Telomeric Repeat Binding Factor Research Articles

Telomere repeat–binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters

The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex–forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex–binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex–dependent fashion.

Structural and functional analysis of parameters governing tankyrase-1 interaction with telomeric repeat-binding factor 1 and GDP-mannose 4,6-dehydratase

Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity.

P2255Senescence associated secretory phenotype exacerbates overload pressure-cardiac hypertrophy

Abstract Background Advanced age is a significant risk factor for cardiovascular diseases such as hypertension and cardiac hypertrophy. The vascular system forms an essential component of cardiac tissue, to provide routes for circulation and transportation of nutrients and oxygen throughout the cardiac muscle. In addition to its function in vascular biology such as vasodilation and neovessel formation, endothelial cell (EC) also provides many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Ageing induces cellular senescence in various cells including EC. Senescent cells produce senescence-messaging secretomes that have deleterious effects on the tissue microenvironment, referred to as the senescence-associated secretory phenotype (SASP). Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related cardiac dysfunction, however, whether and the mechanism by which EC senescence affects age-related cardiac dysfunction remains to be elucidated. Purpose We aimed to investigate the role of senescent ECs in cardiac hypertrophy and heart function. Methods To investigate a contribution of senescent EC in age-related cardiac tissue dysfunction in vivo, we generated EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat-binding factor 2 (TRF2), which play a central role in the protection of chromosome ends, under the control of the vascular endothelial cadherin promoter (VEcad-TRF2DN-Tg). To induce pathological cardiac remodeling, Transverse Aortic Constriction (TAC) was performed in mice at the age of 10–12 weeks old. Cardiac function was assessed using fractional shortening percentage and ejection fraction measured with echocardiography every week until sacrifice day. Mice were sacrificed 4 weeks after TAC, heart tissue was collected for histological analysis, cardiac morphometry analysis, gene expression and protein expression analysis. In vitro, H9C2 rat cardiomyoblast cells were incubated with conditioned medium derived from control or senescent EC in the presence or absence of angiotensin II to induce cardiac hypertrophy. Results The serial echocardiographic analysis after TAC revealed the exacerbated LV dysfunction in VEcad-TRF2DN-Tg compared to that in wild-type mice. Morphometric and histological analysis 4 weeks after TAC showed increased heart weight and aggravated cardiac fibrosis in VEcad-TRF2DN-Tg mice. In vitro studies demonstrated that conditioned medium derived from senescent ECs enhanced cardiomyocyte hypertrophy in H9C2 cells. Of note, we found that treatment with Y2762, a Rho Kinase inhibitor, canceled the exacerbated cardiac hypertrophy caused by endothelial SASP. Conclusion These findings demonstrate for the first time that senescent ECs play causative roles in age-related cardiac disorders through the SASP, potentially by activating Rho-ROCK pathway in cardiomyocytes.

P4500Cellular senescence of endothelial cells impairs angiogenesis by altering energy metabolism through p53-tigar axis

Abstract Background Ischemic disease is prevalent in elderly population due to impaired angiogenesis. Endothelial cell (EC) generates energy largely via glycolysis, which is further activated when angiogenesis actively occurs. PFK-1 is one of the most important regulatory enzymes for glycolysis, which is activated by PFKFB3. On the other hand, TIGAR inhibits PFK-1 under the control of p53. Crucial roles of PFKFB3 in EC functions under physiological and pathological conditions have been reported; however, a role of TIGAR in EC angiogenic functions remains to be elucidated. Furthermore, it remains unknown whether and how cellular senescence affect the energy metabolism in EC. Purpose The purpose of this study is to investigate molecular mechanisms underlying EC dysfunction associated with ageing, especially by focusing on endothelial energy metabolism. Method and result Senescent EC showed reduced glucose consumption assessed by [U-13C]-glucose tracer assay in association with increased expression of p53 and TIGAR. Angiogenic capacity assessed by tube-formation assay was reduced in senescent EC. Of note, either silencing of TIGAR by siRNA or lentivirus-mediated overexpression of PFKFB3 improved angiogenic capacity in senescent EC. These results collectively suggest that senescence impairs glycolysis in EC by activating p53-TIGAR axis, which leads to senescence-associated endothelial dysfunction. To analyze an impact of EC senescence in angiogenesis in vivo, we generated EC-specific progeroid mice in which dominant negative form of telomere repeat-binding factor 2 (TRF2) was overexpressed in EC under the control of the TIE2 promoter. After confirming EC-specific senescence in these endothelial progeroid mice, we generated hind-limb ischemia model. Recovery of blood flow assessed by laser doppler velocimeter was significantly impaired in endothelial progeroid mice, indicating that EC senescence is directly and causally implicated in age-related angiogenic dysfunction. Of note, genetic inactivation of TIGAR completely rescued the impaired ischemia-induced neovessel formation in EC-specific progeroid mice. Conclusion Using unique endothelial progeroid mice, we revealed that EC senescence is a bona fide risk for ischemic disease, largely by reducing glycolysis in EC through p53-TIGAR axis. Our data suggest that endothelial energy metabolism is an attracting therapeutic target for the prevention and/or treatment of ischemic diseases, especially in elderly population.

Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

BackgroundDisturbed flow (d-flow)–induced senescence and activation of endothelial cells (ECs) have been suggested to have critical roles in promoting atherosclerosis. Telomeric repeat-binding factor 2 (TERF2)-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, regulates the senescence-associated secretory phenotype (SASP), in which EC activation and senescence are engendered simultaneously by p90RSK-induced phosphorylation of TERF2IP S205 and subsequent nuclear export of the TERF2IP-TERF2 complex. In this study, we investigated TERF2IP-dependent gene expression and its role in regulating d-flow-induced SASP. MethodsA principal component analysis and hierarchical clustering were used to identify genes whose expression is regulated by TERF2IP in ECs under d-flow conditions. Senescence was determined by reduced telomere length, increased p53 and p21 expression, and increased apoptosis; EC activation was detected by NF-κB activation and the expression of adhesion molecules. The involvement of TERF2IP S205 phosphorylation in d-flow–induced SASP was assessed by depletion of TERF2IP and mutation of the phosphorylation site. ResultsOur unbiased transcriptome analysis showed that TERF2IP caused alteration in the expression of a distinct set of genes, including rapamycin-insensitive companion of mTOR (RICTOR) and makorin-1 (MKRN1) ubiquitin E3 ligase, under d-flow conditions. In particular, both depletion of TERF2IP and overexpression of the TERF2IP S205A phosphorylation site mutant in ECs increased the d-flow and p90RSK-induced MKRN1 expression and subsequently inhibited apoptosis, telomere shortening, and NF-κB activation in ECs via suppression of p53, p21, and telomerase (TERT) induction. ConclusionsMKRN1 and RICTOR belong to a distinct reciprocal gene set that is both negatively and positively regulated by p90RSK. TERF2IP S205 phosphorylation, a downstream event of p90RSK activation, uniquely inhibits MKRN1 expression and contributes to EC activation and senescence, which are key events for atherogenesis.

TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2.

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting thatthe deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

Abstract 3577: A p53-responsive long noncoding RNA GUARDIN is essential for genome integrity

Abstract The list of long non-coding RNAs (lncRNAs) involved in the p53 pathway of the DNA damage response is rapidly expanding, but whether lncRNAs have a role in maintaining the de novo structure of DNA is unknown. Here, we demonstrate that the p53-responsive lncRNA GUARDIN is important for maintaining genomic integrity under steady-state conditions and after exposure to exogenous genotoxic stress. GUARDIN is necessary for preventing chromosome end-to-end fusion through maintaining the expression of telomeric repeat-binding factor 2 (TRF2) by sequestering microRNA-23a. Moreover, GUARDIN also sustains breast cancer 1 (BRCA1) stability by acting as an RNA scaffold to facilitate the heterodimerization of BRCA1 and BRCA1-associated RING domain protein 1 (BARD1). As such, GUARDIN silencing triggered apoptosis and senescence, enhanced cytotoxicity of additional genotoxic stress and inhibited cancer xenograft growth. Thus, GUARDIN may constitute a target for cancer treatment. Citation Format: Lei Jin, Wang Lai Hu, An Xu, Yu Fang Wang, Rick F. Thorne, Xu Dong Zhang, Mian Wu. A p53-responsive long noncoding RNA GUARDIN is essential for genome integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3577.

Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to Shelterin Core Hub TIN2 when TPP1 Is Absent

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1–TIN2–TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1–TIN2–TRF2 core complex.

Disruption of Telomere Integrity and DNA Repair Machineries by KML001 Induces T Cell Senescence, Apoptosis, and Cellular Dysfunctions.

