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Extra-telomeric impact of telomeres: Emerging molecular connections in pluripotency or stemness

  • Soujanya Vinayagamurthy
    Affiliations
    Integrative and Functional Biology Unit, CSIR Institute of Genomics and Integrative Biology, New Delhi, India

    Academy of Scientific and Innovative Research (AcSIR), CSIR Institute of Genomics and Integrative Biology, New Delhi, India
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  • Akansha Ganguly
    Affiliations
    Integrative and Functional Biology Unit, CSIR Institute of Genomics and Integrative Biology, New Delhi, India
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  • Shantanu Chowdhury
    Correspondence
    For correspondence: Shantanu Chowdhury
    Affiliations
    Integrative and Functional Biology Unit, CSIR Institute of Genomics and Integrative Biology, New Delhi, India

    Academy of Scientific and Innovative Research (AcSIR), CSIR Institute of Genomics and Integrative Biology, New Delhi, India

    G.N.R. Knowledge Centre for Genome Informatics, CSIR Institute of Genomics and Integrative Biology, New Delhi, India
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Open AccessPublished:May 22, 2020DOI:https://doi.org/10.1074/jbc.REV119.009710
      Telomeres comprise specialized nucleic acid–protein complexes that help protect chromosome ends from DNA damage. Moreover, telomeres associate with subtelomeric regions through looping. This results in altered expression of subtelomeric genes. Recent observations further reveal telomere length–dependent gene regulation and epigenetic modifications at sites spread across the genome and distant from telomeres. This regulation is mediated through the telomere-binding protein telomeric repeat–binding factor 2 (TRF2). These observations suggest a role of telomeres in extra-telomeric functions. Most notably, telomeres have a broad impact on pluripotency and differentiation. For example, cardiomyocytes differentiate with higher efficacy from induced pluripotent stem cells having long telomeres, and differentiated cells obtained from human embryonic stem cells with relatively long telomeres have a longer lifespan. Here, we first highlight reports on these two seemingly distinct research areas: the extra-telomeric role of telomere-binding factors and the role of telomeres in pluripotency/stemness. On the basis of the observations reported in these studies, we draw attention to potential molecular connections between extra-telomeric biology and pluripotency. Finally, in the context of the nonlocal influence of telomeres on pluripotency and stemness, we discuss major opportunities for progress in molecular understanding of aging-related disorders and neurodegenerative diseases.
      The ends of eukaryotic chromosomes have specialized nucleotide-protein complexes called telomeres. In mammalian cells, they are capped by a complex of six proteins, TRF1, TRF2, POT1, RAP1, TIN2, and TPP1, known as shelterin (
      • Palm W.
      • de Lange T.
      How shelterin protects mammalian telomeres.
      ,
      • Xin H.
      • Liu D.
      • Songyang Z.
      The telosome/shelterin complex and its functions.
      ,
      • Červenák F.
      • Juríková K.
      • Sepšiová R.
      • Neboháčová M.
      • Nosek J.
      • Tomáška L.
      Double-stranded telomeric DNA binding proteins: diversity matters.
      ). The shelterin proteins have distinct roles. TRF1 and TRF2 bind to double-stranded telomeric DNA, whereas POT1 binds to single-stranded telomeric DNA. RAP1 associates with TRF2, whereas TPP1 and TIN2 primarily associate with POT1 (
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ) (Table 1). Together the shelterin proteins form subcomplexes that vary in ssDNA and dsDNA binding. These result in two broad functions: first, protection of telomeres to evade DNA damage repair at chromosome ends (which, when affected, results in chromosome end fusions and genomic instability (
      • Timashev L.A.
      • Babcock H.
      • Zhuang X.
      • de Lange T.
      The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.
      ,
      • de Lange T.
      Shelterin-mediated telomere protection.
      )) and, second, regulation of the recruitment of telomerase (the catalytic reverse transcriptase that synthesizes telomeres) to telomere ends to maintain the length of telomeres (
      • Lim C.J.
      • Zaug A.J.
      • Kim H.J.
      • Cech T.R.
      Reconstitution of human shelterin complexes reveals unexpected stoichiometry and dual pathways to enhance telomerase processivity.
      ,
      • Pike A.M.
      • Strong M.A.
      • Ouyang J.P.T.
      • Greider C.W.
      TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.
      ,
      • Nandakumar J.
      • Cech T.R.
      Finding the end: recruitment of telomerase to telomeres.
      ). The involvement of telomeres in cellular homeostasis, aging, and disease risk (
      • Martínez P.
      • Blasco M.A.
      Replicating through telomeres: a means to an end.
      ,
      • Blackburn E.H.
      • Epel E.S.
      • Lin J.
      Human telomere biology: a contributory and interactive factor in aging, disease risks, and protection.
      ,
      • Shay J.W.
      • Wright W.E.
      Telomeres and telomerase: three decades of progress.
      ); initiation and progression of cancer (
      • Shay J.W.
      Role of telomeres and telomerase in aging and cancer.
      ,
      • Maciejowski J.
      • De Lange T.
      Telomeres in cancer: tumour suppression and genome instability.
      ,
      • Okamoto K.
      • Seimiya H.
      Revisiting telomere shortening in cancer.
      ); and variation of telomere length, during evolution and in different species (
      • Baird D.M.
      Telomeres and genomic evolution.
      ,
      • Monaghan P.
      • Ozanne S.E.
      Somatic growth and telomere dynamics in vertebrates: relationships, mechanisms and consequences.
      ,
      • Tian X.
      • Doerig K.
      • Park R.
      • Can Ran Qin A.
      • Hwang C.
      • Neary A.
      • Gilbert M.
      • Seluanov A.
      • Gorbunova V.
      Evolution of telomere maintenance and tumour suppressor mechanisms across mammals.
      ), have been extensively reviewed (
      • Shay J.W.
      • Wright W.E.
      Telomeres and telomerase: three decades of progress.
      ).
      Table 1Diverse functions of shelterin proteins
      ShelterinsTelomeric DNA bindingChromatin organizationGene regulationMaintenance of the dedifferentiated stateCancer stem cellReferences
      TRF1dsDNA
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • Ohishi T.
      • Hirota T.
      • Tsuruo T.
      • Seimiya H.
      TRF1 mediates mitotic abnormalities induced by Aurora-A overexpression.
      • Ohishi T.
      • Muramatsu Y.
      • Yoshida H.
      • Seimiya H.
      TRF1 ensures the centromeric function of Aurora-B and proper chromosome segregation.
      • Long J.
      • Huang C.
      • Chen Y.
      • Zhang Y.
      • Shi S.
      • Wu L.
      • Liu Y.
      • Liu C.
      • Wu J.
      • Lei M.
      Telomeric TERB1-TRF1 interaction is crucial for male meiosis.
      ,
      • Marión R.M.
      • López de Silanes I.
      • Mosteiro L.
      • Gamache B.
      • Abad M.
      • Guerra C.
      • Megías D.
      • Serrano M.
      • Blasco M.A.
      Common telomere changes during in vivo reprogramming and early stages of tumorigenesis.
      ,
      • Schneider R.P.
      • Garrobo I.
      • Foronda M.
      • Palacios J.A.
      • Marión R.M.
      • Flores I.
      • Ortega S.
      • Blasco M.A.
      TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells.
      ,
      • Varela E.
      • Schneider R.P.
      • Ortega S.
      • Blasco M.A.
      Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells.
      TRF2dsDNA
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ,
      • Biroccio A.
      • Cherfils-Vicini J.
      • Augereau A.
      • Pinte S.
      • Bauwens S.
      • Ye J.
      • Simonet T.
      • Horard B.
      • Jamet K.
      • Cervera L.
      • Mendez-Bermudez A.
      • Poncet D.
      • Grataroli R.
      • de Rodenbeeke C.T.
      • Salvati E.
      • et al.
      TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.
      • El Maï M.
      • Wagner K.-D.
      • Michiels J.-F.
      • Ambrosetti D.
      • Borderie A.
      • Destree S.
      • Renault V.
      • Djerbi N.
      • Giraud-Panis M.-J.
      • Gilson E.
      • Wagner N.
      The telomeric protein TRF2 regulates angiogenesis by binding and activating the PDGFRβ promoter.
      ,
      • Hussain T.
      • Saha D.
      • Purohit G.
      • Kar A.
      • Kishore Mukherjee A.
      • Sharma S.
      • Sengupta S.
      • Dhapola P.
      • Maji B.
      • Vedagopuram S.
      • Horikoshi N.T.
      • Horikoshi N.
      • Pandita R.K.
      • Bhattacharya S.
      • Bajaj A.
      • et al.
      Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
      ,
      • Su C.-H.
      • Cheng C.
      • Tzeng T.-Y.
      • Lin I.-H.
      • Hsu M.-T.
      An H2A histone isotype, H2ac, associates with telomere and maintains telomere integrity.
      • Konishi A.
      • Izumi T.
      • Shimizu S.
      TRF2 protein interacts with core histones to stabilize chromosome ends.
      ,
