Telomere Function Research Articles

Telomere maintenance and the DNA damage response: a paradoxical alliance.

Telomeres are the protective caps at the ends of linear chromosomes of eukaryotic organisms. Telomere binding proteins, including the six components of the complex known as shelterin, mediate the protective function of telomeres. They do this by suppressing many arms of the canonical DNA damage response, thereby preventing inappropriate fusion, resection and recombination of telomeres. One way this is achieved is by facilitation of DNA replication through telomeres, thus protecting against a "replication stress" response and activation of the master kinase ATR. On the other hand, DNA damage responses, including replication stress and ATR, serve a positive role at telomeres, acting as a trigger for recruitment of the telomere-elongating enzyme telomerase to counteract telomere loss. We postulate that repression of telomeric replication stress is a shared mechanism of control of telomerase recruitment and telomere length, common to several core telomere binding proteins including TRF1, POT1 and CTC1. The mechanisms by which replication stress and ATR cause recruitment of telomerase are not fully elucidated, but involve formation of nuclear actin filaments that serve as anchors for stressed telomeres. Perturbed control of telomeric replication stress by mutations in core telomere binding proteins can therefore cause the deregulation of telomere length control characteristic of diseases such as cancer and telomere biology disorders.

Fecal Microbiota Transfer from Young Mice Reverts Vascular Aging Hallmarks and Metabolic Impairments in Aged Mice.

As a major risk factor for cardiometabolic diseases, aging refers to a gradual decline in physiological function, characterized with 12 conspicuous hallmarks, like telomere attrition, chronic inflammation, and dysbiosis. Common vascular aging hallmarks include endothelial dysfunction, telomere dysfunction, and vascular inflammation. In this study, we sought to test the hypothesis that young-derived gut microbiota retards vascular aging hallmarks and metabolic impairments in aged hosts. We also aimed to study the therapeutic efficacy of young microbiota in hosts of different ages. Fecal microbiota transplantation (FMT) from young to aged or middle-aged C57BL/6 mice was conducted for 6 consecutive weeks after antibiotic pretreatment. Endothelium-dependent relaxations (EDRs) in mouse arteries were determined by wire myography. Inflammation and AMPK/SIRT1 signaling in mouse aortas and intestines were studied by biochemical assays. The telomere function of aortas and intestines, in terms of telomerase reverse transcriptase expression, telomerase activity, and relative telomere length, were also studied. FMT significantly reverted vascular dysfunction and metabolic impairments in middle-aged mice than in aged mice. Besides, FMT significantly reverted inflammation and telomere dysfunction in aortas and intestines of middle-aged mice. Improved intestinal barrier function and activated AMPK/SIRT1 signaling potentially underlie benefits of FMT. The findings imply gut-vascular connection as potential target against age-associated cardiometabolic disorders, highlight crosstalk among aging hallmarks, and suggest a critical timepoint for efficacy of anti-aging interventions.

Chapter 6 - Childhood-inherited white matter disorders with calcification

Intracranial calcification (ICC) occurs in many neurologic disorders both acquired and genetic. In some inherited white matter disorders, it is a common or even invariable feature where the presence and pattern of calcification provides an important pointer to the specific diagnosis. This is particularly the case for the Aicardi-Goutières syndrome (AGS) and for Coats plus (CP) and leukoencephalopathy with calcifications and cysts (LCC), which are discussed in detail in this chapter. AGS is a genetic disorder of type 1 interferon regulation, caused by mutations in any of the nine genes identified to date. In its classic form, AGS has very characteristic clinical and neuroimaging features which will be discussed here. LCC is a purely neurologic disorder caused by mutations in the SNORD118 gene, whereas CP is a multisystem disorder of telomere function that may result from mutations in the CTC1, POT1, or STN genes. In spite of the different pathogenetic basis for LCC and CP, they share remarkably similar neuroimaging and neuropathologic features. Cockayne syndrome, in which ICC is usually present, is discussed elsewhere in this volume. ICC may occur as an occasional feature of many other white matter diseases, including Alexander disease, Krabbe disease, X-ALD, and occulodentodigital dysplasia.

