The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.

Transcription-dependent regulation of replication dynamics modulates genome stability.

Common fragile sites (CFSs) are loci that are hypersensitive to replication stress and hotspots for chromosomal rearrangements in cancers. CFSs replicate late in S phase, are cell-type specific and nest in large genes. The relative impact of transcription-replication conflicts versus a low density in initiation events on fragility is currently debated. Here we addressed the relationships between transcription, replication, and instability by manipulating the transcription of endogenous large genes in chicken and human cells. We found that inducing low transcription with a weak promoter destabilized large genes, whereas stimulating their transcription with strong promoters alleviated instability. Notably, strong promoters triggered a switch to an earlier replication timing, supporting a model in which high transcription levels give cells more time to complete replication before mitosis. Transcription could therefore contribute to maintaining genome integrity, challenging the dominant view that it is exclusively a threat.

Fragility Extraordinaire: Unsolved Mysteries of Chromosome Fragile Sites.

Chromosome fragile sites are a fascinating cytogenetic phenomenon now widely implicated in a slew of human diseases ranging from neurological disorders to cancer. Yet, the paths leading to these revelations were far from direct, and the number of fragile sites that have been molecularly cloned with known disease-associated genes remains modest. Moreover, as more fragile sites were being discovered, research interests in some of the earliest discovered fragile sites ebbed away, leaving a number of unsolved mysteries in chromosome biology. In this review we attempt to recount some of the early discoveries of fragile sites and highlight those phenomena that have eluded intense scrutiny but remain extremely relevant in our understanding of the mechanisms of chromosome fragility. We then survey the literature for disease association for a comprehensive list of fragile sites. We also review recent studies addressing the underlying cause of chromosome fragility while highlighting some ongoing debates. We report an observed enrichment for R-loop forming sequences in fragile site-associated genes than genomic average. Finally, we will leave the reader with some lingering questions to provoke discussion and inspire further scientific inquiries.

Oncogenes create a unique landscape of fragile sites.

Recurrent genomic instability in cancer is attributed to positive selection and/or the sensitivity of specific genomic regions to breakage. Among these regions are fragile sites (FSs), genomic regions sensitive to replication stress conditions induced by the DNA polymerase inhibitor aphidicolin. However, the basis for the majority of cancer genomic instability hotspots remains unclear. Aberrant oncogene expression induces replication stress, leading to DNA breaks and genomic instability. Here we map the cytogenetic locations of oncogene-induced FSs and show that in the same cells, each oncogene creates a unique fragility landscape that only partially overlaps with aphidicolin-induced FSs. Oncogene-induced FSs colocalize with cancer breakpoints and large genes, similar to aphidicolin-induced FSs. The observed plasticity in the fragility landscape of the same cell type following oncogene expression highlights an additional level of complexity in the molecular basis for recurrent fragility in cancer.

WWOX, large common fragile site genes, and cancer.

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.

WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

The fragile site WWOX gene and the developing brain.

WWOX was cloned as a tumor suppressor gene mapping to chromosomal fragile site FRA16D. Loss of WWOX is closely related to tumorigenesis, cancer progression, and therapy resistance. Recent studies demonstrate the growing role of WWOX gene in other human pathologies such as metabolic and nervous system-related conditions. The neurologic phenotype of WWOX mutation includes seizures, ataxia, developmental delay, and spasticity of variable severity. WWOX is a ubiquitous protein with high expression in many tissues including brain, cerebellum, brain stem, and spinal cord. WWOX is highly expressed in different brain regions during murine fetal development and remained unchanged in the cortex and the corpus callosum in adult mice. The mechanism or the putative role of WWOX in the nervous system is still unclear but may include abnormal signaling protein, disruption of neuronal pathways, neuronal differentiation, mitochondrial dysfunction, or apoptosis. Homozygous mutations affecting WWOX in humans are likely to be more described in the future using exome sequencing. The described findings highlight that WWOX plays a critical role in normal central nervous system development and disease. The aim of this review is to summarize the roles of WWOX in the developing brain.

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.

A blooming resolvase at chromosomal fragile sites.