      • Wood A.M.
      • Danielsen J.M.R.
      • Lucas C.A.
      • Rice E.L.
      • Scalzo D.
      • Shimi T.
      • Goldman R.D.
      • Smith E.D.
      • Le Beau M.M.
      • Kosak S.T.
      TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends.
      ,
      • Smith E.D.
      • Garza-Gongora A.G.
      • MacQuarrie K.L.
      • Kosak S.T.
      Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.
      ,
      • Alder J.K.
      • Barkauskas C.E.
      • Limjunyawong N.
      • Stanley S.E.
      • Kembou F.
      • Tuder R.M.
      • Hogan B.L.M.
      • Mitzner W.
      • Armanios M.
      Telomere dysfunction causes alveolar stem cell failure.
      • Orun O.
      • Tiber P.M.
      • Serakinci N.
      Partial knockdown of TRF2 increase radiosensitivity of human mesenchymal stem cells.
      ,
      • Serakinci N.
      • Mega Tiber P.
      • Orun O.
      Chromatin modifications of hTERT gene in hTERT-immortalized human mesenchymal stem cells upon exposure to radiation.
      ,
      • Lagunas A.M.
      • Wu J.
      • Crowe D.L.
      Telomere DNA damage signaling regulates cancer stem cell evolution, epithelial mesenchymal transition, and metastasis.
      ,
      • Wu M.
      • Lin Z.
      • Li X.
      • Xin X.
      • An J.
      • Zheng Q.
      • Yang Y.
      • Lu D.
      HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2.
      ,
      • Zhang P.
      • Pazin M.J.
      • Schwartz C.M.
      • Becker K.G.
      • Wersto R.P.
      • Dilley C.M.
      • Mattson M.P.
      Nontelomeric TRF2-REST interaction modulates neuronal gene silencing and fate of tumor and stem cells.
      ,
      • Zhang P.
      • Casaday-Potts R.
      • Precht P.
      • Jiang H.
      • Liu Y.
      • Pazin M.J.
      • Mattson M.P.
      Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor.
      ,
      • Bai Y.
      • Lathia J.D.
      • Zhang P.
      • Flavahan W.
      • Rich J.N.
      • Mattson M.P.
      Molecular targeting of TRF2 suppresses the growth and tumorigenesis of glioblastoma stem cells.
      • Saha A.
      • Roy S.
      • Kar M.
      • Roy S.
      • Thakur S.
      • Padhi S.
      • Akhter Y.
      • Banerjee B.
      Role of telomeric TRF2 in orosphere formation and CSC phenotype maintenance through efficient DNA repair pathway and its correlation with recurrence in OSCC.
      RAP1
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • Martinez P.
      • Thanasoula M.
      • Carlos A.R.
      • Gómez-López G.
      • Tejera A.M.
      • Schoeftner S.
      • Dominguez O.
      • Pisano D.G.
      • Tarsounas M.
      • Blasco M.A.
      Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
      ,
      • Teo H.
      • Ghosh S.
      • Luesch H.
      • Ghosh A.
      • Wong E.T.
      • Malik N.
      • Orth A.
      • de Jesus P.
      • Perry A.S.
      • Oliver J.D.
      • Tran N.L.
      • Speiser L.J.
      • Wong M.
      • Saez E.
      • Schultz P.
      • et al.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
      • Crabbe L.
      • Karlseder J.
      Mammalian Rap1 widens its impact.
      ,
      • Martínez P.
      • Gómez-López G.
      • García F.
      • Mercken E.
      • Mitchell S.
      • Flores J.M.
      • de Cabo R.
      • Blasco M.A.
      RAP1 protects from obesity through its extratelomeric role regulating gene expression.
      ,
      • Yeung F.
      • Ramírez C.M.
      • Mateos-Gomez P.A.
      • Pinzaru A.
      • Ceccarini G.
      • Kabir S.
      • Fernández-Hernando C.
      • Sfeir A.
      Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity.
      ,
      • Cai Y.
      • Kandula V.
      • Kosuru R.
      • Ye X.
      • Irwin M.G.
      • Xia Z.
      Decoding telomere protein Rap1: its telomeric and nontelomeric functions and potential implications in diabetic cardiomyopathy.
      ,
      • Ding Y.
      • Liang X.
      • Zhang Y.
      • Yi L.
      • Shum H.C.
      • Chen Q.
      • Chan B.P.
      • Fan H.
      • Liu Z.
      • Tergaonkar V.
      • Qi Z.
      • Tse H.
      • Lian Q.
      Rap1 deficiency-provoked paracrine dysfunction impairs immunosuppressive potency of mesenchymal stem cells in allograft rejection of heart transplantation.
      • Zhang X.
      • Liu Z.
      • Liu X.
      • Wang S.
      • Zhang Y.
      • He X.
      • Sun S.
      • Ma S.
      • Shyh-Chang N.
      • Liu F.
      • Wang Q.
      • Wang X.
      • Liu L.
      • Zhang W.
      • Song M.
      • et al.
      Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis.
      POT1ssDNA
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • He H.
      • Wang Y.
      • Guo X.
      • Ramchandani S.
      • Ma J.
      • Shen M.-F.
      • Garcia D.A.
      • Deng Y.
      • Multani A.S.
      • You M.J.
      • Chang S.
      Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita.
      ,
      • Hosokawa K.
      • MacArthur B.D.
      • Ikushima Y.M.
      • Toyama H.
      • Masuhiro Y.
      • Hanazawa S.
      • Suda T.
      • Arai F.
      The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age.
      TIN2
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • Kaminker P.G.
      • Kim S.-H.
      • Desprez P.-Y.
      • Campisi J.
      A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.
      ,
      • Chen L.-Y.
      • Zhang Y.
      • Zhang Q.
      • Li H.
      • Luo Z.
      • Fang H.
      • Kim S.H.
      • Qin L.
      • Yotnda P.
      • Xu J.
      • Tu B.P.
      • Bai Y.
      • Songyang Z.
      Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
      TPP1
      • Schmutz I.
      • De Lange T.
      Shelterin.
      ,
      • Tejera A.M.
      • Stagno d'Alcontres M.
      • Thanasoula M.
      • Marion R.M.
      • Martinez P.
      • Liao C.
      • Flores J.M.
      • Tarsounas M.
      • Blasco M.A.
      TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.
      ,
      • Sexton A.N.
      • Regalado S.G.
      • Lai C.S.
      • Cost G.J.
      • O'Neil C.M.
      • Urnov F.D.
      • Gregory P.D.
      • Jaenisch R.
      • Collins K.
      • Hockemeyer D.
      Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.
      Relatively recent work shows association of shelterin proteins outside telomeres across the genome (
      • Martinez P.
      • Thanasoula M.
      • Carlos A.R.
      • Gómez-López G.
      • Tejera A.M.
      • Schoeftner S.
      • Dominguez O.
      • Pisano D.G.
      • Tarsounas M.
      • Blasco M.A.
      Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
      ,
      • Yang D.
      • Xiong Y.
      • Kim H.
      • He Q.
      • Li Y.
      • Chen R.
      • Songyang Z.
      Human telomeric proteins occupy selective interstitial sites.
      ,
      • Simonet T.
      • Zaragosi L.-E.
      • Philippe C.
      • Lebrigand K.
      • Schouteden C.
      • Augereau A.
      • Bauwens S.
      • Ye J.
      • Santagostino M.
      • Giulotto E.
      • Magdinier F.
      • Horard B.
      • Barbry P.
      • Waldmann R.
      • Gilson E.
      The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats.
      ), suggesting functions that are extra-telomeric, or beyond telomeres. Extra-telomeric functions include how telomeres influence gene expression in the subtelomeric regions (∼10 Mb from telomeres (
      • Robin J.D.
      • Ludlow A.T.
      • Batten K.
      • Magdinier F.
      • Stadler G.
      • Wagner K.R.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.
      ,
      • Robin J.D.
      • Ludlow A.T.
      • Batten K.
      • Gaillard M.-C.
      • Stadler G.
      • Magdinier F.
      • Wright W.E.
      • Shay J.W.
      SORBS2 transcription is activated by telomere position effect-over long distance upon telomere shortening in muscle cells from patients with facioscapulohumeral dystrophy.
      )), telomere length–dependent transcriptional activity, and epigenetic modifications at sites distant from telomeres (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). In addition, a large body of work suggests a role of telomeres, particularly telomere length, in self-renewal or pluripotency (
      • Harrington L.
      Does the reservoir for self-renewal stem from the ends?.
      ,
      • Aguado T.
      • Gutiérrez F.J.
      • Aix E.
      • Schneider R.P.
      • Giovinazzo G.
      • Blasco M.A.
      • Flores I.
      Telomere length defines the cardiomyocyte differentiation potency of mouse induced pluripotent stem cells.
      ,
      • Martínez P.
      • Ferrara-Romeo I.
      • Flores J.M.
      • Blasco M.A.
      Essential role for the TRF2 telomere protein in adult skin homeostasis.
      ,
      • Zou Y.
      • Tong H.J.
      • Li M.
      • Tan K.S.
      • Cao T.
      Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies.
      ) (Table 1). Herein, we discuss literature that potentially bridges these two developing aspects, keeping in mind aging-related disorders that involve premature differentiation of stem cells (
      • Meyer K.
      • Feldman H.M.
      • Lu T.
      • Drake D.
      • Lim E.T.
      • Ling K.-H.
      • Bishop N.A.
      • Pan Y.
      • Seo J.
      • Lin Y.-T.
      • Su S.C.
      • Church G.M.
      • Tsai L.-H.
      • Yankner B.A.
      REST and neural gene network dysregulation in iPSC models of Alzheimer's disease.
      ).

