Volume 11
Issue 3
ISSN: 2073-4425
Popis: 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&rsquo
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.
Databáze: OpenAIRE
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