T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Endothelial senescence is induced by phosphorylation and nuclear export of telomeric repeat binding factor 2-interacting protein.

The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity-dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow-induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.

Expression of telomere repeat binding factor 1 and TRF2 in Alzheimer’s disease and correlation with clinical parameters

ABSTRACTObjective: The objective of this study was to investigate the expression of serum telomere repeat binding factor 1 (TRF1) and TRF2 in patients with Alzheimer’s disease (AD) and their correlation with clinicopathological features.Methods: Fifty AD subjects and 50 healthy controls were enrolled in this study. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of TRF1 and TRF2 in the peripheral blood plasma. Correlation analysis was used to evaluate the correlation between the protein expression and AD clinical parameters.Results: The expression of TRF1 in peripheral blood serum of AD patients was significantly higher than that of the control group (t = 5.533, P < 0.01) and TRF2 was lower than the control group (t = 2.627, P = 0.010). The expression of TRF1 was positively correlated with the history of coronary heart disease (Spearman’s r = 0.298，P = 0.035). The expression of TRF1 and TRF2 was positively correlated with age (Pearson’s r1 = 0.830，P1 < 0.01；Pearson’s r2 = 0.942，P2 < 0.01), BMI (Pearson’s r1 = 0.791，P1 < 0.01；Pearson’s r2 = 0.941，P2 < 0.01), Aβ42 (Pearson’s r1 = 0.765，P1 < 0.01；Pearson’s r2 = 0.926，P2 < 0.01) and Tau protein (Pearson’s r1 = 0.648，P1 < 0.01；Pearson’s r2 = 0.691，P2 < 0.01) in blood serum of AD patients. However, there was no significant difference between both proteins expression and the history of hypertension, diabetes or stroke.Conclusion: TRF1 and TRF2 may be both specific to peripheral blood serum of AD, and may be related to the development of AD.

Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.

Telomeres are specialized nucleoprotein complexes that function to protect eukaryotic chromosomes from recombination and erosion. Several telomere binding proteins (TBPs) have been characterized in higher plants, but their detailed in vivo functions at the plant level are largely unknown. In this study, we identified and characterized OsTRFL1 (Oryza sativa Telomere Repeat-binding Factor Like 1) in rice, a monocot model crop. Although OsTRFL1 did not directly bind to telomere repeats (TTTAGGG)4 in vitro, it was associated with telomeric sequences in planta. OsTRFL1 interacted with rice TBPs, such as OsTRBF1 and RTBP1, in yeast and plant cells as well as in vitro. Thus, it seems likely that the association of OsTRFL1 with other TBPs enables OsTRFL1 to bind to telomeres indirectly. T-DNA inserted OsTRFL1 knock-out mutant rice plants displayed significantly longer telomeres (6–25 kb) than those (5–12 kb) in wild-type plants, indicating that OsTRFL1 is a negative factor for telomere lengthening. The reduced levels of OsTRFL1 caused serious developmental defects in both vegetative and reproductive organs of rice plants. These results suggest that OsTRFL1 is an essential factor for the proper maintenance of telomeres and normal development of rice.

Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.

Telomere-binding proteins constituting the shelterin complex have been studied primarily for telomeric functions. However, mounting evidence shows non-telomeric binding and gene regulation by shelterin factors. This raises a key question—do telomeres impact binding of shelterin proteins at distal non-telomeric sites? Here we show that binding of the telomere-repeat-binding-factor-2 (TRF2) at promoters ~60 Mb from telomeres depends on telomere length in human cells. Promoter TRF2 occupancy was depleted in cells with elongated telomeres resulting in altered TRF2-mediated transcription of distal genes. In addition, histone modifications—activation (H3K4me1 and H3K4me3) as well as silencing marks (H3K27me3)—at distal promoters were telomere length-dependent. These demonstrate that transcription, and the epigenetic state, of telomere-distal promoters can be influenced by telomere length. Molecular links between telomeres and the extra-telomeric genome, emerging from findings here, might have important implications in telomere-related physiology, particularly ageing and cancer.

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Buried in PEAT-discovery of a new silencing complex with opposing activities.