      Telomeres: Gene regulation, epigenetics, and genome organization

      The role of telomeres in gene regulation first came to light in 1990. Gottschling et al. (
      • Gottschling D.E.
      • Aparicio O.M.
      • Billington B.L.
      • Zakian V.A.
      Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.
      ) noted heritable silencing of transgenes inserted within 4 kb from telomeric ends in yeast cells and reported this to be due to telomere position effect (TPE). Several years later, TPE was observed at chromosome 22 telomere in human lymphoblastoid cell lines (
      • Baur J.A.
      • Zou Y.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect in human cells.
      ). Extensive research followed to understand the TPE-related silencing of genes in subtelomeric regions of fungi and other organisms, such as Trypanosoma brucei, Plasmodium falciparum, Schizosaccharomyces pombe, Drosophila melanogaster, Pneumocystis carinii, and Candida glabrata (
      • Ottaviani A.
      • Gilson E.
      • Magdinier F.
      Telomeric position effect: from the yeast paradigm to human pathologies?.
      ). It was also observed that genes (e.g. ISG15, DSP, and C1S) positioned ∼10 Mb further from telomeres than found in TPE were down-regulated through physical association of telomeres. This was denoted as TPE-over long distance (TPE-OLD), which involves the long telomeres looping back to the chromatin, causing gene repression and shortening of telomeres, dissociating the loop leading to gene activation (Fig. 1) (
      • Robin J.D.
      • Ludlow A.T.
      • Batten K.
      • Magdinier F.
      • Stadler G.
      • Wagner K.R.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.
      ). Recent work shows telomerase reverse transcriptase gene hTERT is also regulated by TPE-OLD (
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ). TPE or TPE-OLD has been implicated in disorders such as idiopathic mental retardation, ring chromosome 17, and facio-scapulo-humeral dystrophy (
      • Ottaviani A.
      • Gilson E.
      • Magdinier F.
      Telomeric position effect: from the yeast paradigm to human pathologies?.
      ,
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ,
      • Kim W.
      • Shay J.W.
      Long-range telomere regulation of gene expression: telomere looping and telomere position effect over long distances (TPE-OLD).
      ).
      Figure thumbnail gr1
      Figure 1TPE-OLD. Physical association of relatively long telomeres by looping to the subtelomeric regions results in transcriptional repression of genes located in the subtelomeres. In relatively short telomeres, the looping is lost, and genes become transcriptionally active.
      Recent findings show that telomere length influences transcription of genes as far as ∼60 Mb away from telomeres. It was demonstrated that this was because TRF2 binding across the genome (i.e. extra-telomeric sites) depended on telomere length—and TRF2 occupancy at promoters affected expression of target genes (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). TRF2 is known to bind to the G-rich TTAGGG motif present as repeats at the telomeres (
      • Smogorzewska A.
      • van Steensel B.
      • Bianchi A.
      • Oelmann S.
      • Schaefer M.R.
      • Schnapp G.
      • de Lange T.
      Control of human telomere length by TRF1 and TRF2.
      ). Therefore, in human cells with elongated telomeres (i.e. increased number of TTAGGG repeats), telomeric TRF2 binding was enhanced as expected. On the other hand, extra-telomeric TRF2 binding was reduced relative to cells with shorter telomeres (with isogenic background) (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). TRF2 levels in the nucleus, however, remained unaltered in cells with long or short telomeres, consistent with a previous report showing relatively unchanged abundance of nuclear TRF2 in different types of cells with short/long telomeres (
      • Takai K.K.
      • Hooper S.
      • Blackwood S.
      • Gandhi R.
      • de Lange T.
      In vivo stoichiometry of shelterin components.
      ). Based on this, it was postulated that redistribution of TRF2 binding between telomeric and extra-telomeric sites occurs as telomeres elongate (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). This is denoted as the telomere sequestration and partitioning (TSP) model, which describes altered extra-telomeric TRF2 binding in long/short telomeres resulting in differential expression of TRF2-target promoters (Fig. 2). Moreover, altered epigenetic state of the TRF2-target promoters (e.g. modification of histone activation (H3K4Me1 and H3K4Me3) and suppression (H3K27Me3) marks) was evident (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ).
      Figure thumbnail gr2
      Figure 2TSP. The model implies partitioning of TRF2 between telomeric and extra-telomeric sites. Longer telomeres sequester more TRF2, thereby depleting TRF2 binding at extra-telomeric sites. Conversely, when telomeres shorten, an increase in TRF2 binding at promoters influences TRF2-mediated chromatin modifications and transcription.
      Notable in this context are observations of extra-telomeric binding of TRF2 and/or TRF1 constituting 50 TRF2 (
      • Yang D.
      • Xiong Y.
      • Kim H.
      • He Q.
      • Li Y.
      • Chen R.
      • Songyang Z.
      Human telomeric proteins occupy selective interstitial sites.
      ) and 68 sites common to TRF1/TRF2, respectively (
      • Simonet T.
      • Zaragosi L.-E.
      • Philippe C.
      • Lebrigand K.
      • Schouteden C.
      • Augereau A.
      • Bauwens S.
      • Ye J.
      • Santagostino M.
      • Giulotto E.
      • Magdinier F.
      • Horard B.
      • Barbry P.
      • Waldmann R.
      • Gilson E.
      The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats.
      ). A majority of the extra-telomeric sites constituted telomere-like TTAGGG sequences and are therefore called interstitial telomeric sequence. Recent work, however, has revealed more extensive binding of TRF2 across the genome, with more than 20,000 sites from TRF2 ChIP-Seq in HT1080 fibrosarcoma cells. These interstitial TRF2 binding sites comprised G-rich repeat sequences, including interstitial telomeric sequence (
      • Mukherjee A.K.
      • Sharma S.
      • Bagri S.
      • Kutum R.
      • Kumar P.
      • Hussain A.
      • Singh P.
      • Saha D.
      • Kar A.
      • Dash D.
      • Chowdhury S.
      Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.
      ). In addition, ∼12,500 TRF2 peaks mapped within 20 kb of transcription start sites. Several of these promoters were tested and found to be epigenetically modified, and transcriptionally regulated, in a TRF2-dependent way (
      • Mukherjee A.K.
      • Sharma S.
      • Bagri S.
      • Kutum R.
      • Kumar P.
      • Hussain A.
      • Singh P.
      • Saha D.
      • Kar A.
      • Dash D.
      • Chowdhury S.
      Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.
      ).
      Nucleosomes, the basic units of chromatin packaging in cells, comprise a complex of H2A, H2B, H3, and H4 histone proteins. Modifications (e.g. methylation or acetylation) of histone proteins are therefore closely related to how chromatin is packaged. These are known as epigenetic modifications that can impact gene regulation. Short telomeres, and consequent DNA damage, were noted to result in reduced histone biosynthesis (
      • O'Sullivan R.J.
      • Kubicek S.
      • Schreiber S.L.
      • Karlseder J.
      Reduced histone biosynthesis and chromatin changes arising from a damage signal at telomeres.
      ,
      • Hauer M.H.
      • Seeber A.
      • Singh V.
      • Thierry R.
      • Sack R.
      • Amitai A.
      • Kryzhanovska M.
      • Eglinger J.
      • Holcman D.
      • Owen-Hughes T.
      • Gasser S.M.
      Histone degradation in response to DNA damage enhances chromatin dynamics and recombination rates.
      ), affecting the state of chromatin.
      Several studies further show shortened telomeres to be associated with genome-wide altered DNA methylation, nucleosome positioning, and histone modifications (reviewed in Ref.
      • Song S.
      • Johnson F.
      Epigenetic mechanisms impacting aging: a focus on histone levels and telomeres.
      ). Similar observations were also made during stem cell pluripotency, cell senescence, and cancer cell differentiation (
      • Pucci F.
      • Gardano L.
      • Harrington L.
      Short telomeres in ESCs lead to unstable differentiation.
      ,
      • Harrington L.
      • Pucci F.
      In medio stat virtus: unanticipated consequences of telomere dysequilibrium.
      ).
      Another line of investigation implicated the shelterin factor RAP1 more directly. On telomere shortening RAP1 was found to affect nucleosome occupancy, down-regulate histone genes, and increase expression of senescence-associated genes (
      • Song S.
      • Johnson F.
      Epigenetic mechanisms impacting aging: a focus on histone levels and telomeres.
      ,
      • Pucci F.
      • Gardano L.
      • Harrington L.
      Short telomeres in ESCs lead to unstable differentiation.
      ,
      • Platt J.M.
      • Ryvkin P.
      • Wanat J.J.
      • Donahue G.
      • Ricketts M.D.
      • Barrett S.P.
      • Waters H.J.
      • Song S.
      • Chavez A.
      • Abdallah K.O.
      • Master S.R.
      • Wang L.S.
      • Johnson F.B.
      Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence.
      ,
      • Song S.
      • Perez J.V.
      • Svitko W.
      • Ricketts M.D.
      • Dean E.
      • Schultz D.
      • Marmorstein R.
      • Johnson F.B.
      Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence.
      ). Together, these suggest a broader role of telomeres, particularly short telomeres, in the epigenetic state of the genome.

