Chromosome Fragility Research Articles

MicroRNA and Metastasis.

Noncoding RNAs are important regulatory molecules of cellular processes. MicroRNAs (miRNAs) are small noncoding RNAs that bind to complementary sequences in the 3' untranslated region of target mRNAs, leading to degradation of the target mRNAs and/or inhibition of their translation. Some miRNAs are essential for normal animal development; however, many other miRNAs are dispensable for development but play a critical role in pathological conditions, including tumorigenesis and metastasis. miRNA genes often reside at fragile chromosome sites and are deregulated in cancer. Some miRNAs function as oncogenes or tumor suppressors, collectively termed "oncomirs." Specific metastasis-regulating miRNAs, collectively termed "metastamirs," govern molecular processes and pathways in malignant progression in either a tumor cell-autonomous or a cell-nonautonomous manner. Recently, exosome-transferred miRNAs have emerged as mediators of the tumor-stroma cross talk. In this chapter, we focus on the functions, mechanisms of action, and therapeutic potential of miRNAs, particularly oncomirs and metastamirs.

Molecular characterization of X chromosome fragility in idiopathic mental retardation

BackgroundFragile X syndrome (FXS) is the most common form of inherited mental retardation. Frequency of fragile X syndrome among male siblings and relatives of mentally retarded patients is relatively high. Cytogenetic diagnosis of FXS is unreliable since it is ineffective for the diagnosis of premutated males or females. Proper molecular diagnosis is a pre-requisite for providing proper counseling advice. Subjects and methodsSixty-four males with idiopathic mental retardation, ranging in age from 4.2 to 19years (10.92±4.00) were clinically pre-selected, based on scoring protocol comprising eight features of the syndrome, before molecular testing. A rapid polymerase chain reaction-based screening was applied for detection of expanded FMR1 alleles. Samples that did not yield the normal band lengths were subjected to a second PCR screen. The secondary screen utilizes a chimeric primer demonstrating the presence or absence of an expanded allele. ResultsAmplification of FMRI gene by PCR of tested patients revealed that 8 cases (12.5%) have full mutation and 6 cases (9.4%) have premutation. A wide range of Fra X-scoring ranging from 1 to 7 features was detected in examined cases. Significant clinical features included large prominent ears, hyperextensibility of joints and macroorchidism in post pubertal males. ConclusionsA simplified checklist of fragile X should be used for patients with idiopathic MR and those patients above score 3 should be tested for FXS. The diagnostic assay may be used as a screening method for fragile X syndrome being rapid and cost effective compared to other techniques. In addition, screening of all relatives of proven patients should be performed to detect clinically unidentified cases for provision of proper counseling and optimal management of detected cases.

Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance

SummaryThe essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.

Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events—disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia

Friedreich ataxia (FRDA) is a member of the Repeat Expansion Diseases, a group of genetic conditions resulting from an increase/expansion in the size of a specific tandem array. FRDA results from expansion of a GAA/TTC-tract in the first intron of the frataxin gene (FXN). The disease-associated tandem repeats all form secondary structures that are thought to contribute to the propensity of the repeat to expand. The subset of these diseases that result from a CGG/CCG-repeat expansion, such as Fragile X syndrome, also express a folate-sensitive fragile site coincident with the repeat on the affected chromosome. This chromosome fragility involves the generation of chromosome/chromatid gaps or breaks, or the high frequency loss of one or both copies of the affected gene when cells are grown under folate stress or as we showed previously, in the presence of an inhibitor of the ATM checkpoint kinase. Whether Repeat Expansion Disease loci containing different repeats form similar fragile sites was not known. We show here that the region of chromosome 9 that contains the FXN locus is intrinsically prone to breakage in vivo even in control cells. However, like FXS alleles, FRDA alleles show significantly elevated levels of chromosome abnormalities in the presence of an ATM inhibitor, consistent with the formation of a fragile site.