Common fragile sites (CFSs) are chromosomal regions that are prone to form breaks or gaps during mitosis, in particular following replication stress. The mechanisms modulating CFS expression and promoting safe chromatid transmission to daughter cells are not clear. Now CFS expression is shown to reflect the activity of the MUS81-EME1 resolvase complex which cooperates with the dissolving action of the BLM helicase to prevent uncontrolled chromosome breakage and to promote genome integrity.

ERCC1 and MUS81-EME1 promote sister chromatid separation by processing late replication intermediates at common fragile sites during mitosis.

Chromosomal instability (CIN) is a hallmark of tumour initiation and progression. Some genomic regions are particularly unstable under replication stress, notably common fragile sites (CFSs) whose rearrangements in tumour cells contribute to cancer development. Recent work has shown that the Fanconi anaemia (FANC) pathway plays a role in preventing defective chromosome segregation and CIN under conditions of replication stress. Strikingly, FANCD2 is recruited to regions hosting CFSs on metaphase chromosomes. To decipher the mechanisms protecting CFSs in G2/M, we searched for proteins that co-localize with FANCD2 on mitotic chromosomes, and identified XPF-ERCC1 and MUS81-EME1, two structure-specific endonucleases. We show that depletion of either ERCC1 or MUS81-EME1 affects accurate processing of replication intermediates or under-replicated DNA that persist at CFSs until mitosis. Depletion of these endonucleases also leads to an increase in the frequency of chromosome bridges during anaphase that, in turn, favours accumulation of DNA damage in the following G1 phase.

Prenatal detection of del(10)(q11.2) mosaicism in chorionic villus specimens likely caused by a common chromosomal fragile site FRA10G is associated with a normal phenotype.

OBJECTIVE: To summarize the pregnancy outcomes of cases with mosaicism for chromosome 10q11.2 deletion detected by chorionic villus sampling (CVS) and determine whether extensive cytogenetic work-up and follow-up amniocentesis are necessary in such cases. METHODS: CVS was performed at 10-12 weeks of gestation. Chromosome analysis of chorionic villi was performed by standard G-banding techniques. RESULTS: Mosaicism of chromosome 10q11.2 deletion was observed in 24 out of 6063 CVS cases (0.39%). A common fragile site, FRA10G is located at the breakpoint region. The level of mosaicism ranged from 4% to 25%. No evidence of mosaic 10q11.2 deletion was found in follow-up amniocentesis, maternal peripheral blood cells, or from cytogenetic studies of other pregnancies from the same group of patients. All these cases resulted in the live birth of normal healthy infants. CONCLUSION: The presence of del(10)(q11.2) mosaicism in chorionic villus specimens most likely represents an in vitro culture artifact due to FRA10G fragile site in this region without any clinical consequences. If ultrasound results are normal, it is not necessary to perform follow-up amniocenteses and additional laboratory work-up for such cases.

Common chromosomal fragile sites (CFS) may be involved in normal and traumatic cognitive stress memory consolidation and altered nervous system immunity.

Previous reports of specific patterns of increased fragility at common chromosomal fragile sites (CFS) found in association with certain neurobehavioural disorders did not attract attention at the time due to a shift towards molecular approaches to delineate neuropsychiatric disorder candidate genes. Links with miRNA, altered methylation and the origin of copy number variation indicate that CFS region characteristics may be part of chromatinomic mechanisms that are increasingly linked with neuroplasticity and memory. Current reports of large-scale double-stranded DNA breaks in differentiating neurons and evidence of ongoing DNA demethylation of specific gene promoters in adult hippocampus may shed new light on the dynamic epigenetic changes that are increasingly appreciated as contributing to long-term memory consolidation. The expression of immune recombination activating genes in key stress-induced memory regions suggests the adoption by the brain of this ancient pattern recognition and memory system to establish a structural basis for long-term memory through controlled chromosomal breakage at highly specific genomic regions. It is furthermore considered that these mechanisms for management of epigenetic information related to stress memory could be linked, in some instances, with the transfer of the somatically acquired information to the germline. Here, rearranged sequences can be subjected to further selection and possible eventual retrotranscription to become part of the more stable coding machinery if proven to be crucial for survival and reproduction. While linkage of cognitive memory with stress and fear circuitry and memory establishment through structural DNA modification is proposed as a normal process, inappropriate activation of immune-like genomic rearrangement processes through traumatic stress memory may have the potential to lead to undesirable activation of neuro-inflammatory processes. These theories could have a significant impact on the interpretation of risks posed by heredity and the environment and the search for neuropsychiatric candidate genes.