Posttranslational histone modifications serve critical roles in gene regulation by determining the functional status of chromatin. Histone‐modifying enzymes often work in large multiprotein complexes. A paper in this issue of The EMBO Journal describes a new chromatin‐modifying complex called PEAT that acts via histone deacetylation. The PEAT complex is involved in heterochromatin formation and gene repression but also appears to have a locus‐specific activating role, possibly through promoting histone acetylation.

Inhibition of TRF2 accelerates telomere attrition and DNA damage in na\xefve CD4 T cells during HCV infection

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

The miR-590/Acvr2a/Terf1 Axis Regulates Telomere Elongation and Pluripotency of Mouse iPSCs.

SummaryDuring reprogramming, telomere re-elongation is important for pluripotency acquisition and ensures the high quality of induced pluripotent stem cells (iPSCs), but the regulatory mechanism remains largely unknown. Our study showed that fully reprogrammed mature iPSCs or mouse embryonic stem cells expressed higher levels of miR-590-3p and miR-590-5p than pre-iPSCs. Ectopic expression of either miR-590-3p or miR-590-5p in pre-iPSCs improved telomere elongation and pluripotency. Activin receptor II A (Acvr2a) is the downstream target and mediates the function of miR-590. Downregulation of Acvr2a promoted telomere elongation and pluripotency. Overexpression of miR-590 or inhibition of ACTIVIN signaling increased telomeric repeat binding factor 1 (Terf1) expression. The p-SMAD2 showed increased binding to the Terf1 promoter in pre-iPSCs compared with mature iPSCs. Downregulation of Terf1 blocked miR-590- or shAcvr2a-mediated promotion of telomere elongation and pluripotency in pre-iPSCs. This study elucidated the role of the miR-590/Acvr2a/Terf1 signaling pathway in modulating telomere elongation and pluripotency in pre-iPSCs.

Nucleolar TRF2 attenuated nucleolus stress-induced HCC cell-cycle arrest by altering rRNA synthesis

The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.

Abstract 613: Disturbed Flow-induced Telomeric Repeat Binding Factor 2 (terf2)-interacting Protein (terf2ip) K240 Sumoylation is Critical for Endothelial Senescence but Not Inflammation

Upregulation of both inflammation and senescence (Sen) of endothelial cells (ECs) in the disturbed flow area within the aortic arch is well established, but the exact mechanisms and the relationship between inflammation and senescence remain unclear. TERF2IP is involved in regulation of DNA double strand break (DSB) repair of telomeres (TLs) as a component of the shelterin complex and in IκB kinase (IKK)-NF-kB signaling in the cytosol. TERF2IP and TERF2 form a stable heterodimer to protect the duplex region of TLs. Recent studies showed that TERF2IP or TERF2 deficiency caused TL shortening in mice. We studied how TERF2IP SUMOylation is regulated in ECs and what role it may play in EC inflammation and Sen. We found that disturbed flow (d-flow) activated p90RSK and increased TERF2IP S205 phosphorylation and subsequently K240 SUMOylation. Overexpression of dominant negative p90RSK inhibited d-flow-induced TERF2IP S205 phosphorylation and K240 SUMOylation, and also inhibited EC inflammation and Sen provoked by d-flow. TERF2IP S205 phosphorylation induced TERF2IP nuclear export, which then caused EC inflammation and Sen. In contrast, TERF2IP K240 SUMOylation had no effect on TERF2IP nuclear export, and the TERF2IP K240R SUMOylation site mutant failed to inhibit d-flow-induced NF-kB activation. Interestingly, this point mutation inhibited d-flow-induced Sen with reduced d-flow-induced TL shortening, 8-OHdG level, and apoptosis. Overexpression of a de-SUMOylation enzyme SENP2 and SENP2 T368 mutant significantly inhibited d-flow-induced TERF2IP SUMOylation, suggesting that SENP2 regulates nuclear TERF2IP SUMOylation. Lastly, EC Sen increased when SENP2 was specifically knocked out in ECs in mice, suggesting that TERF2IP K240 SUMOylation may play a role in d-flow-induced EC Sen. These data suggest that TERF2IP post-translational modifications play roles in regulating EC inflammation and Sen, which are differentially regulated by S205 phosphorylation and K240 SUMOylation. TERF2IP K240R knock-in mice will be used to determine the role of TERF2IP K240 SUMOylation in d-flow-induced atherogenesis. We suggest that TERF2IP SUMOylation promotes TL dysfunction, but it does not interfere with TERF2IP-mediated IKK-NF-kB activation in the cytosol.