Pathway choice in the alternative telomere lengthening in neoplasia is dictated by replication fork processing mediated by EXD2's nuclease activity.

Telomerase-independent cancer proliferation via the alternative lengthening of telomeres (ALT) relies upon two distinct, largely uncharacterized, break-induced-replication (BIR) processes. How cancer cells initiate and regulate these terminal repair mechanisms is unknown. Here, we establish that the EXD2 nuclease is recruited to ALT telomeres to direct their maintenance. We demonstrate that EXD2 loss leads to telomere shortening, elevated telomeric sister chromatid exchanges, C-circle formation as well as BIR-mediated telomeric replication. We discover that EXD2 fork-processing activity triggers a switch between RAD52-dependent and -independent ALT-associated BIR. The latter is suppressed by EXD2 but depends specifically on the fork remodeler SMARCAL1 and the MUS81 nuclease. Thus, our findings suggest that processing of stalled replication forks orchestrates elongation pathway choice at ALT telomeres. Finally, we show that co-depletion of EXD2 with BLM, DNA2 or POLD3 confers synthetic lethality in ALT cells, identifying EXD2 as a potential druggable target for ALT-reliant cancers.

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence.

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53-TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.

Desmin deficiency affects the microenvironment of the cardiac side population and Sca1+ stem cell population of the adult heart and impairs their cardiomyogenic commitment.

The heart's limited regenerative capacity raises the need for novel stem cell-based therapeutic approaches for cardiac regeneration. However, the use of stem cells is restrictive due to poor determination of their properties and the factors that regulate them. Here, we investigated the role of desmin, the major muscle-specific intermediate filament protein, in the characteristics and differentiation capacity of cardiac side population (CSP) and Sca1+ stem cells of adult mice. We found that desmin deficiency affects the microenvironment of the cells and leads to increased numbers of CSP but not Sca1+ cells; CSP subpopulation composition is altered, the expression of the senescence marker p16INK4a in Sca1+ cells is increased, and early cardiomyogenic commitment is impaired. Specifically, we found that mRNA levels of the cardiac transcription factors Mef2c and Nkx2.5 were significantly reduced in des-/- CSP and Sca1+ cells, while differentiation of CSP and Sca1+ cells demonstrated that in the absence of desmin, the levels of Nkx2.5, Mef2c, Tnnt2, Hey2, and Myh6 mRNA are differentially affected. Thus, desmin deficiency restricts the regenerative potential of CSP and Sca1+ cells, both directly and indirectly through their microenvironment.

HN1 interacts with γ-tubulin to regulate centrosomes in advanced prostate cancer cells.

Prostate cancer is one of the most common cancer for men worldwide with advanced forms showing supernumerary or clustered centrosomes. Hematological and neurological expressed 1 (HN1) also known as Jupiter Microtubule Associated Homolog 1 (JPT1) belongs to a small poorly understood family of genes that are evolutionarily conserved across vertebrate species. The co-expression network of HN1 from the TCGA PRAD dataset indicates the putative role of HN1 in centrosome-related processes in the context of prostate cancer. HN1 expression is low in normal RWPE-1 cells as compared to cancerous androgen-responsive LNCaP and androgen insensitive PC-3 cells. HN1 overexpression resulted in differential response for cell proliferation and cell cycle changes in RWPE-1, LNCaP, and PC-3 cells. Since HN1 overexpression increased the proliferation rate in PC-3 cells, these cells were used for functional characterization of HN1 in advanced prostate carcinogenesis. Furthermore, alterations in HN expression led to an increase in abnormal to normal nuclei ratio and increased chromosomal aberrations in PC-3 cells. We observed the co-localization of HN1 with γ-tubulin foci in prostate cancer cells, further validated by immunoprecipitation. HN1 was observed as physically associated with γ-tubulin and its depletion led to increased γ-tubulin foci and disruption in microtubule spindle assembly. Higher HN1 expression was correlated with prostate cancer as compared to normal tissues. The restoration of HN1 expression after silencing suggested that it has a role in centrosome clustering, implicating a potential role of HN1 in cell division as well as in prostate carcinogenesis warranting further studies.

AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

Detection of a novel unbalanced X;21 translocation in a girl with Turner syndrome phenotype.

