scholarly journals Large Intronic Deletion of the Fragile Site Gene PRKN Dramatically Lowers Its Fragility Without Impacting Gene Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Sebastian H. N. Munk ◽  
Vasileios Voutsinos ◽  
Vibe H. Oestergaard
Keyword(s):  
Gene Expression ◽  
Reverse Genetics ◽  
Fragile Site ◽  
Fragile Sites ◽  
Metaphase Chromosomes ◽  
Large Gene ◽  
Large Intron ◽  
Chromosomal Fragile Sites ◽  
Large Genes ◽  
Genomic Regions

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

scholarly journals FANCD2 tunes the UPR preventing mitochondrial stress-­induced common fragile site instability

2019 ◽  
Author(s):  
Philippe Fernandes ◽  
Benoit Miotto ◽  
Claude Saint-Ruf ◽  
Viola Nähse ◽  
Silvia Ravera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Instability ◽  
Fragile Site ◽  
Transcriptional Response ◽  
Genomic Analysis ◽  
Fragile Sites ◽  
The Unfolded Protein Response ◽  
And Function ◽  
Large Genes ◽  
Genomic Regions

AbstractCommon fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability relies on transcription- and replication-dependent mechanisms. Here, we uncover a role for the UBL5-dependent branch of the unfolded protein response pathway (UPR) in the maintenance of CFS stability. We show that genetic or pharmacological UPR activation induces CFS gene expression and concomitant relocalization of FANCD2, a master regulator of CFS stability, to CFSs. Furthermore, a genomic analysis of FANCD2 binding sites identified an enrichment for mitochondrial UPR transcriptional response elements in FANCD2 bound regions. We demonstrated that depletion of FANCD2 increases CFS gene transcription and their instability while also inducing mitochondrial dysfunction and triggering the activation of the UPR pathway. Depletion of UBL5, a mediator of the UPR, but not ATF4, reduces CFS gene expression and breakage in FANCD2-depleted cells. We thus demonstrate that FANCD2 recruitment and function at CFSs depends on transcription and UPR signaling, and in absence of transcription or UBL5, FANCD2 is dispensable for CFS stability. We propose that FANCD2 coordinates nuclear and mitochondrial activities by tuning the UPR to prevent genome instability.

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.

scholarly journals FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Philippe Fernandes ◽  
Benoit Miotto ◽  
Claude Saint-Ruf ◽  
Maha Said ◽  
Viviana Barra ◽  
...  
Keyword(s):  
Stress Response ◽  
Genome Instability ◽  
Fragile Site ◽  
Fragile Sites ◽  
Human Cells ◽  
Mitochondrial Stress ◽  
Stress Response Pathway ◽  
Stress Dependent ◽  
Large Genes ◽  
Genomic Regions

AbstractCommon fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability.

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.

scholarly journals Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

2019 ◽  
Vol 47 (15) ◽  
pp. 8004-8018 ◽  
Author(s):  
David Pladevall-Morera ◽  
Stephanie Munk ◽  
Andreas Ingham ◽  
Lorenza Garribba ◽  
Eliene Albers ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Fragile Site ◽  
Global Analysis ◽  
Fragile Sites ◽  
Replication Proteins ◽  
Chromosomal Breaks ◽  
Associated Proteins ◽  
Genomic Regions ◽  
Conserved Genomic Regions

Abstract Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.

scholarly journals Common Fragile Site Profiling in Epithelial and Erythroid Cells Reveals that Most Recurrent Cancer Deletions Lie in Fragile Sites Hosting Large Genes

Cell Reports ◽  
2013 ◽  
Vol 4 (3) ◽  
pp. 420-428 ◽  
Author(s):  
Benoît Le Tallec ◽  
Gaël Armel Millot ◽  
Marion Esther Blin ◽  
Olivier Brison ◽  
Bernard Dutrillaux ◽  
...  
Keyword(s):  
Fragile Site ◽  
Fragile Sites ◽  
Common Fragile Site ◽  
Erythroid Cells ◽  
Recurrent Cancer ◽  
Large Genes

The FRA12E Fragile Site Is Localized within the SMRT Gene at 12q24.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4274-4274
Author(s):  
Lionel Coignet ◽  
Jean Dolcce ◽  
Pushpankur Ghoshal ◽  
Alain Nganga ◽  
Timothy Johnson ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Nuclear Receptors ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Fragile Site ◽  
Fragile Sites ◽  
Lymphoblastoid Cell Lines ◽  
Metaphase Chromosomes ◽  
Chromosome 3P