      Functions of shelterin proteins independent of chromosome-end protection

      In 2010, Martinez et al. (
      • Martinez P.
      • Thanasoula M.
      • Carlos A.R.
      • Gómez-López G.
      • Tejera A.M.
      • Schoeftner S.
      • Dominguez O.
      • Pisano D.G.
      • Tarsounas M.
      • Blasco M.A.
      Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
      ) found extra-telomeric binding of RAP1 to the TTAGGGTTAGGG consensus motif in mice; 70% of RAP1 binding was intragenic or proximal to coding regions. Based on this, they described potential RAP1 target genes in the mouse genome. In RAP1-deficient mice, about one-third of the deregulated genes were noted to be RAP1 target genes, implicating RAP1-mediated gene regulation. In a later report, genome-wide RAP1 ChIP-Seq in telomerase-deficient mice showed altered RAP1 binding on telomere shortening (
      • Martínez P.
      • Gómez-López G.
      • Pisano D.G.
      • Flores J.M.
      • Blasco M.A.
      A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.
      ). Further, 63 genes in the human genome were reported to have RAP1 occupancy (
      • Yang D.
      • Xiong Y.
      • Kim H.
      • He Q.
      • Li Y.
      • Chen R.
      • Songyang Z.
      Human telomeric proteins occupy selective interstitial sites.
      ).
      It was reported that RAP1 interacted with the IκB kinase, a function not expected of shelterin. This resulted in activation of NF-κB, leading to up-regulation of NF-κB target genes (
      • Teo H.
      • Ghosh S.
      • Luesch H.
      • Ghosh A.
      • Wong E.T.
      • Malik N.
      • Orth A.
      • de Jesus P.
      • Perry A.S.
      • Oliver J.D.
      • Tran N.L.
      • Speiser L.J.
      • Wong M.
      • Saez E.
      • Schultz P.
      • et al.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
      ). It was therefore postulated that expression of NF-κB targets might be telomere length–dependent (Fig. 3) (
      • Crabbe L.
      • Karlseder J.
      Mammalian Rap1 widens its impact.
      ) (Table 1).
      Figure thumbnail gr3
      Figure 3Extra-telomeric functions of shelterin proteins independent of telomeres. Examples of noncanonical function(s) of shelterin proteins are shown. Extra-telomeric binding of TRF2 to G-quadruplex–forming sequences across the genome induces epigenetic and transcriptional changes. NF-κB signaling is modulated through the shelterin factor RAP1: telomere-independent interaction of RAP1 with the IKK complex results in phosphorylation of the NF-κB p65 subunit, leading to activation of NF-κB target gene(s). Mitochondrial localization of TIN2, another shelterin protein, was reported to negatively regulate oxidative phosphorylation.
      Extra-telomeric function of RAP1 was also noted in positive regulation of PPARα and PGC1α genes in mice, which affected cellular metabolism related to obesity (
      • Martínez P.
      • Gómez-López G.
      • García F.
      • Mercken E.
      • Mitchell S.
      • Flores J.M.
      • de Cabo R.
      • Blasco M.A.
      RAP1 protects from obesity through its extratelomeric role regulating gene expression.
      ,
      • Yeung F.
      • Ramírez C.M.
      • Mateos-Gomez P.A.
      • Pinzaru A.
      • Ceccarini G.
      • Kabir S.
      • Fernández-Hernando C.
      • Sfeir A.
      Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity.
      ). RAP1 interaction with other co-factors was also reported in mesenchymal stem cell–based therapy for myocardial infarction and inflammation-dependent disorders (
      • Cai Y.
      • Kandula V.
      • Kosuru R.
      • Ye X.
      • Irwin M.G.
      • Xia Z.
      Decoding telomere protein Rap1: its telomeric and nontelomeric functions and potential implications in diabetic cardiomyopathy.
      ,
      • Ding Y.
      • Liang X.
      • Zhang Y.
      • Yi L.
      • Shum H.C.
      • Chen Q.
      • Chan B.P.
      • Fan H.
      • Liu Z.
      • Tergaonkar V.
      • Qi Z.
      • Tse H.
      • Lian Q.
      Rap1 deficiency-provoked paracrine dysfunction impairs immunosuppressive potency of mesenchymal stem cells in allograft rejection of heart transplantation.
      ). Recently, Zhang et al. (
      • Zhang X.
      • Liu Z.
      • Liu X.
      • Wang S.
      • Zhang Y.
      • He X.
      • Sun S.
      • Ma S.
      • Shyh-Chang N.
      • Liu F.
      • Wang Q.
      • Wang X.
      • Liu L.
      • Zhang W.
      • Song M.
      • et al.
      Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis.
      ) observed another key function of RAP1 in epigenetic regulation of the RELN promoter in hematopoiesis (Table 1). Together, these show RAP1 functions that are clearly independent of its canonical role in the protection of telomeres.
      Noncanonical extra-telomeric function was also observed for another shelterin protein, TIN2. A truncated isoform of TIN2, hTIN2S, was found to localize outside telomeres and affect heterochromatin organization (
      • Kaminker P.G.
      • Kim S.-H.
      • Desprez P.-Y.
      • Campisi J.
      A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.
      ). However, in cells with elongated telomeres, the dual localization was lost, such that hTIN2S redistributed from nontelomeric chromatin to telomeres exclusively. This is consistent with the TSP model described above (Fig. 2), where redistribution of a shelterin protein with change in telomere length would be expected. TIN2 was also shown to localize to mitochondria and regulate oxidative phosphorylation and glucose metabolism (Fig. 3) (
      • Chen L.-Y.
      • Zhang Y.
      • Zhang Q.
      • Li H.
      • Luo Z.
      • Fang H.
      • Kim S.H.
      • Qin L.
      • Yotnda P.
      • Xu J.
      • Tu B.P.
      • Bai Y.
      • Songyang Z.
      Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
      ) (Table 1).
      Noncanonical function of TRF1 was noted in phosphorylation of nontelomeric TRF1 by Aurora-A, which resulted in mitotic abnormality (
      • Ohishi T.
      • Hirota T.
      • Tsuruo T.
      • Seimiya H.
      TRF1 mediates mitotic abnormalities induced by Aurora-A overexpression.
      ). This was also suggested by the role of TRF1 in chromosome segregation by positively regulating Aurora-B's centromeric function (
      • Ohishi T.
      • Muramatsu Y.
      • Yoshida H.
      • Seimiya H.
      TRF1 ensures the centromeric function of Aurora-B and proper chromosome segregation.
      ). Further, a crystallographic study showed that TRF1 interacts with TERB1, crucial for X-Y chromosome pairing, during meiosis (
      • Long J.
      • Huang C.
      • Chen Y.
      • Zhang Y.
      • Shi S.
      • Wu L.
      • Liu Y.
      • Liu C.
      • Wu J.
      • Lei M.
      Telomeric TERB1-TRF1 interaction is crucial for male meiosis.
      ) (Table 1).
      A telomere-independent role(s) was also reported for TRF2 in the transcriptional activation of HS3ST4 (
      • Biroccio A.
      • Cherfils-Vicini J.
      • Augereau A.
      • Pinte S.
      • Bauwens S.
      • Ye J.
      • Simonet T.
      • Horard B.
      • Jamet K.
      • Cervera L.
      • Mendez-Bermudez A.
      • Poncet D.
      • Grataroli R.
      • de Rodenbeeke C.T.
      • Salvati E.
      • et al.
      TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.
      ) and PDGFRβ (
      • El Maï M.
      • Wagner K.-D.
      • Michiels J.-F.
      • Ambrosetti D.
      • Borderie A.
      • Destree S.
      • Renault V.
      • Djerbi N.
      • Giraud-Panis M.-J.
      • Gilson E.
      • Wagner N.
      The telomeric protein TRF2 regulates angiogenesis by binding and activating the PDGFRβ promoter.
      ) and repression of the cell cycle–dependent kinase inhibitor CDKN1A (p21) (
      • Hussain T.
      • Saha D.
      • Purohit G.
      • Kar A.
      • Kishore Mukherjee A.
      • Sharma S.
      • Sengupta S.
      • Dhapola P.
      • Maji B.
      • Vedagopuram S.
      • Horikoshi N.T.
      • Horikoshi N.
      • Pandita R.K.
      • Bhattacharya S.
      • Bajaj A.
      • et al.
      Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
      ). TRF2 was further found to associate with core histone proteins (
      • Su C.-H.
      • Cheng C.
      • Tzeng T.-Y.
      • Lin I.-H.
      • Hsu M.-T.
      An H2A histone isotype, H2ac, associates with telomere and maintains telomere integrity.
      ,
      • Konishi A.
      • Izumi T.
      • Shimizu S.
      TRF2 protein interacts with core histones to stabilize chromosome ends.
      ), including the RE-1–silencing factor (REST) repressor complex (
      • Hussain T.
      • Saha D.
      • Purohit G.
      • Kar A.
      • Kishore Mukherjee A.
      • Sharma S.
      • Sengupta S.
      • Dhapola P.
      • Maji B.
      • Vedagopuram S.
      • Horikoshi N.T.
      • Horikoshi N.
      • Pandita R.K.
      • Bhattacharya S.
      • Bajaj A.
      • et al.
      Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
      ). In addition to these, a role of telomere-independent TRF2 was reported in natural killer cell activation, angiogenesis, and intrinsic aspects like nucleosome formation and chromatin compaction (
      • Kaur P.
      • Wu D.
      • Lin J.
      • Countryman P.
      • Bradford K.C.
      • Erie D.A.
      • Riehn R.
      • Opresko P.L.
      • Wang H.
      Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.
      ) (Table 1).
      As discussed above, recent work also showed TRF2-mediated regulation of genes spread across the genome. This involved interaction of TRF2 with DNA secondary structures called G-quadruplexes within promoters (
      • Mukherjee A.K.
      • Sharma S.
      • Bagri S.
      • Kutum R.
      • Kumar P.
      • Hussain A.
      • Singh P.
      • Saha D.
      • Kar A.
      • Dash D.
      • Chowdhury S.
      Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.
      ). It is important to mention here that sequences with potential to form G-quadruplex structures are enriched in regulatory regions throughout the genome across genera (
      • Rawal P.
      • Kummarasetti V.B.R.
      • Ravindran J.
      • Kumar N.
      • Halder K.
      • Sharma R.
      • Mukerji M.
      • Das S.K.
      • Chowdhury S.
      Genome-wide prediction of G4 DNA as regulatory motifs: role in Escherichia coli global regulation.
      ,
      • Huppert J.L.
      • Balasubramanian S.
      G-quadruplexes in promoters throughout the human genome.
      ,
      • Verma A.
      • Halder K.
      • Halder R.
      • Yadav V.K.
      • Rawal P.
      • Thakur R.K.
      • Mohd F.
      • Sharma A.
      • Chowdhury S.
      Genome-wide computational and expression analyses reveal G-quadruplex DNA motifs as conserved cis-regulatory elements in human and related species.
      ,
      • Yadav V.K.
      • Abraham J.K.
      • Mani P.
      • Kulshrestha R.
      • Chowdhury S.
      QuadBase: genome-wide database of G4 DNA–occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes.
      ,
      • Dhapola P.
      • Chowdhury S.
      QuadBase2: web server for multiplexed guanine quadruplex mining and visualization.
      ,
      • Yadav V.
      • Hemansi
      • Kim N.
      • Tuteja N.
      • Yadav P.
      G quadruplex in plants: a ubiquitous regulatory element and its biological relevance.
      ), and evidence suggests that G-quadruplexes might influence local epigenetic modifications (
      • Saha D.
      • Singh A.
      • Hussain T.
      • Srivastava V.
      • Sengupta S.
      • Kar A.
      • Dhapola P.
      • Dhople V.
      • Ummanni R.
      • Chowdhury S.
      Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.
      ,
      • Guilbaud G.
      • Murat P.
      • Recolin B.
      • Campbell B.C.
      • Maiter A.
      • Sale J.E.
      • Balasubramanian S.
      Local epigenetic reprogramming induced by G-quadruplex ligands.
      ,
      • Mukherjee A.K.
      • Sharma S.
      • Chowdhury S.
      Non-duplex G-quadruplex structures emerge as mediators of epigenetic modifications.
      ).

      Telomeres in 3D—correlation of telomere architecture and cell state

      Telomeres are known to be organized within the nuclear matrix through interactions with lamin. Further telomeric association with distant parts of chromatin by looping are also known. Several studies show that these three-dimensional associations (i.e. nonlocal or extra-telomeric interactions) can impact cellular functions.
      Early work from the Blackburn group (
      • Wang X.
      • Kam Z.
      • Carlton P.M.
      • Xu L.
      • Sedat J.W.
      • Blackburn E.H.
      Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy.
      ) found telomeres to be motile with rapid motions of the telomere ends within the nucleus. Moreover, individual telomeres in a nucleus showed heterogeneity in motility. Relatively short uncapped telomeres in cancer cells had more motility than cells with long telomeres, possibly due to untethering of telomeres from the nuclear matrix (
      • Wang X.
      • Kam Z.
      • Carlton P.M.
      • Xu L.
      • Sedat J.W.
      • Blackburn E.H.
      Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy.
      ,
      • Burla R.
      • La Torre M.
      • Saggio I.
      Mammalian telomeres and their partnership with lamins.
      ,
      • Gonzalo S.
      • Eissenberg J.C.
      Tying up loose ends: telomeres, genomic instability and lamins.
      ). Furthermore, TRF2 association with lamin A/C proteins was observed to promote physical association of telomeres with interstitial chromatin through looping (
      • Wood A.M.
      • Danielsen J.M.R.
      • Lucas C.A.
      • Rice E.L.
      • Scalzo D.
      • Shimi T.
      • Goldman R.D.
      • Smith E.D.
      • Le Beau M.M.
      • Kosak S.T.
      TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends.
      ,
      • Smith E.D.
      • Garza-Gongora A.G.
      • MacQuarrie K.L.
      • Kosak S.T.
      Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.
      ) (Table 1).
      Telomere-associated change in chromatin organization was noticed in other cell types also. A study in 2014 reported altered 3D telomeric architecture in buccal cells derived from Alzheimer's disease (AD) patients of mild, moderate, and extreme pathology using three-dimensional (3D) microscopy and quantitative fluorescence in situ hybridization. Telomere aggregates and overall numbers increased in mild to severe AD along with a decrease in telomere length (
      • Mathur S.
      • Glogowska A.
      • McAvoy E.
      • Righolt C.
      • Rutherford J.
      • Willing C.
      • Banik U.
      • Ruthirakuhan M.
      • Mai S.
      • Garcia A.
      Three-dimensional quantitative imaging of telomeres in buccal cells identifies mild, moderate, and severe Alzheimer's disease patients.
      ). A follow-up study of the same parameters between AD and control group buccal cell samples found similar results (
      • Garcia A.
      • Mathur S.
      • Kalaw M.C.
      • McAvoy E.
      • Anderson J.
      • Luedke A.
      • Itorralba J.
      • Mai S.
      Quantitative 3D telomeric imaging of buccal cells reveals Alzheimer's disease-specific signatures.
      ).
      Tichy et al. (
      • Tichy E.D.
      • Sidibe D.K.
      • Tierney M.T.
      • Stec M.J.
      • Sharifi-Sanjani M.
      • Hosalkar H.
      • Mubarak S.
      • Johnson F.B.
      • Sacco A.
      • Mourkioti F.
      Single stem cell imaging and analysis reveals telomere length differences in diseased human and mouse skeletal muscles.
      ) reported altered telomere length and inconsistency in telomeric foci in the muscle stem cells of Duchenne muscular dystrophy patients when compared with the control group. On the other hand, muscle-stem cell telomere length remained unchanged between young and old healthy mice (
      • Tichy E.D.
      • Sidibe D.K.
      • Tierney M.T.
      • Stec M.J.
      • Sharifi-Sanjani M.
      • Hosalkar H.
      • Mubarak S.
      • Johnson F.B.
      • Sacco A.
      • Mourkioti F.
      Single stem cell imaging and analysis reveals telomere length differences in diseased human and mouse skeletal muscles.
      ). This was similar to what was previously noted for humans and macaques (
      • Gardner J.P.
      • Kimura M.
      • Chai W.
      • Durrani J.F.
      • Tchakmakjian L.
      • Cao X.
      • Lu X.
      • Li G.
      • Peppas A.P.
      • Skurnick J.
      • Wright W.E.
      • Shay J.W.
      • Aviv A.
      Telomere dynamics in macaques and humans.
      ), although mouse somatic tissues have longer telomeres and higher telomerase activity than humans and other primates.
      Similar observations were noted in other cases. For example, esophageal squamous cell carcinoma cells have altered 3D telomere organization compared with normal epithelial cells from the same patient with relatively long telomeres (
      • Sunpaweravong S.
      • Sunpaweravong P.
      • Sathitruangsak C.
      • Mai S.
      Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.
      ), and thyrospheres constituting stem cells from four subgroups of papillary thyroid carcinoma patients were found to have telomeric localization that was unique for each subgroup (
      • Caria P.
      • Dettori T.
      • Frau D.V.
      • Lichtenzstejn D.
      • Pani F.
      • Vanni R.
      • Mai S.
      Characterizing the three-dimensional organization of telomeres in papillary thyroid carcinoma cells.
      ).
      These data, describing studies of telomere-dependent gene expression and chromatin folding mediated through telomeres or telomere-associated factors, provide a clear view of how extra-telomeric functions may contribute to the role of telomeres in basic biological processes. Now we turn to the functions and implications of telomeres in integrated systems during health and disease.