Abstract A020: Neutralizing oxidative damage at telomeres prevents T cell dysfunction and improves adoptive cell therapy

Abstract Background: Failure of adoptive cell therapies (ACTs) is associated with insufficient persistence within the patient, inability to infiltrate tumor sites, and cell-intrinsic loss of functionality. One of the key factors responsible for T cell dysfunction are metabolic barriers such as nutrient competition, low oxygen tension and damaging byproducts in the tumor microenvironment. This leads to a T cell metabolic deregulation affecting their ability to find and kill cancer cell targets properly and effectively. Therefore, elucidating the metabolic pressures experienced in the TME could provide new therapeutic targets to improve ACTs. Reactive Oxidative Species (ROS) accumulation in the TME have detrimental effects on T cell function and anti-tumor response, although the precise targets of ROS are unclear. There are accumulating data showing that mitochondrial ROS can have profound effects on the telomere status of cells. However, there is little evidence describing the role or oxidative stress on telomere health, or the importance of telomere function in immune cells. Our current study demonstrates that tumor infiltrating lymphocytes (TIL) accumulate DNA damage at telomeres, most prominently in terminally exhausted T cells. Furthermore, inducing ROS accumulation at telomeres alone drives T cell dysfunction. Importantly we discovered that alleviating ROS specifically at telomeres improves the response to adoptive cell therapies in a mouse tumor model. Methods: In this study we perform telomeric and centromeric FISH assays to analyze TIL for DNA damage accumulation. We used a chemo-optogenetic FAPS-TAPS to generate singlet oxygen and consequent 8-oxo-guanine lesions specifically at telomeres.1 We tethered the antioxidant protein GPX1 to TRF1, subunit of the Shelterin Complex at telomeres, to generate a telomere-guided ROS scavenger. Results: Telo-FISH analysis demonstrates an accumulation of telomeric DNA damage in TIL from B16 mouse tumors shown by the presence of 53BP1 and ƔH2AX at telomeres. Our data show that mitochondria and telomeric ROS cause the accumulation of DNA damage at telomeres, as well as the development of telomere fragility. These cells ultimately become dysfunctional showing a diminished capability for cytokine production. Importantly, localizing the ROS scavenger GPX1 directly to telomeres reduced telomere fragility and improved the function of therapeutic T cells in the B16 melanoma. Conclusions: Our data suggest that dysfunctional T cells in cancer are not classically senescent, bearing short telomeres, but rather harbor damaged telomeres due to exposure to oxidative stress. Telomeric damage is sufficient to drive a dysfunctional state in newly activated T cells. Protecting telomeres through expression of a telomere-targeted antioxidant protein may preserve T cell function in the tumor microenvironment and drive superior responses to adoptive cell therapies. Citation Format: Dayana Rivadeneira, Sanjana Thosar, Victoria Dean, William Gunn, Konstantinos Lontos, Marcel Bruchez, Patricia Opresko, Greg Delgoffe. Neutralizing oxidative damage at telomeres prevents T cell dysfunction and improves adoptive cell therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A020.

Germline Variant in ZCCHC8 Is Associated with Pulmonary Fibrosis, Bone Marrow Failure, Liver Inflammation, Early Grey Hair and Short Telomer Length