Activation of the Fanconi anemia/BRCA pathway at low doses of ionization radiation

Fanconi anemia (FA) is a rare, clinically heterogeneous autosomal recessive or X-linked genetic disease characterized by chromosome fragility, congenital malformations and cancer susceptibility. FA patients are usually radiosensitive when exposed to radiotherapy but the role of the FA in response to ionizing radiation (IR) is controversial. Here we have investigated IR-induced activation of the FA pathway by systematically analyzing monoubiquitination of the central protein FANCD2 and subsequent recruitment to stalled replication forks in primary fibroblasts. We developed an immunolabelling method to simultaneously visualize IR-induced FANCD2 and γH2AX foci in S-phase. We observed FANCD2 foci formation in a subset of IR-induced γH2AX foci in S-phase cells. This was observed at doses of IR ranging from 0.1 to 5.0Gy in a dose dependent non-threshold fashion. Our results indicate that minimum doses of IR can produce replication fork stalling and FA pathway activation during S-phase in primary cells.

Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein.

There are many layers of regulation governing DNA replication to ensure that genetic information is accurately transmitted from mother cell to daughter cell. While much of the control occurs at the level of origin selection and firing, less is known about how replication fork progression is controlled throughout the genome. In Drosophila polytene cells, specific regions of the genome become repressed for DNA replication, resulting in underreplication and decreased copy number. Importantly, underreplicated domains share properties with common fragile sites. The Suppressor of Underreplication protein SUUR is essential for this repression. Recent work established that SUUR functions by directly inhibiting replication fork progression, raising several interesting questions as to how replication fork progression and stability can be modulated within targeted regions of the genome. Here we discuss potential mechanisms by which replication fork inhibition can be achieved and the consequences this has on genome stability and copy number control.

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability invitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive invitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

The hidden side of unstable DNA repeats: Mutagenesis at a distance.

Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e., deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur up to ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon.

CHROMOSOMAL ABERRATIONS IN MEN IN THE MARRIED COUPLES WITH THE COMPROMISED REPRODUCTION FNCTION.

Nearly 10-15% of married couples suffer from infertility. Cytogenetic analysis makes it possible to diagnose chromosomal aetiology of infertility and to select respective system of treatment. The aim of the work was to determine karyotype of couples with reproductive problems. Preparations of GTG-differentially stained metaphase chromosomes, prepared in accordance with the standard method, were analysed. In all analysed infertile married couples chromosomal aberrations were identified only in males. Among the identified disorders were: Klinefelter syndrome, Robertsonian translocations between acrocentric chromosomes – 14, 15 and 13, 21, balanced translocations between 16 and 18 chromosomes, pericentric inversions of 8, 9 and 10 chromosomes, de la Chapelle syndrome (46, XX), Y chromosome long arm macrodeletion, fragile chromosomes and presence of enlarged pericentromeric heterochromatin of 16 chromosome. The identified abnormalities resulted in spermatogenesis impairment or non-developing pregnancy in a healthy wife. The performed karyotyping makes it possible to select the optimal tactics for the management of the couple to give birth to a healthy baby

Association of human papillomavirus with Fanconi anemia promotes carcinogenesis in Fanconi anemia patients.

Fanconi anemia (FA) is a rare recessive disorder associated with chromosomal fragility. FA patients are at very high risk of cancers, especially head and neck squamous cell carcinomas and squamous cell carcinomas caused by infection of human papillomaviruses (HPVs). By integrating into the host genome, HPV oncogenes E6 and E7 drive the genomic instability to promote DNA damage and gene mutations necessary for carcinogenesis in FA patients. Furthermore, E6 and E7 oncoproteins not only inhibit p53 and retinoblastoma but also impair the FANC/BRCA signaling pathway to prevent DNA damage repair and alter multiple signals including cell-cycle checkpoints, telomere function, cell proliferation, and interference of the host immune system leading to cancer development in FA patients. In this review, we summarize recent advances in unraveling the molecular mechanisms of FA susceptibility to HPV-induced cancers, which facilitate rational preventive and therapeutic strategies.

Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription

We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.

BackgroundThe histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells.ResultsTo investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors.ConclusionsOur data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-8935-8-2) contains supplementary material, which is available to authorized users.