      Telomeres in cellular pluripotency and “stemness”—emerging observations

      Induced pluripotent stem cells (iPSCs) have rapidly gained significance in basic and applied biological sciences (
      • Takahashi K.
      • Yamanaka S.
      Induced pluripotent stem cells in medicine and biology.
      ) and serve as a facile model system for cellular differentiation and development. The important role of telomere elongation and homeostasis in formation/maintenance of iPSCs, including their implications in aging, is known (
      • Harrington L.
      Does the reservoir for self-renewal stem from the ends?.
      ,
      • Liu L.
      Linking telomere regulation to stem cell pluripotency.
      ). In the following sections, we discuss the importance of telomeres in self-renewal and chromosome stability in iPSCs and consider emerging literature on how extra-telomeric function of telomere-associated factors might influence pluripotency.

      Telomere elongation and pluripotency

      The presence of relatively long telomeres has been generally observed in pluripotent cells. For example, reprogramed iPSCs obtained from mice showed elongated telomeres in the pluripotent cells (
      • Marión R.M.
      • López de Silanes I.
      • Mosteiro L.
      • Gamache B.
      • Abad M.
      • Guerra C.
      • Megías D.
      • Serrano M.
      • Blasco M.A.
      Common telomere changes during in vivo reprogramming and early stages of tumorigenesis.
      ); iPSCs generated from dyskeratosis congenita patient samples had relatively long telomeres (
      • Agarwal S.
      • Loh Y.-H.
      • McLoughlin E.M.
      • Huang J.
      • Park I.-H.
      • Miller J.D.
      • Huo H.
      • Okuka M.
      • dos Reis R.M.
      • Loewer S.
      • Ng H.-H.
      • Keefe D.L.
      • Goldman F.D.
      • Klingelhutz A.J.
      • Liu L.
      • et al.
      Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
      ), and human fibroblast TIG-1 cells reprogrammed to iPSCs, telomeres increased from 6 to 8 Kb (
      • Kamada M.
      • Mitsui Y.
      • Matsuo T.
      • Takahashi T.
      Reversible transformation and de-differentiation of human cells derived from induced pluripotent stem cell teratomas.
      ). Similarly, in many cancers like liposarcoma, hepatocellular carcinoma, and in pliocytic astrocytoma, elongated telomeres were noted in the dedifferentiated or pluripotent cells (
      • Wood L.D.
      • Heaphy C.M.
      • Daniel H.D.-J.
      • Naini B.V.
      • Lassman C.R.
      • Arroyo M.R.
      • Kamel I.R.
      • Cosgrove D.P.
      • Boitnott J.K.
      • Meeker A.K.
      • Torbenson M.S.
      Chromophobe hepatocellular carcinoma with abrupt anaplasia: a proposal for a new subtype of hepatocellular carcinoma with unique morphological and molecular features.
      ,
      • Lee J.-C.
      • Jeng Y.-M.
      • Liau J.-Y.
      • Tsai J.-H.
      • Hsu H.-H.
      • Yang C.-Y.
      Alternative lengthening of telomeres and loss of ATRX are frequent events in pleomorphic and dedifferentiated liposarcomas.
      ,
      • Rodriguez F.J.
      • Brosnan-Cashman J.A.
      • Allen S.J.
      • Vizcaino M.A.
      • Giannini C.
      • Camelo-Piragua S.
      • Webb M.
      • Matsushita M.
      • Wadhwani N.
      • Tabbarah A.
      • Hamideh D.
      • Jiang L.
      • Chen L.
      • Arvanitis L.D.
      • Alnajar H.H.
      • et al.
      Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia.
      ).
      Consistent with this, shortening of telomeres was associated with differentiation. Telomere attrition was associated with loss of stemness markers in cardiac progenitor cells isolated from adult human heart failure cases (
      • Hariharan N.
      • Quijada P.
      • Mohsin S.
      • Joyo A.
      • Samse K.
      • Monsanto M.
      • De La Torre A.
      • Avitabile D.
      • Ormachea L.
      • McGregor M.J.
      • Tsai E.J.
      • Sussman M.A.
      Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging.
      ); unstable differentiation was observed in ESCs with telomere dysfunction because of critically shorter telomeres (
      • Pucci F.
      • Gardano L.
      • Harrington L.
      Short telomeres in ESCs lead to unstable differentiation.
      ); regenerative capacity of stem cells declined because of progressive telomere attrition in aging cells (
      • Diao D.
      • Wang H.
      • Li T.
      • Shi Z.
      • Jin X.
      • Sperka T.
      • Zhu X.
      • Zhang M.
      • Yang F.
      • Cong Y.
      • Shen L.
      • Zhan Q.
      • Yan J.
      • Song Z.
      • Ju Z.
      Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan.
      ); and reduced proliferation and differentiation to osteoblasts was observed in mesenchymal stromal cells isolated from adults compared with those isolated from children, suggesting the impact of telomere attrition (
      • Choumerianou D.M.
      • Martimianaki G.
      • Stiakaki E.
      • Kalmanti L.
      • Kalmanti M.
      • Dimitriou H.
      Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults.
      ). On the other hand, longer life span was observed in cells that differentiated from human ESCs possessing relatively long telomeres (
      • Zou Y.
      • Tong H.J.
      • Li M.
      • Tan K.S.
      • Cao T.
      Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies.
      ) and cardiomyocytes differentiated with improved efficacy from iPSCs that had relatively long telomeres (
      • Aguado T.
      • Gutiérrez F.J.
      • Aix E.
      • Schneider R.P.
      • Giovinazzo G.
      • Blasco M.A.
      • Flores I.
      Telomere length defines the cardiomyocyte differentiation potency of mouse induced pluripotent stem cells.
      ).
      Furthermore, impaired differentiation due to poor telomere maintenance was observed in keratinocytes (
      • Martínez P.
      • Ferrara-Romeo I.
      • Flores J.M.
      • Blasco M.A.
      Essential role for the TRF2 telomere protein in adult skin homeostasis.
      ), and telomere elongation was found to be key for telomere length homeostasis in mouse embryonic stem cells (
      • Dan J.
      • Rousseau P.
      • Hardikar S.
      • Veland N.
      • Wong J.
      • Autexier C.
      • Chen T.
      Zscan4 inhibits maintenance DNA methylation to facilitate telomere elongation in mouse embryonic stem cells.
      ). Together, these studies show the importance of telomere length maintenance in stem cells and how telomere shortening or attrition (with aging) impacts differentiation (Fig. 4).
      Figure thumbnail gr4
      Figure 4Role of telomeres in stem cell homeostasis. Importance of telomere length in maintaining stemness. Stem cells with relatively long telomeres are reported to retain stemness, whereas reduction of telomere length is generally observed during differentiation and/or in differentiated cells.