Introduction Telomere biology disorders (TBD) encompass rare genetic conditions that affect telomere function. TBD is associated with a wide range of symptoms, including hematologic, dermatologic, pulmonary, hepatic manifestations. Research is continuously identifying novel variants and genes causative of TBD. In a recent study, Gable et al. (2019) detected a germline missense variant in ZCCHC8 (NM_017612.5:c.557C>T; p.(Pro186Leu)) in a single family with pulmonary fibrosis. This ZCCHC8 variant was associated with short telomere length (TL) and pulmonary fibrosis. To the best of our knowledge, no other families or individuals have been reported to have pathogenic variants in ZCCHC8 and TBD . However, in this abstract, we present data from a second family discovered to harbor a pathogenic variant in ZCCHC8 and to have symptoms of TBD. Methods Family members provided informed consent for genetic testing and underwent trio-based whole-genome sequencing (WGS). DNA isolated from fkin-derived fibroblasts was analyzed in the proband, while DNA from whole blood was used for the parents. To measure TL, we employed Computel (Nersisyan L. et al., 2015) and compared the mean TL to a cohort of patients who also underwent WGS to estimate a relative mean TL. Results: The proband was a 14-year-old male who had pancytopenia and elevated ALAT levels at presentation. A bone marrow biopsy showed a hypocellular bone marrow. A liver biopsy revealed mild centrilobular, pericellular, and portal fibrosis, along with mild inflammation in the portal space, these findings were interpreted as liver regeneration following acute hepatitis. Despite thorough investigations, no explanation for the findings was found. The proband had a history of psychogenic non-epileptic seizures. When the patient was around 20 years of age his hair started to gray. At present, the proband is 23 years old and has not experienced any progression of symptoms. Family history revealed that the proband's father and two second-degree relatives on the paternal side of the family had developed pulmonary fibrosis in the fifth and sixth decade of life as well as early grey hair. Thus, the inheritance pattern of disease related to TBD was autosomal dominant. WGS revealed a heterozygous pathogenic variant in ZCCHC8 (NM_017612.5c.557C>T (p.P186L)) present in the proband and the father which was absent in the mother. The variant was the same as reported by Gable et al. (2019). The variant is not present in gnomAD or COSMIC and it is the only reported pathogenic or likely pathogenic ZCCHC8 missense variant in ClinVar to date. Both the proband and the father exhibited short relative mean TL by Computel estimation. Subsequently, Flow-FISH analysis confirmed that the proband's telomere length was below the 1st percentile. Conclusion Here we present a family with a heterozygous germline variant in ZCCHC8 and TBD including pulmonary fibrosis, pancytopenia, early graying of the hair, and liver disease. Thus, we provide strong evidence of an association between TBD and ZCCHC8.

Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase.

The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.

Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase

The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.

Beyond tradition: exploring the non-canonical functions of telomeres in meiosis.

The telomere bouquet is a specific chromosomal configuration that forms during meiosis at the zygotene stage, when telomeres cluster together at the nuclear envelope. This clustering allows cytoskeleton-induced movements to be transmitted to the chromosomes, thereby facilitating homologous chromosome search and pairing. However, loss of the bouquet results in more severe meiotic defects than can be attributed solely to recombination problems, suggesting that the bouquet's full function remains elusive. Despite its transient nature and the challenges in performing in vivo analyses, information is emerging that points to a remarkable suite of non-canonical functions carried out by the bouquet. Here, we describe how new approaches in quantitative cell biology can contribute to establishing the molecular basis of the full function and plasticity of the bouquet, and thus generate a comprehensive picture of the telomeric control of meiosis.

Urinary essential and toxic metal mixtures, and type 2 diabetes mellitus: Telomere shortening as an intermediary factor?

The joint effect of metal mixtures on telomere function and type 2 diabetes mellitus (T2DM) is unclear. This large-scale cross-sectional study sought to assess the role of telomere length (TL) in the relationship between urinary essential and toxic metal mixtures, and T2DM in 7410 Chinese adults ≥ 60 years of age. Essential (Cr, Cu, Zn, Se) and non-essential metals (V, Al, Sb, Sn, Cd, Pb) in urine samples were quantified, while leukocyte TL was measured from blood samples. Restricted cubic splines regression showed nonlinear relationships between single metal and T2DM, and between TL and T2DM. Bayesian kernel machine regression and quantile-based g-computation showed that the overall status of urinary metals was positively associated with risk of developing T2DM, which was mainly explained by exposure to Pb, Cd, and Sb, excessive Se intake, and high excretion of Zn. Mediation analyses showed that shortened TL mediated 27.9% of the overall positive effect of metal exposure on T2DM, and this mediation was mainly explained by toxic metal exposure and excessive Se intake. Tobacco smoke exposure, extensive cooking at home, and black tea consumption were found to be important contributors of toxic metal exposures. Further studies are needed to explore the recommended Zn dosage for T2DM patients at different stages, which may ameliorate pancreatic senescence and glycemic progression.