Human DNA polymerase eta is required for common fragile site stability during unperturbed DNA replication.

Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol eta in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol eta from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol eta-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol iota, the closest relative of Pol eta. Our results identify a new role for Pol eta in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol eta in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.

Association between decreased WWOX protein expression and thyroid cancer development.

CONTEXT: Chromosomal fragile sites are often related to cancer development. The WW domain-containing oxidoreductase gene (WWOX) spans the second most common chromosomal fragile site (FRA16D) and encodes an important proapoptotic protein. OBJECTIVE: To verify our hypothesis that underexpression of WWOX could contribute to malignant transformation of the thyroid cells. METHOD: We compared WWOX expression among follicular adenomas (FAs) and differentiated thyroid carcinomas [follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs)] in 53 thyroid tumors resected from patients submitted to total thyroidectomy. DESIGN: Multiple fields of tumor areas of FAs, FTCs, and PTCs as well as normal thyroid tissue were stained with WWOX antiserum, and classified by the extent of staining (percentage of cells staining) and staining intensity. MAIN OUTCOME: PTCs showed a significantly decreased expression of WWOX when compared to FAs and FTCs. Further, using a unique model of comparison in patients in whom FAs and PTCs were concomitantly present, we detected the same result (i.e., no expression in PTCs). CONCLUSION: We conclude that WWOX underexpression is an important step that might increase the vulnerability to the carcinogenesis process in PTCs.

Roles of FHIT and WWOX fragile genes in cancer.

It was hypothesized as early as 1986, that the recently discovered common fragile sites could facilitate recombination events, such as deletions and translocations, that result in clonally expanded cancer cell populations with specific chromosome alterations in specific cancer types. A natural extension of this hypothesis is that the clonal expansion must be driven by alteration of genes at recombination breakpoints whose altered functions actually drive clonal expansion. Nevertheless, when the FHIT gene was discovered at FRA3B, the most active common chromosome fragile region, and proposed as an example of a tumor suppressor gene altered by chromosome translocations and deletions, a wave of reports suggested that the FHIT gene was altered in cancer simply because it was in a fragile region and not because it had contributed to the clonal expansion, thus turning the original hypothesis upside down. Now, after nearly ten years and more than 500 FHIT reports, it is apparent that FHIT is an important tumor suppressor gene and that there are genes at other fragile regions that contribute significantly to development of cancer. A second fragile gene with a demonstrated role in cancer development is the WWOX gene on chromosome 16q; alterations to the WWOX gene contribute to development of hormone responsive and other cancers. Results of our recent studies of these two fragile tumor suppressor genes were summarized at the first Fragilome meeting in Heidelberg, Feb. 2005.

Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability.

Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.

Mechanisms of common fragile site instability.

The study of common fragile sites has its roots in the early cytogenetic investigations of the fragile X syndrome. Long considered an interesting component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to certain forms of replication stress, which are frequently rearranged in cancer cells. In recent years, much has been learned about the genomic structure at fragile sites and the cellular checkpoint functions that monitor their stability. Recent findings suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early stages of tumorigenesis. Thus, the study of common fragile sites can provide insight not only into the nature of fragile sites, but also into the broader consequences of replication stress on DNA damage and cancer. However, despite recent advances, many questions remain regarding the normal functional significance of these conserved regions and the basis of their fragility.

Chromosomal translocations in yeast induced by low levels of DNA polymerase a model for chromosome fragile sites.

In the yeast Saccharomyces cerevisiae, reduced levels of the replicative alpha DNA polymerase result in greatly elevated frequencies of chromosome translocations and chromosome loss. We selected translocations in a small region of chromosome III and found that they involve homologous recombination events between yeast retrotransposons (Ty elements) on chromosome III and retrotransposons located on other chromosomes. One of the two preferred sites of these translocations on chromosome III involve two Ty elements arrayed head-to-head; disruption of this site substantially reduces the rate of translocations. We demonstrate that this pair of Ty elements constitutes a preferred site for double-strand DNA breaks when DNA replication is compromised, analogous to the fragile sites observed in mammalian chromosomes.

The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability.

Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response.