      Role of telomere-associated factors in pluripotency or “stemness” and disease

      The pluripotency factor Oct3/4 was reported to positively regulate TRF1 during the induction and maintenance of pluripotency (
      • Schneider R.P.
      • Garrobo I.
      • Foronda M.
      • Palacios J.A.
      • Marión R.M.
      • Flores I.
      • Ortega S.
      • Blasco M.A.
      TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells.
      ). Consistent with this, TRF1 was observed to be up-regulated in ESCs and iPSCs (
      • Sunpaweravong S.
      • Sunpaweravong P.
      • Sathitruangsak C.
      • Mai S.
      Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.
      ,
      • Hariharan N.
      • Quijada P.
      • Mohsin S.
      • Joyo A.
      • Samse K.
      • Monsanto M.
      • De La Torre A.
      • Avitabile D.
      • Ormachea L.
      • McGregor M.J.
      • Tsai E.J.
      • Sussman M.A.
      Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging.
      ), and in the presence of the small molecule ETP-47037, which inhibits TRF1, reprograming efficiency in mice was reduced (
      • Marión R.M.
      • López de Silanes I.
      • Mosteiro L.
      • Gamache B.
      • Abad M.
      • Guerra C.
      • Megías D.
      • Serrano M.
      • Blasco M.A.
      Common telomere changes during in vivo reprogramming and early stages of tumorigenesis.
      ). Moreover, increase in TRF1 expression was observed during in vitro derivation of ESCs from the inner cell mass (
      • Varela E.
      • Schneider R.P.
      • Ortega S.
      • Blasco M.A.
      Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells.
      ) (Table 1).
      Several reports implicated TRF2 in pluripotency. Self-renewal and maintenance potential was perturbed when TRF2 was deleted in alveolar stem cells (
      • Alder J.K.
      • Barkauskas C.E.
      • Limjunyawong N.
      • Stanley S.E.
      • Kembou F.
      • Tuder R.M.
      • Hogan B.L.M.
      • Mitzner W.
      • Armanios M.
      Telomere dysfunction causes alveolar stem cell failure.
      ), and human mesenchymal stem cells showed increased sensitivity to irradiation when TRF2 was knocked down (
      • Orun O.
      • Tiber P.M.
      • Serakinci N.
      Partial knockdown of TRF2 increase radiosensitivity of human mesenchymal stem cells.
      ,
      • Serakinci N.
      • Mega Tiber P.
      • Orun O.
      Chromatin modifications of hTERT gene in hTERT-immortalized human mesenchymal stem cells upon exposure to radiation.
      ). Further, in TRF2-null mice, terminal differentiation was triggered during skin carcinogenesis (
      • Lagunas A.M.
      • Wu J.
      • Crowe D.L.
      Telomere DNA damage signaling regulates cancer stem cell evolution, epithelial mesenchymal transition, and metastasis.
      ), and increase in TRF2 was implicated in aggressive proliferation of liver cancer stem cells (
      • Wu M.
      • Lin Z.
      • Li X.
      • Xin X.
      • An J.
      • Zheng Q.
      • Yang Y.
      • Lu D.
      HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2.
      ).
      In the above studies, it was not clear whether the function of TRF2 was involved as a telomeric and/or extra-telomeric factor. However, we noted further work suggesting extra-telomeric function of TRF2 in stemness. This includes nuclear interaction of TRF2 with REST, which was reported to be important in maintenance of the neural stem cell population (
      • Zhang P.
      • Pazin M.J.
      • Schwartz C.M.
      • Becker K.G.
      • Wersto R.P.
      • Dilley C.M.
      • Mattson M.P.
      Nontelomeric TRF2-REST interaction modulates neuronal gene silencing and fate of tumor and stem cells.
      ,
      • Zhang P.
      • Casaday-Potts R.
      • Precht P.
      • Jiang H.
      • Liu Y.
      • Pazin M.J.
      • Mattson M.P.
      Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor.
      ). Further, TRF2 depletion resulted in reduced proliferation and enhanced differentiation of glioblastoma stem cells due to both telomeric dysfunction and loss of REST-mediated repression (
      • Bai Y.
      • Lathia J.D.
      • Zhang P.
      • Flavahan W.
      • Rich J.N.
      • Mattson M.P.
      Molecular targeting of TRF2 suppresses the growth and tumorigenesis of glioblastoma stem cells.
      ), and silencing of TRF2 resulted in the reduction of the Yamanaka factors in oral cancer stem cells (
      • Saha A.
      • Roy S.
      • Kar M.
      • Roy S.
      • Thakur S.
      • Padhi S.
      • Akhter Y.
      • Banerjee B.
      Role of telomeric TRF2 in orosphere formation and CSC phenotype maintenance through efficient DNA repair pathway and its correlation with recurrence in OSCC.
      ) (Table 1). In addition to these, computational modeling indicated high binding affinity of TRF2 to the stem-cell factor KLF4 (
      • Saha A.
      • Roy S.
      • Kar M.
      • Roy S.
      • Thakur S.
      • Padhi S.
      • Akhter Y.
      • Banerjee B.
      Role of telomeric TRF2 in orosphere formation and CSC phenotype maintenance through efficient DNA repair pathway and its correlation with recurrence in OSCC.
      ).
      TPP1-mediated recruitment of the reverse transcriptase telomerase (TERT) for telomere elongation was observed. Here, abrogation of TPP1 affected the reprograming of mouse embryonic fibroblasts (
      • Tejera A.M.
      • Stagno d'Alcontres M.
      • Thanasoula M.
      • Marion R.M.
      • Martinez P.
      • Liao C.
      • Flores J.M.
      • Tarsounas M.
      • Blasco M.A.
      TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.
      ). Later TPP1 was also shown to be important in maintaining the length of telomeres in human ESCs (
      • Sexton A.N.
      • Regalado S.G.
      • Lai C.S.
      • Cost G.J.
      • O'Neil C.M.
      • Urnov F.D.
      • Gregory P.D.
      • Jaenisch R.
      • Collins K.
      • Hockemeyer D.
      Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.
      ).
      Depletion of POT1, on the other hand, triggered DNA damage response and thereby telomeric dysfunction, resulting in reduced survival of hematopoietic stem cells (HSCs) and bone marrow failure that mimicked the phenotypes of dyskeratosis congenita (
      • He H.
      • Wang Y.
      • Guo X.
      • Ramchandani S.
      • Ma J.
      • Shen M.-F.
      • Garcia D.A.
      • Deng Y.
      • Multani A.S.
      • You M.J.
      • Chang S.
      Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita.
      ). Exogenous expression of POT1 induced self-renewal of human HSCs by inhibiting generation of reactive oxygen species (Table 1) (
      • Hosokawa K.
      • MacArthur B.D.
      • Ikushima Y.M.
      • Toyama H.
      • Masuhiro Y.
      • Hanazawa S.
      • Suda T.
      • Arai F.
      The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age.
      ). In addition, POT1-mediated metabolic control and transcriptional regulation in HSCs was shown (
      • Hosokawa K.
      • Arai F.
      The role of telomere binding molecules for normal and abnormal hematopoiesis.
      ). Together, these implicate extra-telomeric functions of POT1 in pluripotency.
      Furthermore, mutations within shelterin genes were found to be associated with hematological malignancies due to telomere deprotection, showing the importance of the shelterin factors in hematopoiesis and self-renewal (
      • Hosokawa K.
      • Arai F.
      The role of telomere binding molecules for normal and abnormal hematopoiesis.
      ).
      The role of the noncoding RNA transcribed from telomeres called telomeric repeat–containing RNA (TERRA) in pluripotency is also notable. TERRA was found to be overexpressed and contributed to the self-renewal of mesenchymal stem cells (
      • Xu X.
      • Guo M.
      • Zhang N.
      • Ye S.
      Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity.
      ). In addition, decline in TERRA resulted in differentiation, and overexpression resulted in rescue of the self-renewal activity (
      • Xu X.
      • Guo M.
      • Zhang N.
      • Ye S.
      Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity.
      ). TERRA foci formation (i.e. clustered presence of TERRA molecules as seen in microscopy) due to elevated expression and aggregation of TERRA was reported to occur in both developing cerebellar neural progenitors and medulloblastoma (
      • Deng Z.
      • Wang Z.
      • Xiang C.
      • Molczan A.
      • Baubet V.
      • Conejo-Garcia J.
      • Xu X.
      • Lieberman P.M.
      • Dahmane N.
      Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma.
      ). A more recent study showed how TERRA through a TRF1-dependent mode regulates the transcriptional state of ESCs such that a naive state is maintained (
      • Marión R.M.
      • Montero J.J.
      • López de Silanes I.
      • Graña-Castro O.
      • Martínez P.
      • Schoeftner S.
      • Palacios-Fábrega J.A.
      • Blasco M.A.
      TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2.
      ).
      Decrease in telomere length with age and associated telomere dysfunction contributes to initiation and progress of cancer (
      • Martínez P.
      • Blasco M.A.
      Replicating through telomeres: a means to an end.
      ,
      • Gunes C.
      • Avila A.I.
      • Rudolph K.L.
      Telomeres in cancer.
      ). It is also widely known that in more than 90% of human cancers, telomerase (hTERT)—the enzyme necessary for telomere synthesis—is reactivated, and as a result telomeres are maintained, unlike in normal adult somatic cells (
      • Shay J.W.
      • Wright W.E.
      Telomeres and telomerase: three decades of progress.
      ). However, despite reactivation, most cancer cells and cancer stem cells have shorter telomeres than surrounding normal cells (
      • Shay J.W.
      • Wright W.E.
      Telomeres and telomerase: three decades of progress.
      ,
      • Shay J.W.
      • Wright W.E.
      Telomeres and telomerase in normal and cancer stem cells.
      ,
      • Barthel F.P.
      • Wei W.
      • Tang M.
      • Martinez-Ledesma E.
      • Hu X.
      • Amin S.B.
      • Akdemir K.C.
      • Seth S.
      • Song X.
      • Wang Q.
      • Lichtenberg T.
      • Hu J.
      • Zhang J.
      • Zheng S.
      • Verhaak R.G.W.
      Systematic analysis of telomere length and somatic alterations in 31 cancer types.
      ).
      Expression of hTERT was shown to involve TPE-OLD (
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ). In normal cells, the chromosome 5p telomere folds backs and associates with the hTERT loci ∼1.3 Mb away. Kim et al. (
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ) concluded that through this interaction telomeric TRF2 associates with the hTERT promoter. Further, the TRF2 interaction was lost in cells with relatively short telomeres where the telomeric loop was unable to form (
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ). Loss of TRF2 from the hTERT promoter correlated with increased hTERT expression. However, it was not clear whether this involved TRF2-mediated regulation or was a result of telomere-induced gene silencing as noted for several genes in earlier studies (
      • Robin J.D.
      • Ludlow A.T.
      • Batten K.
      • Magdinier F.
      • Stadler G.
      • Wagner K.R.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.
      ,
      • Gottschling D.E.
      • Aparicio O.M.
      • Billington B.L.
      • Zakian V.A.
      Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.
      ).
      More recent work, on the other hand, suggested that hTERT regulation is under direct transcriptional control of TRF2 (118). Here, TRF2 presence on the hTERT promoter was independent of telomeres (i.e. there was involvement of extra-telomeric TRF2). This was also clear from TRF2 occupancy at the exogenously inserted hTERT promoter ∼46 Mb away from telomeres (
      • Sharma S.
      • Mukherjee A.K.
      • Roy S.S.
      • Bagri S.
      • Lier S.
      • Verma M.
      • Sengupta A.
      • Kumar M.
      • Nesse G.
      • Pandey D.P.
      • Chowdhury S.
      Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2.
      )—where looping due to physical proximity like the 5p telomere end was unlikely. Together, these leads suggest the involvement of the TSP model discussed above in hTERT regulation, where the presence of extra-telomeric TRF2 on the hTERT promoter is of interest and depends on how much TRF2 is free or sequestered at the telomere ends.
      How might aspects of extra-telomeric biology impact stem cells? Stem cells, that replenish “worn out” cells, undergo telomere shortening, as reviewed earlier (
      • Xu X.
      • Guo M.
      • Zhang N.
      • Ye S.
      Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity.
      ,
      • Deng Z.
      • Wang Z.
      • Xiang C.
      • Molczan A.
      • Baubet V.
      • Conejo-Garcia J.
      • Xu X.
      • Lieberman P.M.
      • Dahmane N.
      Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma.
      ), suggesting that many of the mechanisms described above could be in play. It must be mentioned here that although the literature suggests a potential role of extra-telomeric function(s) in pluripotency/stemness, evidence supporting direct causal links remains to be established to the best of our knowledge.
      The platelet-derived growth factor receptor (PDGFR) was found to be significantly abrogated in the myocardium of people with increasing age, suggesting the role of PDGFR signaling in cardiomyocyte regeneration and proliferation (
      • Yue Z.
      • Chen J.
      • Lian H.
      • Pei J.
      • Li Y.
      • Chen X.
      • Song S.
      • Xia J.
      • Zhou B.
      • Feng J.
      • Zhang X.
      • Hu S.
      • Nie Y.
      PDGFR-β signaling regulates cardiomyocyte proliferation and myocardial regeneration.
      ). This was consistent with the telomere length–dependent differentiation of cardiomyocytes observed frequently (reviewed in Ref.
      • Aguado T.
      • Gutiérrez F.J.
      • Aix E.
      • Schneider R.P.
      • Giovinazzo G.
      • Blasco M.A.
      • Flores I.
      Telomere length defines the cardiomyocyte differentiation potency of mouse induced pluripotent stem cells.
      ). As mentioned above, PDGFR-β is a transcriptional target of extra-telomeric TRF2 (
      • El Maï M.
      • Wagner K.-D.
      • Michiels J.-F.
      • Ambrosetti D.
      • Borderie A.
      • Destree S.
      • Renault V.
      • Djerbi N.
      • Giraud-Panis M.-J.
      • Gilson E.
      • Wagner N.
      The telomeric protein TRF2 regulates angiogenesis by binding and activating the PDGFRβ promoter.
      ). Furthermore, it was demonstrated that PDGFR-β is regulated epigenetically by TRF2 in a telomere length-dependent fashion (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). Therefore, it is likely that regulation of PDGFR-β by extra-telomeric TRF2, which depends on telomere length (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ) (described above as TSP), plays a more direct role in telomere-dependent cardiomyocyte differentiation described above.
      A recent study demonstrated the increased expression of genes related to neurogenesis and neuronal maturation in sporadic Alzheimer's disease, suggesting a potential link between neuronal differentiation and this debilitating neurodegenerative disease (
      • Meyer K.
      • Feldman H.M.
      • Lu T.
      • Drake D.
      • Lim E.T.
      • Ling K.-H.
      • Bishop N.A.
      • Pan Y.
      • Seo J.
      • Lin Y.-T.
      • Su S.C.
      • Church G.M.
      • Tsai L.-H.
      • Yankner B.A.
      REST and neural gene network dysregulation in iPSC models of Alzheimer's disease.
      ). Telomere shortening, a hallmark of aging, is also widely observed in neurodegenerative diseases like AD (
      • Yue Z.
      • Chen J.
      • Lian H.
      • Pei J.
      • Li Y.
      • Chen X.
      • Song S.
      • Xia J.
      • Zhou B.
      • Feng J.
      • Zhang X.
      • Hu S.
      • Nie Y.
      PDGFR-β signaling regulates cardiomyocyte proliferation and myocardial regeneration.
      ,
      • Hiyama E.
      • Hiyama K.
      Telomere and telomerase in stem cells.
      ,
      • Hou Y.
      • Dan X.
      • Babbar M.
      • Wei Y.
      • Hasselbalch S.G.
      • Croteau D.L.
      • Bohr V.A.
      Ageing as a risk factor for neurodegenerative disease.
      ). Like the other cell types discussed above, short telomeres affect the proliferative capacity of neural stem cells and reduce the self-renewal potential of progenitors required for normal adult neurogenesis (
      • Ferrón S.
      • Mira H.
      • Franco S.
      • Cano-Jimenez M.
      • Bellmunt E.
      • Ramírez C.
      • Fariñas I.
      • Blasco M.A.
      Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells.
      ). Could a telomere function, and particularly an extra-telomeric function, serve as a molecular trigger underlying the pathophysiology of the disease (
      • 2019 Alzheimer's disease facts and figures
      )?
      The recent study on pathophysiology in AD also showed that accelerated differentiation of neural stem cells in AD was associated with deregulated levels of REST (
      • Meyer K.
      • Feldman H.M.
      • Lu T.
      • Drake D.
      • Lim E.T.
      • Ling K.-H.
      • Bishop N.A.
      • Pan Y.
      • Seo J.
      • Lin Y.-T.
      • Su S.C.
      • Church G.M.
      • Tsai L.-H.
      • Yankner B.A.
      REST and neural gene network dysregulation in iPSC models of Alzheimer's disease.
      ). Notably, earlier work had reported that extra-telomeric TRF2-mediated stabilization of REST was critical for neuronal differentiation (
      • Ovando-Roche P.
      • Yu J.S.L.
      • Testori S.
      • Ho C.
      • Cui W.
      TRF2-mediated stabilization of hREST4 is critical for the differentiation and maintenance of neural progenitors.
      ). Furthermore, recent findings showed that REST binding to nontelomeric chromatin was also dependent on extra-telomeric TRF2 (
      • Hussain T.
      • Saha D.
      • Purohit G.
      • Kar A.
      • Kishore Mukherjee A.
      • Sharma S.
      • Sengupta S.
      • Dhapola P.
      • Maji B.
      • Vedagopuram S.
      • Horikoshi N.T.
      • Horikoshi N.
      • Pandita R.K.
      • Bhattacharya S.
      • Bajaj A.
      • et al.
      Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
      ). As described above, in TSP (Fig. 2), the presence of extra-telomeric TRF2 depends on telomere length. Therefore, these findings suggest a direct causal link between telomere length, extra-telomeric TRF2, and REST in neural stem cells, which might be key to neuronal differentiation. It will be fascinating to determine whether cellular renewal, rather than the more canonical aggregation hypothesis, might play a driving role in this and other degenerative diseases.