Mouse HP1γ regulates TRF1 expression and telomere stability

AimsTelomeric repeat-containing RNAs are long non-coding RNAs generated from the telomeres. TERRAs are essential for the establishment of heterochromatin marks at telomeres, which serve for the binding of members of the heterochromatin protein 1 (HP1) protein family of epigenetic modifiers involved with chromatin compaction and gene silencing. While HP1γ is enriched on gene bodies of actively transcribed human and mouse genes, it is unclear if its transcriptional role is important for HP1γ function in telomere cohesion and telomere maintenance. We aimed to study the effect of mouse HP1γ on the transcription of telomere factors and molecules that can affect telomere maintenance. Main methodsWe investigated the telomere function of HP1γ by using HP1γ deficient mouse embryonic fibroblasts (MEFs). We used gene expression analysis of HP1γ deficient MEFs and validated the molecular and mechanistic consequences of HP1γ loss by telomere FISH, immunofluorescence, RT-qPCR and DNA-RNA immunoprecipitation (DRIP). Key findingsLoss of HP1γ in primary MEFs led to a downregulation of various telomere and telomere-accessory transcripts, including the shelterin protein TRF1. Its downregulation is associated with increased telomere replication stress and DNA damage (γH2AX), effects more profound in females. We suggest that the source for the impaired telomere maintenance is a consequence of increased telomeric DNA-RNA hybrids and TERRAs arising at and from mouse chromosomes 18 and X. SignificanceOur results suggest an important transcriptional control by mouse HP1γ of various telomere factors including TRF1 protein and TERRAs that has profound consequences on telomere stability, with a potential sexually dimorphic nature.

Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin

Noncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells.

Predicted leukocyte telomere length and risk of myeloid neoplasms.

Maintenance of telomere length has long been established to play a role in the biology of cancer and several studies suggest that it may be especially important in myeloid malignancies. To overcome potential bias in confounding and reverse causation of observational studies, we use both a polygenic risk score (PRS) and inverse-variance weighted (IVW) Mendelian randomization (MR) analyses to estimate the relationship between genetically predicted leukocyte telomere length (LTL) and acute myeloid leukemia (AML) risk in 498 cases and 2099 controls and myelodysplastic syndrome (MDS) risk in 610 cases and 1759 controls. Genetic instruments derived from four recent studies explaining 1.23-4.57% of telomere variability were considered. We used multivariable logistic regression to estimate odds ratios (OR, 95% confidence intervals [CI]) as the measure of association between individual single-nucleotide polymorphisms and myeloid malignancies. We observed a significant association between a PRS of longer predicted LTL and AML using three genetic instruments (OR = 4.03 per ~1200 base pair [bp] increase in LTL, 95% CI: 1.65, 9.85 using Codd etal. [Codd, V., Nelson, C.P., Albrecht, E., Mangino, M., Deelen, J., Buxton, J.L., Hottenga, J.J., Fischer, K., Esko, T., Surakka, I. etal. (2013) Identification of seven loci affecting mean telomere length and their association with disease. Nat. Genet., 45, 422-427 427e421-422.], OR = 3.48 per one-standard deviation increase in LTL, 95% CI: 1.74, 6.97 using Li etal. [Li, C., Stoma, S., Lotta, L.A., Warner, S., Albrecht, E., Allione, A., Arp, P.P., Broer, L., Buxton, J.L., Alves, A.D.S.C. etal. (2020) Genome-wide association analysis in humans links nucleotide metabolism to leukocyte telomere length. Am. J. Hum. Genet., 106, 389-404.] and OR = 2.59 per 1000bp increase in LTL, 95% CI: 1.03, 6.52 using Taub etal. [Taub, M.A., Conomos, M.P., Keener, R., Iyer, K.R., Weinstock, J.S., Yanek, L.R., Lane, J., Miller-Fleming, T.W., Brody, J.A., Raffield, L.M. etal. (2022) Genetic determinants of telomere length from 109,122 ancestrally diverse whole-genome sequences in TOPMed. Cell Genom., 2.] genetic instruments). MR analyses further indicated an association between LTL and AML risk (PIVW ≤ 0.049) but not MDS (all PIVW ≥ 0.076). Findings suggest variation in genes relevant to telomere function and maintenance may be important in the etiology of AML but not MDS.

