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

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 326 ◽  
Author(s):  
Klizia Maccaroni ◽  
Elisa Balzano ◽  
Federica Mirimao ◽  
Simona Giunta ◽  
Franca Pelliccia
Keyword(s):  
Dna Replication ◽  
Medical Research ◽  
Replication Timing ◽  
Fragile Sites ◽  
Chromosome 1 ◽  
Metaphase Chromosomes ◽  
Specific Expression ◽  
Common Fragile Sites ◽  
Tissue Specific ◽  
Long Genes

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.

scholarly journals Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

2019 ◽  
Author(s):  
Olivier Brison ◽  
Sami EL-Hilali ◽  
Dana Azar ◽  
Stéphane Koundrioukoff ◽  
Mélanie Schmidt ◽  
...  
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Common Fragile Sites ◽  
Large Gene ◽  
Genome Wide ◽  
Underlying Mechanisms ◽  
Large Genes ◽  
Nascent Transcripts

ABSTRACTCommon Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

Differential expression of the closely linked KISS1, REN, and FLJ10761 genes in transgenic mice

2004 ◽  
Vol 17 (1) ◽  
pp. 4-10 ◽  
Author(s):  
Ravi Nistala ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Transgenic Mice ◽  
Human Chromosome ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Artificial Chromosome ◽  
Chromosome 1 ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Human Chromosome 1

We previously reported the development and characterization of transgenic mice containing a large 160-kb P1 artificial chromosome (PAC) encompassing the renin (REN) locus from human chromosome 1. Here we demonstrate that PAC160 not only encodes REN, but also complete copies of the next upstream (KISS1) and downstream ( FLJ10761 ) gene along human chromosome 1. Incomplete copies of the second upstream (PEPP3) and downstream (SOX13) genes are also present. The gene order PEPP3-KISS1-REN-FLJ10761-SOX13 is conserved in mice containing either one or two copies of the REN locus. Despite the close localization of KISS1, REN, and FLJ10761 , they each exhibit distinct, yet overlapping tissue-specific expression profiles in humans. The tissue-specific expression patterns of REN and FLJ10761 were retained in transgenic mice containing PAC160. Expression of REN and FLJ10761 were also proportional to copy number. Expression of KISS1 in PAC160 mice showed both similarities and differences to humans. These data suggest that expression of gene blocks encoded on large genomic clones are retained when the clones are used to generate transgenic mice. Genomic elements which act to insulate genes from their neighbors are also apparently retained.

scholarly journals Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

2019 ◽  
Author(s):  
Dan Sarni ◽  
Takayo Sasaki ◽  
Karin Miron ◽  
Michal Irony Tur-Sinai ◽  
Juan Carlos Rivera-Mulia ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosomal Instability ◽  
Temporal Order ◽  
Replication Timing ◽  
Replication Stress ◽  
Fragile Sites ◽  
Precise Mapping ◽  
Chromosomal Fragility ◽  
Transcriptional Changes ◽  
Large Genes

AbstractCommon fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

scholarly journals Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

2021 ◽  
Vol 118 (48) ◽  
pp. e2106477118
Author(s):  
Shyam Twayana ◽  
Albino Bacolla ◽  
Angelica Barreto-Galvez ◽  
Ruth B. De-Paula ◽  
William C. Drosopoulos ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Single Molecule ◽  
Human Population ◽  
Repetitive Sequences ◽  
Fragile Sites ◽  
Metaphase Chromosomes ◽  
Healthy Human ◽  
Common Fragile Sites ◽  
Dna Structures

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

scholarly journals Sites of chromosomal instability in the context of nuclear architecture and function

2020 ◽  
Author(s):  
Constanze Pentzold ◽  
Miriam Kokal ◽  
Stefan Pentzold ◽  
Anja Weise
Keyword(s):  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Replication Timing ◽  
Fragile Sites ◽  
Chromosomal Breakage ◽  
Metaphase Chromosomes ◽  
Chromosomal Fragile Sites ◽  
And Function ◽  
Genomic Regions ◽  
Mitotic Chromatin

AbstractChromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication–transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.