      Conclusions and future perspectives

      The notion that telomeres influence function beyond chromosome ends is relatively recent. Findings from many research groups, including ours, reveal this to be through two primary modes: (a) physical looping of telomeres (mostly within subtelomeric regions) (
      • Robin J.D.
      • Ludlow A.T.
      • Batten K.
      • Magdinier F.
      • Stadler G.
      • Wagner K.R.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.
      ) or (b) extra-telomeric function of shelterin factor(s) (
      • Mukherjee A.K.
      • Sharma S.
      • Bagri S.
      • Kutum R.
      • Kumar P.
      • Hussain A.
      • Singh P.
      • Saha D.
      • Kar A.
      • Dash D.
      • Chowdhury S.
      Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.
      ,
      • Crabbe L.
      • Karlseder J.
      Mammalian Rap1 widens its impact.
      ,
      • Chen L.-Y.
      • Zhang Y.
      • Zhang Q.
      • Li H.
      • Luo Z.
      • Fang H.
      • Kim S.H.
      • Qin L.
      • Yotnda P.
      • Xu J.
      • Tu B.P.
      • Bai Y.
      • Songyang Z.
      Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
      ), which in the case of TRF2 depends on telomere length, as described above in the TSP model (Fig. 2) (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). The role of telomeres, particularly telomere length, has been observed closely during both pluripotency and stem cell differentiation. However, the underlying molecular processes that link telomeres to pluripotency are only beginning to emerge.
      Notably, recent work has shown that TRF2 binding throughout the genome results in epigenetic modifications. Further, this depends on telomere length (
      • Mukherjee A.K.
      • Sharma S.
      • Sengupta S.
      • Saha D.
      • Kumar P.
      • Hussain T.
      • Srivastava V.
      • Roy S.D.
      • Shay J.W.
      • Chowdhury S.
      Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
      ). Together, these findings contribute to a new understanding of telomeric factors. Moreover, these data suggest that a novel set of protein-protein interactions are possibly induced instead of the canonical shelterin complex at telomeres. It will be interesting to explore how these interactions with TRF2 and other telomeric factors are regulated (e.g. with distinct post-translational modifications that direct nontelomeric binding).
      Based on RAP1 and NF-κB interactions (
      • Teo H.
      • Ghosh S.
      • Luesch H.
      • Ghosh A.
      • Wong E.T.
      • Malik N.
      • Orth A.
      • de Jesus P.
      • Perry A.S.
      • Oliver J.D.
      • Tran N.L.
      • Speiser L.J.
      • Wong M.
      • Saez E.
      • Schultz P.
      • et al.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
      ), association of telomeric factors with proteins independent of DNA binding is another molecular aspect that might be worthwhile to study. Contextually, whether other telomeric factors associate with nuclear or cytoplasmic factors—and how such interactions are affected as telomere length changes—would be of interest.
      Telomerase—the only protein that synthesizes telomeres—is overexpressed in most cancers. Recent findings suggest that telomeres exert control over telomerase through telomeric or extra-telomeric mechanisms (
      • Kim W.
      • Ludlow A.T.
      • Min J.
      • Robin J.D.
      • Stadler G.
      • Mender I.
      • Lai T.-P.
      • Zhang N.
      • Wright W.E.
      • Shay J.W.
      Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
      ,
      • Sharma S.
      • Mukherjee A.K.
      • Roy S.S.
      • Bagri S.
      • Lier S.
      • Verma M.
      • Sengupta A.
      • Kumar M.
      • Nesse G.
      • Pandey D.P.
      • Chowdhury S.
      Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2.
      ). Teasing out molecular details of these controls, including whether and how the TSP model might be involved in telomere-dependent control of telomerase, remains to be studied in further detail, considering its broad and significant implications.
      Taken together, these new aspects of extra-telomeric biology—dependent on telomere length (and thereby aging)—may reveal a novel understanding of the molecular processes underlying pluripotency. Moreover, whether and how, particularly in what context, premature differentiation is linked to aging through telomeres would be of interest in improving our understanding of diseases associated with aging.

      Acknowledgments

      We acknowledge all members of the Chowdhury group for fruitful discussions on many aspects of the review and Dr. Munia Ganguli for assisting with editing of the manuscript.