OBJECTIVE: To describe a novel unbalanced X;21 translocation resulting in a derivative pseudodicentric chromosome X;21 lacking the critical region for ovarian development and function, in a 16-year-old girl referred for cytogenetic analysis due to primary amenorrhea and Turner-like features. METHODS: Cytogenetic analysis of the proband and her parents was performed on peripheral blood lymphocytes by GTG banding. Molecular cytogenetic FISH analysis was performed on metaphase preparations, using X chromosome centromeric probe and telomeric and pancentromeric peptide nucleic acid (PNA) analog probes. The HUMARA assay as well as methylation studies for PCSK1N and FMR-1 loci were performed. RESULTS: Cytogenetic analysis revealed a de novo unbalanced X;21 translocation, described as 45,X,der(X)t(X;21)(q22.2;p11.2),-21. FISH analysis showed that the derivative X chromosome carried both the X and 21 centromeres, as well as, the Xp and 21q telomeres. The karyotype was thus reevaluated as 45,X,psu dic(21;X)(21qter→21p13::Xq22.2→Xpter),-21. X inactivation studies revealed that the derivative chromosome was of paternal origin and confirmed the selective inactivation of the derivative X segment of the pseudodicentric chromosome. CONCLUSIONS: Primary amenorrhea and other Turner-like characteristics of the proband are apparently due to the loss of the Xq22.2→Xqter critical region which contains critical genes for the ovarian development and function. The chromosome X segment of the derivative pseudodicentric chromosome is selectively inactivated, but inactivation does not seem to spread onto the translocated chromosome 21, accounting probably for the lack of severe clinical consequences which would result from monosomy 21.

Chromosome extremities under the microscopy lens: molecular cytogenetics in telomere research.

At the crossroads of DNA damage repair and genomic instability, telomere research significantly expands our knowledge on fundamental mechanisms involved in cancer initiation and progression, pledging novel tools for targeted and universal onco-therapies. Molecular cytogenetics through the application of a battery of fluorescent hybridization technologies plays an important role toward understanding telomere homeostasis. Herein, we review distinct molecular cytogenetic phenotypes associated with telomere repair, functionality, and elongation. We discuss the underlying mechanisms responsible for their formation or repair, focusing on Break-induced-Replication (BIR)-mediated conservative telomeric neo-synthesis, recently shown to drive the enigmatic Alternative Lengthening of Telomeres in neoplasia.

Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes.

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

Childhood obesity and leucocyte telomere length.

BACKGROUND: Obesity in adulthood is associated with decreased leucocyte telomere length (LTL), which is associated with cardiovascular disease and diabetes mellitus type 2. The aim of our study was to investigate whether increased body mass index (BMI) is associated with decreased LTL in children and adolescents, and to identify other risk factors of shorter LTL in this population. MATERIALS AND METHODS: A cross-sectional study was conducted among 919 Greek children aged 9-13 years (The Healthy Growth Study). Participants were classified as obese (n = 124), overweight (n = 276) or of normal BMI (n = 519). LTL was determined by monochrome multiplex quantitative real-time polymerase chain reaction. Univariate and multivariable linear regression analyses were applied to determine the predictive factors of LTL. RESULTS: Both overweight and obese children had significantly shorter LTL than their normal-BMI counterparts. Following adjustment for age, sex, total daily energy intake and average weekly physical activity (average total steps per day), increasing weight category was inversely associated with LTL in children and adolescents (ß: -0.110 ± 0.035; P = .002). CONCLUSION: Overweight and obesity in childhood and adolescence are associated with shorter LTL, even following adjustment for potential confounding effects. Therefore, the increased BMI in childhood and adolescence may be associated with accelerated biological ageing and may have an adverse impact on future health in adulthood.

SMUG1 Promotes Telomere Maintenance through Telomerase RNA Processing.

Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3'-polyadenylated and 3'-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.

Unbalanced X;9 translocation in an infertile male with de novo duplication Xp22.31p22.33.