Parental Folate Deficiency Inhibits Proliferation and Increases Apoptosis of Neural Stem Cells in Rat Offspring: Aggravating Telomere Attrition as a Potential Mechanism

The effect of maternal folate status on the fetal central nervous system (CNS) is well recognized, while evidence is emerging that such an association also exists between fathers and offspring. The biological functions of telomeres and telomerase are also related to neural cell proliferation and apoptosis. The study aimed to investigate the effect of parental folate deficiency on the proliferation and apoptosis of neural stem cells (NSCs) in neonatal offspring and the role of telomeres in this effect. In this study, rats were divided into four groups: maternal folate-deficient and paternal folate-deficient diet (D-D) group; maternal folate-deficient and paternal folate-normal diet (D-N) group; maternal folate-normal and paternal folate-deficient diet (N-D) group; and the maternal folate-normal and paternal folate-normal diet (N-N) group. The offspring were sacrificed at postnatal day 0 (PND0), and NSCs were cultured from the hippocampus and striatum tissues of offspring for future assay. The results revealed that parental folate deficiency decreased folate levels, increased homocysteine (Hcy) levels of the offspring’s brain tissue, inhibited proliferation, increased apoptosis, shortened telomere length, and aggravated telomere attrition of offspring NSCs in vivo and in vitro. In vitro experiments further showed that offspring NSCs telomerase activity was inhibited due to parental folate deficiency. In conclusion, parental folate deficiency inhibited the proliferation and increased apoptosis of offspring NSCs, maternal folate deficiency had more adverse effects than paternal, and the mechanisms may involve the telomere attrition of NSCs.

Obstructive sleep apnea syndrome: nocturnal intermittent hypoxia and telomerase complex activity

Obstructive sleep apnea syndrome (OSAS) is a common pathology of the respiratory system characterized by upper airway blockage during sleep. The blockage is caused by intermittent throat collapse and thereby no lung ventilation with preserved respiratory efforts. Earlier we demonstrated that telomeres at the end of chromosomes shorten during intermittent nocturnal hypoxia and sleep fragmentation in OSA patients; the elimination of OSA triggers contributed to an increase in relative telomere length (RTL). The search for the relationship between telomeres and the telomerase complex activity with the sleep stages, as well as indicators of blood oxygen saturation in OSA, seems relevant.Aim. To evaluate the activity of the telomerase complex main components and to determine its relationship with the sleep phases and the level of desaturation in patients with OSA.Methods. The main group included 32 men (age 51.2 ± 3.1 years) with complaints of snoring, respiratory arrest during sleep, and increased daytime sleep. The control group consisted of 26 matched volunteers without clinical manifestations of OSA. Patients of both groups did not have any exacerbations of their chronic conditions at baseline. The study included questionnaires, polysomnography monitoring, analysis of the RLT by PCR, ELISA to determine activity of the telomerase complex components, and statistical analysis.Results. Differences were revealed in the functioning of telomeres and the TERT, TEP1. A positive relationship between the oxygen blood saturation, telomere length and TER1 activity was found in OSA patients.Conclusion. We have identified the decrease in RTL and the telomerase complex activity and proved their positive relationship with the oxygen saturation during intermittent nocturnal hypoxia in OSA patients.

Exercise as a Therapy to Maintain Telomere Function and Prevent Cellular Senescence.

Exercise transiently impacts the expression, regulation, and activity of TERT/telomerase to maintain telomeres and protect the genome from insults. By protecting the telomeres (chromosome ends) and the genome, telomerase promotes cellular survival and prevents cellular senescence. By increasing cellular resiliency, via the actions of telomerase and TERT, exercise promotes healthy aging.

