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 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 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 A Drosophila Insulator Interacting Protein Suppresses Enhancer-Blocking Function and Modulates Replication Timing

2019 ◽  
Author(s):  
Emily C. Stow ◽  
Ran An ◽  
Todd A. Schoborg ◽  
Nastasya M. Davenport ◽  
James R. Simmons ◽  
...  
Keyword(s):  
Genome Structure ◽  
Nuclear Lamina ◽  
Replication Timing ◽  
Interacting Protein ◽  
Insulator Protein ◽  
Protein Partners ◽  
Insulator Proteins ◽  
Enhancer Blocking ◽  
And Function ◽  
Partner Proteins

AbstractInsulators play important roles in genome structure and function in Drosophila and mammals. More than six different insulator proteins are required in Drosophila for normal genome function, whereas CTCF is the only identified protein contributing to insulator function in mammals. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. Functional analysis of insulator partner proteins in Drosophila is necessary to understand how genomes are compartmentalized and the roles that different insulators play in genome function. In Drosophila, the Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the Gypsy retrotransposon. The properties that the insulator confers to these sites rely on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)-67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a recently identified partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulators has not yet been elucidated. Here, we find that mutations in the HIPP1 crotonase-like domain have no impact on the function of Su(Hw) enhancer-blocking activity but do exhibit an impaired ability to repair double-strand breaks. Additionally, we find that the overexpression of each HIPP1 and Su(Hw) causes defects in cell proliferation by limiting the progression of DNA replication. We also find that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function.

scholarly journals Comprehensive characterization of neutrophil genome topology

2017 ◽  
Author(s):  
Yina Zhu ◽  
Ke Gong ◽  
Matthew Denholtz ◽  
Vivek Chandra ◽  
Mark P. Kamps ◽  
...  
Keyword(s):  
Large Scale ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Population Based ◽  
Receptor Expression ◽  
Lamin B ◽  
Topologically Associating Domains ◽  
Lamin B Receptor ◽  
Genomic Regions ◽  
Neutrophil Differentiation

Neutrophils are responsible for the first line of defense against invading pathogens. Their nuclei are uniquely structured as multiple lobes that establish a highly constrained nuclear environment. Here we found that neutrophil differentiation was not associated with large-scale changes in the number and sizes of topologically associating domains. However, neutrophil genomes were enriched for long-range genomic interactions that spanned multiple topologically associating domains. Population-based simulation of spherical and toroid genomes revealed declining radii of gyration for neutrophil chromosomes. We found that neutrophil genomes were highly enriched for heterochromatic genomic interactions across vast genomic distances, a process named super-contraction. Super-contraction involved genomic regions located in the heterochromatic compartment in both progenitors and neutrophils or genomic regions that switched from the euchromatic to the heterochromatic compartment during neutrophil differentiation. Super-contraction was accompanied by the repositioning of centromeres, pericentromeres and Long-Interspersed Nuclear Elements (LINEs) to the neutrophil nuclear lamina. We found that Lamin-B Receptor expression was required to attach centromeric and pericentromeric repeats but not LINE-1 elements to the lamina. Differentiating neutrophils also repositioned ribosomal DNA and mini-nucleoli to the lamina: a process that was closely associated with sharply reduced ribosomal RNA expression. We propose that large-scale chromatin reorganization involving super-contraction and recruitment of heterochromatin and nucleoli to the nuclear lamina facilitate the folding of the neutrophil genome into a confined geometry imposed by a multi–lobed nuclear architecture.

scholarly journals The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 266
Author(s):  
Shin-ichiro Takebayashi ◽  
Tyrone Ryba ◽  
Kelsey Wimbish ◽  
Takuya Hayakawa ◽  
Morito Sakaue ◽  
...  
Keyword(s):  
Dna Replication ◽  
Temporal Order ◽  
Expression System ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Dna Methyltransferases ◽  
Dna Hypomethylation ◽  
Transcriptional Changes ◽  
A Cell ◽  
Genomic Regions

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

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.

Cutis verticis gyrata and chromosomal fragile sites

1991 ◽  
Vol 38 (2-3) ◽  
pp. 249-250 ◽  
Author(s):  
S. A. Musumeci ◽  
R. Ferri ◽  
M. Viglianesi ◽  
M. Elia ◽  
R. M. Ragusa ◽  
...  
Keyword(s):  
Fragile Sites ◽  
Cutis Verticis Gyrata ◽  
Chromosomal Fragile Sites
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