WWOX: a fragile tumor suppressor.

WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3-q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox(+/-) mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of "classical" tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility associated with the FRA16D locus.

Endogenous florendoviruses are major components of plant genomes and hallmarks of virus evolution.

The extent and importance of endogenous viral elements have been extensively described in animals but are much less well understood in plants. Here we describe a new genus of Caulimoviridae called ‘Florendovirus’, members of which have colonized the genomes of a large diversity of flowering plants, sometimes at very high copy numbers (>0.5% total genome content). The genome invasion of Oryza is dated to over 1.8 million years ago (MYA) but phylogeographic evidence points to an even older age of 20–34 MYA for this virus group. Some appear to have had a bipartite genome organization, a unique characteristic among viral retroelements. In Vitis vinifera, 9% of the endogenous florendovirus loci are located within introns and therefore may influence host gene expression. The frequent colocation of endogenous florendovirus loci with TA simple sequence repeats, which are associated with chromosome fragility, suggests sequence capture during repair of double-stranded DNA breaks.

Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress.

Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.

Karyotype structure and chromosome fragility in the grass Phleum echinatum Host.

Phleum echinatum Host (2n = 2x = 10) is an annual Mediterranean species which differs from other representatives of the genus Phleum by reduced chromosome number, asymmetric karyotype and unusually high amount of DNA in the genome. Chromosomes of this plant were studied using conventional acetic-orcein staining and fluorescence in situ hybridization (FISH). FISH showed the major 35S ribosomal DNA (rDNA) site at the secondary constriction of satellite chromosome (3) and the minor 35S rDNA site near 5S rDNA cluster in the monobrachial chromosome 5. Telomeric repeats were detected at all chromosome ends within secondary constriction in satellited chromosome 3 and at the centromeric regions of chromosomes 1 and 2. Intrachromosomally located telomeric repeats are probably traces of chromosomal rearrangements that have shaped P.echinatum genome; they were prone to breakage which was manifested in chromosome fragmentation. The most distinct telomeric signals, suggesting massive amplification of interstitial telomeric sequences (ITRs), were observed at the nucleolar organizer region (NOR) of the third chromosome pair. Double FISH confirmed co-localization of telomeric and 35S rDNA repeats in this locus characterized by the biggest fragility in the karyotype. Fragile sites of P.echinatum, composed of amplified telomeric repeats, may bear a resemblance to metazoan rare fragile sites enriched in microsatellite repeats.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-014-0681-5) contains supplementary material, which is available to authorized users.

Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders.

The Fragile X-related disorders are a group of genetic conditions that include the neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS), the fertility disorder, Fragile X-associated primary ovarian insufficiency (FXPOI) and the intellectual disability, Fragile X syndrome (FXS). The pathology in all these diseases is related to the number of CGG/CCG-repeats in the 5′ UTR of the Fragile X mental retardation 1 (FMR1) gene. The repeats are prone to continuous expansion and the increase in repeat number has paradoxical effects on gene expression increasing transcription on mid-sized alleles and decreasing it on longer ones. In some cases the repeats can simultaneously both increase FMR1 mRNA production and decrease the levels of the FMR1 gene product, Fragile X mental retardation 1 protein (FMRP). Since FXTAS and FXPOI result from the deleterious consequences of the expression of elevated levels of FMR1 mRNA and FXS is caused by an FMRP deficiency, the clinical picture is turning out to be more complex than once appreciated. Added complications result from the fact that increasing repeat numbers make the alleles somatically unstable. Thus many individuals have a complex mixture of different sized alleles in different cells. Furthermore, it has become apparent that the eponymous fragile site, once thought to be no more than a useful diagnostic criterion, may have clinical consequences for females who inherit chromosomes that express this site. This review will cover what is currently known about the mechanisms responsible for repeat instability, for the repeat-mediated epigenetic changes that affect expression of the FMR1 gene, and for chromosome fragility. It will also touch on what current and future options are for ameliorating some of these effects.