STABILIZATION OF TELOMERE BY THE ANTI-AGING PROPERTY OF METFORMIN MITIGATES OLEIC ACID-INDUCED ATHEROSCLEROTIC PATHOGENESIS

Objective: Factors associated with the atherosclerotic process, including inflammation, senescence-associated secretory phenotypes and telomere dysfunction are closely linked to senescence in VSMCs. However, whether senescent VSMCs are sufficient to cause atherosclerotic process and whether the phenotypes and risk factors of senescent VSMCs affect atherosclerotic pathogenesis are unclear. We investigated the mechanism of inhibition of atherosclerosis by the anti-aging agent metformin and whether attenuating risk factors senescent VSMCs affects atherogenesis. Design and method: We performed in vitro studies including western blot, immunoprecipitation, RT-PCR, flow cytometry, immunofluorescence, FISH analysis, MMP zymography, and transfection of TERT siRNA. In vivo, to test the effect metformin on atherosclerotic lesion formation in ApoE-/- mice, male mice were divided into three groups (each, n = 5). After feeding the high-fat diet for 16 weeks, mice were sacrificed and the formation of atherosclerotic lesions in the aortas and the aortic sinuses were analyzed. Results: Oleic acid (OA)-induced atherosclerotic pathogenesis in normal VSMCs showed marked senescence-associated features, manifested as increased b-gal positive cells and phenotypic switching of VSMCs in vitro; however, these phenomena were shown to be reversed by metformin. To determine what mechanism by metformin alleviates atherogenesis and senescent VSMC, we studied in the enhancement of telomere function by phosphorylating AMPK (Ser485) and PGC1 alpha. In our results, metformin showed increased the expression of p-AMPK (Ser485) as well as the phosphorylation of PGC1 alpha-dependent TERT expression and telomerase activity. Importantly, the depletion of TERT did not reduce risk factors for atherosclerosis and aged VSMCs despites metformin treatment in OA-induced atherosclerotic pathogenesis. We also found in the aortas of high-fat diet ApoE-/- mice, metformin treatment significantly attenuated atherosclerotic plaques and senescent VSMCs, and upregulated the expression of p-AMPK (Ser485)/p-PGC1 alpha and the enhancement of telomere function. Conclusions: The reduction of atherosclerotic pathogenesis in VSMCs and ApoE-/- mice is associated with significantly inhibited senescent phenotypes and risk factors. Increasing p-AMPK (Ser485)/p-PGC1 alpha by metformin the enhancement of telomere function mitigates senescent risk factors and may be a promising therapeutic strategy in atherosclerosis.

Mono-(2-ethylhexyl) Phthalate (MEHP)-Induced Telomere Structure and Function Disorder Mediates Cell Cycle Dysregulation and Apoptosis via c-Myc and Its Upstream Transcription Factors in a Mouse Spermatogonia-Derived (GC-1) Cell Line.

As a typical environmental endocrine disrupting chemical (EDC), di-(2-ethylhexyl) phthalate (DEHP) is thought to be related to reproductive disorders, especially in males. Growing evidence suggests that various EDCs may result in an impaired telomere structure and function, which is associated with male infertility. However, the adverse effect of DEHP on telomeres in male reproductive cells has rarely been studied, and the related mechanisms remain unclear. In this study, we tested the effects of mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP, on telomere dysfunction in mouse spermatogonia-derived cells (GC-1) and the potential role of TERT and c-Myc in MEHP-induced spermatogenic cell damage. Results showed that MEHP induced cell viability inhibition, G0/G1 phase cell cycle arrest, and apoptosis in GC-1 cells in a dose-dependent manner. Shortened telomeres, reduced telomerase activity, and decreased expression of TERT, c-Myc, and upstream transcription factors of c-Myc were also observed in the MEHP-treated cells. In conclusion, it can be concluded that TERT-mediated telomere dysfunction may contribute to MEHP-induced G0/G1 phase cell cycle arrest and apoptosis in GC-1 cells through the impairment of c-Myc and its upstream transcription factors.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