Replication timing of two human common fragile sites: FRA1H and FRA2G

2008 ◽  
Vol 121 (3-4) ◽  
pp. 196-200 ◽  
Author(s):  
F. Pelliccia ◽  
N. Bosco ◽  
A. Curatolo ◽  
A. Rocchi
Keyword(s):  
Replication Timing ◽  
Fragile Sites ◽  
Common Fragile Sites

scholarly journals Genomic Structure of the Porcine CYP19 Locus and Expression of the CYP19A3 Paralog

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 533
Author(s):  
Jens Vanselow ◽  
Alan J. Conley ◽  
Cynthia J. Corbin ◽  
Trish Berger
Keyword(s):  
Genomic Structure ◽  
In Silico Analysis ◽  
Chromosome 1 ◽  
Promoter Regions ◽  
Specific Expression ◽  
Tissue Specific ◽  
Additional Layer ◽  
Estrogen Synthesis ◽  
Key Enzyme ◽  
Specific Regulation

Proper, tissue-specific regulation of CYP19, the gene encoding aromatase, the key enzyme of estrogen synthesis, is essential for reproductive processes. Here, we analyzed transcriptional regulation of the porcine CYP19 in female and male gonads and brain by 5’RACE and RT-PCR and comprehensively mapped the pig CYP19 locus by in silico analysis. Our data revealed that the complete locus, including three paralogous copies, CYP19A1, CYP19A2 and CYP19A3, spans approximately 330 kb of the porcine chromosome 1. The locus also harbors the first exon of the Gliomedin gene (GLDN) in reverse orientation. Only transcripts of the CYP19A3 paralog were substantially expressed in gonads and hypothalamus. We identified CYP19A3-associated untranslated exons approximately 160 kb and 50 kb distal from the first codon. The 5´ untranslated regions of transcripts were derived from either a proximal or from one of these distal untranslated exons. Transcripts including only untranslated exons could be amplified from testis, thus suggesting long non-coding transcripts. The data revealed an additional layer of complexity in the regulation of the porcine CYP19 locus. Tissue-specific expression is not only achieved by tissue- and stage-specific expression of the three different CYP19 paralogs, but also by directing the expression of CYP19A3 from different, proximal and distal promoter regions.

scholarly journals Pathways for maintenance of telomeres and common fragile sites during DNA replication stress

Open Biology ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 180018 ◽  
Author(s):  
Özgün Özer ◽  
Ian D. Hickson
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
Replication Stress ◽  
Fragile Sites ◽  
Repair Synthesis ◽  
Common Fragile Sites ◽  
The Face ◽  
Dna Replication Stress ◽  
Dna Repair Synthesis ◽  
Insight Into

Oncogene activation during tumour development leads to changes in the DNA replication programme that enhance DNA replication stress. Certain regions of the human genome, such as common fragile sites and telomeres, are particularly sensitive to DNA replication stress due to their inherently ‘difficult-to-replicate’ nature. Indeed, it appears that these regions sometimes fail to complete DNA replication within the period of interphase when cells are exposed to DNA replication stress. Under these conditions, cells use a salvage pathway, termed ‘mitotic DNA repair synthesis (MiDAS)’, to complete DNA synthesis in the early stages of mitosis. If MiDAS fails, the ensuing mitotic errors threaten genome integrity and cell viability. Recent studies have provided an insight into how MiDAS helps cells to counteract DNA replication stress. However, our understanding of the molecular mechanisms and regulation of MiDAS remain poorly defined. Here, we provide an overview of how DNA replication stress triggers MiDAS, with an emphasis on how common fragile sites and telomeres are maintained. Furthermore, we discuss how a better understanding of MiDAS might reveal novel strategies to target cancer cells that maintain viability in the face of chronic oncogene-induced DNA replication stress.

scholarly journals Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Olivier Brison ◽  
Sami El-Hilali ◽  
Dana Azar ◽  
Stéphane Koundrioukoff ◽  
Mélanie Schmidt ◽  
...  
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Fragile Sites ◽  
Replication Initiation ◽  
Common Fragile Sites ◽  
Large Gene ◽  
Genome Wide ◽  
Underlying Mechanisms ◽  
Large Genes ◽  
Nascent Transcripts

AbstractCommon fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription–replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription–replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

scholarly journals High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

Cell Research ◽  
2020 ◽  
Vol 30 (11) ◽  
pp. 997-1008 ◽  
Author(s):  
Morgane Macheret ◽  
Rahul Bhowmick ◽  
Katarzyna Sobkowiak ◽  
Laura Padayachy ◽  
Jonathan Mailler ◽  
...  
Keyword(s):  
Dna Replication ◽  
High Resolution ◽  
Dna Synthesis ◽  
Direct Sequencing ◽  
Replication Stress ◽  
Fragile Sites ◽  
Common Fragile Sites ◽  
Dna Replication Stress ◽  
Genomic Regions ◽  
In Cells

Abstract DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

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