PURPOSE: Male carriers of an X-autosome translocation are generally infertile, regardless of the position of the breakpoint on the X chromosome while the pathogenicity of Xp22.3 subtelomeric duplications is under debate. To shed light into this controversy, we present a rare case, of an azoospermic male with no other significant clinical findings, in whom classical cytogenetics revealed additional unbalanced chromosomal material, at the telomere of the long arm of one homolog of chromosome 9. METHODS: In peripheral blood specimens of the index case and his parents, we performed GBanding, Inverted-DAPI Banding, AgNOR staining, Telomere specific Fluorescence in Situ Hybridization (FISH), Molecular karyotyping by Multi-color FISH, whole genome SNP microarrays, sub-telomeric MLPA, and transcription analysis of the expression of KAL1 gene by RT-PCR. RESULTS: Multi-color FISH revealed an unbalanced translocation involving the short arm of chromosome X. SNP microarray analysis combined to classical cytogenetics and MLPA demonstrated a de novo 8.796 Mb duplication of Xp22.31-p22.33. Compared to three control specimens, the patient presented significantly elevated expression levels of KAL1 mRNA in peripheral blood, suggesting transcriptional functionality of the duplicated segment. CONCLUSIONS: The duplicated segment contains the pseudo-autosomal region PAR1 and more than 30 genes including SHOX, ARSE, STS, KAL1, and FAM9A and is not listed as polymorphic. Our data advocate that duplications of the Xp22.3 region may not be associated with a clinical consequence.

The effect of intrauterine growth on leukocyte telomere length at birth.

Objective: Telomeres are specialized nucleoprotein structures located at the ends of chromosomes, which play a crucial role in genomic stability. Telomere shortening has been proposed as a biomarker for the onset of age-related diseases. This study aimed to determine whether restricted or increased intrauterine growth affects leukocyte telomere length (LTL) at birth. Materials and methods: One hundred sixty-five (n = 165) full-term neonates participated in the study. Fetuses were classified as intrauterine growth restriction (IUGR, n = 21), large-for-gestational-age (LGA, n = 15), or appropriate-for-gestational-age (AGA, n = 129), based on customized birth-weight standards. Mixed arteriovenous cord blood samples were collected for isolation of leukocyte DNA. The LTL was measured using multiplex monochrome quantitative real-time PCR and telomeric restriction fragments through Southern blot analysis (terminal restriction fragment [TRF]). Results: Despite differences among groups in birth weight, length and head circumference, LTL did not differ among AGA (6.78 ± 0.58), IUGR (10.54 ± 1.80), and LGA (11.95 ± 2.42) neonates (p = .098). Cord blood IGF-1 and IGFBP-3 concentrations were higher in the LGA group. LTL positively correlated with birth length (r = 0.176, p = .032). Conclusions: Intrauterine growth does not seem to affect LTL at birth. Further studies, comprising a larger sample size of IUGR, LGA, and AGA neonates, are required to determine whether growth at birth influences LTL.

A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence.

BACKGROUND: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. METHODS: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. RESULTS: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.

Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication.

Human malignancies overcome replicative senescence either by activating the reverse-transcriptase telomerase or by utilizing a homologous recombination-based mechanism, referred to as alternative lengthening of telomeres (ALT). In budding yeast, ALT exhibits features of break-induced replication (BIR), a repair pathway for one-ended DNA double-strand breaks (DSBs) that requires the non-essential subunit Pol32 of DNA polymerase delta and leads to conservative DNA replication. Here, we examined whether ALT in human cancers also exhibits features of BIR A telomeric fluorescence in situ hybridization protocol involving three consecutive staining steps revealed the presence of conservatively replicated telomeric DNA in telomerase-negative cancer cells. Furthermore, depletion of PolD3 or PolD4, two subunits of human DNA polymerase delta that are essential for BIR, reduced the frequency of conservatively replicated telomeric DNA ends and led to shorter telomeres and chromosome end-to-end fusions. Taken together, these results suggest that BIR is associated with conservative DNA replication in human cells and mediates ALT in cancer.

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

Nuclear-receptor-mediated telomere insertion leads to genome instability in ALT cancers.

The breakage-fusion-bridge cycle is a classical mechanism of telomere-driven genome instability in which dysfunctional telomeres are fused to other chromosomal extremities, creating dicentric chromosomes that eventually break at mitosis. Here, we uncover a distinct pathway of telomere-driven genome instability, specifically occurring in cells that maintain telomeres with the alternative lengthening of telomeres mechanism. We show that, in these cells, telomeric DNA is added to multiple discrete sites throughout the genome, corresponding to regions regulated by NR2C/F transcription factors. These proteins drive local telomere DNA addition by recruiting telomeric chromatin. This mechanism, which we name targeted telomere insertion (TTI), generates potential common fragile sites that destabilize the genome. We propose that TTI driven by NR2C/F proteins contributes to the formation of complex karyotypes in ALT tumors.

Rif1 maintains telomere length homeostasis of ESCs by mediating heterochromatin silencing.

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Alternative lengthening of telomeres: recurrent cytogenetic aberrations and chromosome stability under extreme telomere dysfunction.

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.