Abstract The Silencing Mediator of retinoid and Thyroid hormone (SMRT) is a transcription co-repressor whose association with nuclear receptors both in solution and bound to DNA response elements is destabilized by ligand. SMRT and a related co-repressor N-CoR are recruiting a transcriptional repression complex, that contains sin3A/B protein and histone deacetylases (HDAC1/2) to nuclear receptors. It is now well established that SMRT can interact with non-receptor proteins such as BCL6, for its repression activities. The ability of the “silencing complex” to deacetylate histones results in a condensed chromatin state that is inhibitory to transcription. We have previously shown that SMRT was localized at chromosome 12q24. In addition, down-regulation of the SMRT protein due to 12q24 rearrangements was recurrently associated with NHL transformation, supporting a tumor suppressor function for SMRT (Cancer Res, 65(11):4554–4561, 2005). We further investigated the reasons why the 12q24 region and SMRT was targeted by chromosomal rearrangements. Genetic breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which genetic breakage may be induced are known as fragile sites. Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3. Two fragile sites exist on the long arm of chromosome 12. FRA12B is located at 12q24.13 and FRA12E has been located at 12q24.2–3. Interestingly the FRA12E region corresponds to the site of SMRT (12q24.2). To assess whether the FRA12E fragile site is localized within the SMRT gene, we studied normal lymphocytes cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared following classical cytogenetics protocols. These preparations were then subjected to fluorescence in situ hybridization (FISH) using a set of SMRT-specific RPCI BAC clones. Using this approach, we were able to demonstrate that the FRA12E fragile site is localized within the SMRT gene. We further characterize several lymphoblastoid cell lines that were either carrier or not of the FRA12E fragile site. This provides us with a mechanistic explanation for recurrent 12q24 rearrangements seen in transformed NHLs. The FRA12E-carrier lymphoblastoid cell lines will provide us the opportunity to characterize the structure of this fragile site.

scholarly journals Molecular Basis for Expression of Common and Rare Fragile Sites

2003 ◽  
Vol 23 (20) ◽  
pp. 7143-7151 ◽  
Author(s):  
Eitan Zlotorynski ◽  
Ayelet Rahat ◽  
Jennifer Skaug ◽  
Neta Ben-Porat ◽  
Efrat Ozeri ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Fragile Site ◽  
Secondary Structures ◽  
Fragile Sites ◽  
Entire Genome ◽  
Common Fragile Sites ◽  
Dna Structures ◽  
Significant Difference ◽  
Minisatellite Repeats ◽  
Genomic Regions

ABSTRACT Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.

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 Chromosomal Instability at Fragile Sites in Blue Foxes, Silver Foxes, and Their Interspecific Hybrids

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1743
Author(s):  
Marta Kuchta-Gładysz ◽  
Ewa Wójcik ◽  
Anna Grzesiakowska ◽  
Katarzyna Rymuza ◽  
Olga Szeleszczuk
Keyword(s):  
Interspecific Hybrids ◽  
Genome Stability ◽  
Diploid Number ◽  
Fragile Sites ◽  
B Chromosomes ◽  
Silver Fox ◽  
Chromosomal Breaks ◽  
Blue Fox ◽  
Chromosomal Fragile Sites ◽  
Karyotypic Variation

A cytogenetic assay based on fragile sites (FS) enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. Genome stability in these species is affected by Robertsonian translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The aims of the study were to characterise the karyotype of blue foxes, silver foxes, and their hybrids and to identify chromosomal fragile sites used to evaluate genome stability. The diploid number of A chromosomes in blue foxes ranged from 48 to 50, while the number of B chromosomes in silver foxes varied from one to four, with a constant number of A chromosomes (2n = 34). In interspecific hybrids, both types of karyotypic variation were identified, with the diploid number of A chromosomes ranging from 40 to 44 and the number of B chromosomes varying from 0 to 3. The mean frequency of FS in foxes was 4.06 ± 0.19: 4.61 ± 0.37 in blue foxes, 3.46 ± 0.28 in silver foxes, and 4.12 ± 0.22 in hybrids. A relationship was identified between an increased number of A chromosomes in the karyotype of the hybrids and the frequency of chromosomal breaks. The FS assay was used as a biomarker for the evaluation of genomic stability in the animals in the study.

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