The genome-wide mutational consequences of DNA hypomethylation

DNA methylation is important for establishing and maintaining cell identity and for genomic stability. This is achieved by regulating the accessibility of regulatory and transcriptional elements and the compaction of subtelomeric, centromeric, and other inactive genomic regions. Carcinogenesis is accompanied by a global loss in DNA methylation, which facilitates the transformation of cells. Cancer hypomethylation may also cause genomic instability, for example through interference with the protective function of telomeres and centromeres. However, understanding the role(s) of hypomethylation in tumor evolution is incomplete because the precise mutational consequences of global hypomethylation have thus far not been systematically assessed. Here we made genome-wide inventories of all possible genetic variation that accumulates in single cells upon the long-term global hypomethylation by CRISPR interference-mediated conditional knockdown of DNMT1. Depletion of DNMT1 resulted in a genomewide reduction in DNA methylation. The degree of DNA methylation loss was similar to that observed in many cancer types. Hypomethylated cells showed reduced proliferation rates, increased transcription of genes, reactivation of the inactive X-chromosome and abnormal nuclear morphologies. Prolonged hypomethylation was accompanied by increased chromosomal instability. However, there was no increase in mutational burden, enrichment for certain mutational signatures or accumulation of structural variation to the genome. In conclusion, the primary consequence of hypomethylation is genomic instability, which in cancer leads to increased tumor heterogeneity and thereby fuels cancer evolution.

Abstract 5811: Epigenetic regulation of chromosomal instability in triple-negative breast cancer

Abstract Metastasis is the leading cause of cancer-related death among women with breast cancer. Chromosomal instability (CIN) has emerged as a hallmark of triple-negative breast cancer (TNBC) as it has recently been shown to promote metastasis. However, the underlying molecular mechanisms by which CIN drives metastasis are not completely understood. We have identified a discrete population of highly metastatic SOX2/OCT4+ cells expressing elevated levels of epigenetic regulator EZH2 associated with increased CIN in both human and mouse TNBC. EZH2 histone methyl transferase (HMT) is the catalytic subunit of the Polycomb repressive complex 2 (PRC2), represses target genes through trimethylation of Histone 3 at lysine 27 (H3K27me3). Importantly, genetic and pharmacologic inhibition of EZH2 lead to reduction of CIN and impaired metastasis. These findings led to the hypothesis that EZH2-mediated CIN constitutes a novel mechanism of metastasis regulation. To directly demonstrate epigenetic regulation of CIN, we used genome wide-Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in parallel with RNA-seq. Gene set enrichment of EZH2-repressed target genes directly implicated the spindle formation pathway network. The central core of this network comprised of tankyrase (TNKS), a multifunctional poly (ADP-ribose) polymerase (PARP) previously implicated in DNA repair, telomere function and centrosome maturation. ChIP-PCR confirmed that EZH2 directly binds to the TNKS promoter, and CRISPR knockout of TNKS abrogated the ability of EZH2 inhibition in suppressing CIN, consistent with pharmacological inhibition of TNKS. More specifically, dysregulation of TNKS by EZH2 in OCT4/SOX2+ cells lead to increased numbers of centrosomes and multipolar mitosis. To directly demonstrate the role of aberrant H3K27me3 in regulating chromosomal segregation during mitosis, we used dCas9-EZH2 or dCas9-EZH2 catalytically dead mutant together with chromosome-specific CRISPR guides to ectopically enhance H3K27 trimethylation at the pericentromeric region of specific chromosomes. Ectopic deposition of EZH2 at pericentromeric regions lead to increased CIN. Conceptually, our work provides an unappreciated link between epigenetic regulation and CIN which have been hitherto studied in isolation. From a clinical perspective, demonstrating epigenetic regulation of CIN has opened the possibility for the development of first CIN suppressive therapeutic strategies targeting TNBC metastasis. Citation Format: Yang Bai, Albert Agustinus, Cem Meydan, Dylan R. McNally, Shira Yomtoubian, Liron Yoffe, Ari M. Melnick, Samuel Bakhoum, Vivek Mittal. Epigenetic regulation of chromosomal instability in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5811.