      References

        • Palm W.
        • de Lange T.
        How shelterin protects mammalian telomeres.
        Annu. Rev. Genet. 2008; 42 (18680434): 301-334
        • Xin H.
        • Liu D.
        • Songyang Z.
        The telosome/shelterin complex and its functions.
        Genome Biol. 2008; 9 (18828880): 232
        • Červenák F.
        • Juríková K.
        • Sepšiová R.
        • Neboháčová M.
        • Nosek J.
        • Tomáška L.
        Double-stranded telomeric DNA binding proteins: diversity matters.
        Cell Cycle. 2017; 16 (28749196): 1568-1577
        • Schmutz I.
        • De Lange T.
        Shelterin.
        Curr. Biol. 2016; 26 (27218840): R397-R399
        • Timashev L.A.
        • Babcock H.
        • Zhuang X.
        • de Lange T.
        The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.
        Genes Dev. 2017; 31 (28381412): 578-589
        • de Lange T.
        Shelterin-mediated telomere protection.
        Annu. Rev. Genet. 2018; 52 (30208292): 223-247
        • Lim C.J.
        • Zaug A.J.
        • Kim H.J.
        • Cech T.R.
        Reconstitution of human shelterin complexes reveals unexpected stoichiometry and dual pathways to enhance telomerase processivity.
        Nat. Commun. 2017; 8 (29057866): 1075
        • Pike A.M.
        • Strong M.A.
        • Ouyang J.P.T.
        • Greider C.W.
        TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.
        Mol. Cell Biol. 2019; 39 (31383750): e00518-e00593
        • Nandakumar J.
        • Cech T.R.
        Finding the end: recruitment of telomerase to telomeres.
        Nat. Rev. Mol. Cell Biol. 2013; 14 (23299958): 69-82
        • Martínez P.
        • Blasco M.A.
        Replicating through telomeres: a means to an end.
        Trends Biochem. Sci. 2015; 40 (26188776): 504-515
        • Blackburn E.H.
        • Epel E.S.
        • Lin J.
        Human telomere biology: a contributory and interactive factor in aging, disease risks, and protection.
        Science. 2015; 350 (26785477): 1193-1198
        • Shay J.W.
        • Wright W.E.
        Telomeres and telomerase: three decades of progress.
        Nat. Rev. Genet. 2019; 20 (30760854): 299-309
        • Shay J.W.
        Role of telomeres and telomerase in aging and cancer.
        Cancer Discov. 2016; 6 (27029895): 584-593
        • Maciejowski J.
        • De Lange T.
        Telomeres in cancer: tumour suppression and genome instability.
        Nat. Rev. Mol. Cell Biol. 2017; 18 (28096526): 175-186
        • Okamoto K.
        • Seimiya H.
        Revisiting telomere shortening in cancer.
        Cells. 2019; 8 (30709063): 107
        • Baird D.M.
        Telomeres and genomic evolution.
        Philos. Trans. R. Soc. B Biol. Sci. 2018; 373 (29335376): 20160437
        • Monaghan P.
        • Ozanne S.E.
        Somatic growth and telomere dynamics in vertebrates: relationships, mechanisms and consequences.
        Philos. Trans. R. Soc. B Biol. Sci. 2018; 373 (29335370): 20160446
        • Tian X.
        • Doerig K.
        • Park R.
        • Can Ran Qin A.
        • Hwang C.
        • Neary A.
        • Gilbert M.
        • Seluanov A.
        • Gorbunova V.
        Evolution of telomere maintenance and tumour suppressor mechanisms across mammals.
        Philos. Trans. R. Soc. B Biol. Sci. 2018; 373 (29335367): 20160443
        • Martinez P.
        • Thanasoula M.
        • Carlos A.R.
        • Gómez-López G.
        • Tejera A.M.
        • Schoeftner S.
        • Dominguez O.
        • Pisano D.G.
        • Tarsounas M.
        • Blasco M.A.
        Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
        Nat. Cell Biol. 2010; 12 (20622869): 768-780
        • Yang D.
        • Xiong Y.
        • Kim H.
        • He Q.
        • Li Y.
        • Chen R.
        • Songyang Z.
        Human telomeric proteins occupy selective interstitial sites.
        Cell Res. 2011; 21 (21423278): 1013-1027
        • Simonet T.
        • Zaragosi L.-E.
        • Philippe C.
        • Lebrigand K.
        • Schouteden C.
        • Augereau A.
        • Bauwens S.
        • Ye J.
        • Santagostino M.
        • Giulotto E.
        • Magdinier F.
        • Horard B.
        • Barbry P.
        • Waldmann R.
        • Gilson E.
        The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats.
        Cell Res. 2011; 21 (21423270): 1028-1038
        • Robin J.D.
        • Ludlow A.T.
        • Batten K.
        • Magdinier F.
        • Stadler G.
        • Wagner K.R.
        • Shay J.W.
        • Wright W.E.
        Telomere position effect: regulation of gene expression with progressive telomere shortening over long distances.
        Genes Dev. 2014; 28 (25403178): 2464-2476
        • Robin J.D.
        • Ludlow A.T.
        • Batten K.
        • Gaillard M.-C.
        • Stadler G.
        • Magdinier F.
        • Wright W.E.
        • Shay J.W.
        SORBS2 transcription is activated by telomere position effect-over long distance upon telomere shortening in muscle cells from patients with facioscapulohumeral dystrophy.
        Genome Res. 2015; 25 (26359233): 1781-1790
        • Mukherjee A.K.
        • Sharma S.
        • Sengupta S.
        • Saha D.
        • Kumar P.
        • Hussain T.
        • Srivastava V.
        • Roy S.D.
        • Shay J.W.
        • Chowdhury S.
        Telomere length-dependent transcription and epigenetic modifications in promoters remote from telomere ends.
        PLoS Genet. 2018; 14 (30439955): e1007782
        • Harrington L.
        Does the reservoir for self-renewal stem from the ends?.
        Oncogene. 2004; 23 (15378088): 7283-7289
        • Aguado T.
        • Gutiérrez F.J.
        • Aix E.
        • Schneider R.P.
        • Giovinazzo G.
        • Blasco M.A.
        • Flores I.
        Telomere length defines the cardiomyocyte differentiation potency of mouse induced pluripotent stem cells.
        Stem Cells. 2017; 35 (27612935): 362-373
        • Martínez P.
        • Ferrara-Romeo I.
        • Flores J.M.
        • Blasco M.A.
        Essential role for the TRF2 telomere protein in adult skin homeostasis.
        Aging Cell. 2014; 13 (24725274): 656-668
        • Zou Y.
        • Tong H.J.
        • Li M.
        • Tan K.S.
        • Cao T.
        Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies.
        Biogerontology. 2017; 18 (27757766): 69-84
        • Meyer K.
        • Feldman H.M.
        • Lu T.
        • Drake D.
        • Lim E.T.
        • Ling K.-H.
        • Bishop N.A.
        • Pan Y.
        • Seo J.
        • Lin Y.-T.
        • Su S.C.
        • Church G.M.
        • Tsai L.-H.
        • Yankner B.A.
        REST and neural gene network dysregulation in iPSC models of Alzheimer's disease.
        Cell Rep. 2019; 26 (30699343): 1112-1127.e9
        • Gottschling D.E.
        • Aparicio O.M.
        • Billington B.L.
        • Zakian V.A.
        Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.
        Cell. 1990; 63 (2225075): 751-762
        • Baur J.A.
        • Zou Y.
        • Shay J.W.
        • Wright W.E.
        Telomere position effect in human cells.
        Science. 2001; 292 (11408657): 2075-2077
        • Ottaviani A.
        • Gilson E.
        • Magdinier F.
        Telomeric position effect: from the yeast paradigm to human pathologies?.
        Biochimie. 2008; 90 (17868970): 93-107
        • Kim W.
        • Ludlow A.T.
        • Min J.
        • Robin J.D.
        • Stadler G.
        • Mender I.
        • Lai T.-P.
        • Zhang N.
        • Wright W.E.
        • Shay J.W.
        Regulation of the human telomerase gene TERT by telomere position effect—over long distances (TPE-OLD): implications for aging and cancer.
        PLoS Biol. 2016; 14 (27977688): e2000016
        • Kim W.
        • Shay J.W.
        Long-range telomere regulation of gene expression: telomere looping and telomere position effect over long distances (TPE-OLD).
        Differentiation. 2018; 99 (29197683): 1-9
        • Smogorzewska A.
        • van Steensel B.
        • Bianchi A.
        • Oelmann S.
        • Schaefer M.R.
        • Schnapp G.
        • de Lange T.
        Control of human telomere length by TRF1 and TRF2.
        Mol. Cell Biol. 2000; 20 (10669743): 1659-1668
        • Takai K.K.
        • Hooper S.
        • Blackwood S.
        • Gandhi R.
        • de Lange T.
        In vivo stoichiometry of shelterin components.
        J. Biol. Chem. 2010; 285 (19864690): 1457-1467
        • Mukherjee A.K.
        • Sharma S.
        • Bagri S.
        • Kutum R.
        • Kumar P.
        • Hussain A.
        • Singh P.
        • Saha D.
        • Kar A.
        • Dash D.
        • Chowdhury S.
        Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters.
        J. Biol. Chem. 2019; 294 (31575660): 17709-17722
        • O'Sullivan R.J.
        • Kubicek S.
        • Schreiber S.L.
        • Karlseder J.
        Reduced histone biosynthesis and chromatin changes arising from a damage signal at telomeres.
        Nat. Struct. Mol. Biol. 2010; 17 (20890289): 1218-1225
        • Hauer M.H.
        • Seeber A.
        • Singh V.
        • Thierry R.
        • Sack R.
        • Amitai A.
        • Kryzhanovska M.
        • Eglinger J.
        • Holcman D.
        • Owen-Hughes T.
        • Gasser S.M.
        Histone degradation in response to DNA damage enhances chromatin dynamics and recombination rates.
        Nat. Struct. Mol. Biol. 2017; 24 (28067915): 99-107
        • Song S.
        • Johnson F.
        Epigenetic mechanisms impacting aging: a focus on histone levels and telomeres.
        Genes (Basel). 2018; 9 (29642537): 201
        • Pucci F.
        • Gardano L.
        • Harrington L.
        Short telomeres in ESCs lead to unstable differentiation.
        Cell Stem Cell. 2013; 12 (23561444): 479-486
        • Harrington L.
        • Pucci F.
        In medio stat virtus: unanticipated consequences of telomere dysequilibrium.
        Philos. Trans. R. Soc. B Biol. Sci. 2018; 373 (29335368): 20160444
        • Platt J.M.
        • Ryvkin P.
        • Wanat J.J.
        • Donahue G.
        • Ricketts M.D.
        • Barrett S.P.
        • Waters H.J.
        • Song S.
        • Chavez A.
        • Abdallah K.O.
        • Master S.R.
        • Wang L.S.
        • Johnson F.B.
        Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence.
        Genes Dev. 2013; 27 (23756653): 1406-1420
        • Song S.
        • Perez J.V.
        • Svitko W.
        • Ricketts M.D.
        • Dean E.
        • Schultz D.
        • Marmorstein R.
        • Johnson F.B.
        Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence.
        Aging Cell. 2020; 19 (31742863): e13061
        • Martínez P.
        • Gómez-López G.
        • Pisano D.G.
        • Flores J.M.
        • Blasco M.A.
        A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.
        Aging Cell. 2016; 15 (27586969): 1113-1125
        • Teo H.
        • Ghosh S.
        • Luesch H.
        • Ghosh A.
        • Wong E.T.
        • Malik N.
        • Orth A.
        • de Jesus P.
        • Perry A.S.
        • Oliver J.D.
        • Tran N.L.
        • Speiser L.J.
        • Wong M.
        • Saez E.
        • Schultz P.
        • et al.
        Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
        Nat. Cell Biol. 2010; 12 (20622870): 758-767
        • Crabbe L.
        • Karlseder J.
        Mammalian Rap1 widens its impact.
        Nat. Cell Biol. 2010; 12 (20622867): 733-735
        • Martínez P.
        • Gómez-López G.
        • García F.
        • Mercken E.
        • Mitchell S.
        • Flores J.M.
        • de Cabo R.
        • Blasco M.A.
        RAP1 protects from obesity through its extratelomeric role regulating gene expression.
        Cell Rep. 2013; 3 (23791526): 2059-2074
        • Yeung F.
        • Ramírez C.M.
        • Mateos-Gomez P.A.
        • Pinzaru A.
        • Ceccarini G.
        • Kabir S.
        • Fernández-Hernando C.
        • Sfeir A.
        Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity.
        Cell Rep. 2013; 3 (23791522): 1847-1856
        • Cai Y.
        • Kandula V.
        • Kosuru R.
        • Ye X.
        • Irwin M.G.
        • Xia Z.
        Decoding telomere protein Rap1: its telomeric and nontelomeric functions and potential implications in diabetic cardiomyopathy.
        Cell Cycle. 2017; 16 (28853973): 1765-1773
        • Ding Y.
        • Liang X.
        • Zhang Y.
        • Yi L.
        • Shum H.C.
        • Chen Q.
        • Chan B.P.
        • Fan H.
        • Liu Z.
        • Tergaonkar V.
        • Qi Z.
        • Tse H.
        • Lian Q.
        Rap1 deficiency-provoked paracrine dysfunction impairs immunosuppressive potency of mesenchymal stem cells in allograft rejection of heart transplantation.
        Cell Death Dis. 2018; 9 (29515165): 386
        • Zhang X.
        • Liu Z.
        • Liu X.
        • Wang S.
        • Zhang Y.
        • He X.
        • Sun S.
        • Ma S.
        • Shyh-Chang N.
        • Liu F.
        • Wang Q.
        • Wang X.
        • Liu L.
        • Zhang W.
        • Song M.
        • et al.
        Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis.
        Protein Cell. 2019; 10 (30796637): 649-667
        • Kaminker P.G.
        • Kim S.-H.
        • Desprez P.-Y.
        • Campisi J.
        A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.
        Cell Cycle. 2009; 8 (19229133): 931-939
        • Chen L.-Y.
        • Zhang Y.
        • Zhang Q.
        • Li H.
        • Luo Z.
        • Fang H.
        • Kim S.H.
        • Qin L.
        • Yotnda P.
        • Xu J.
        • Tu B.P.
        • Bai Y.
        • Songyang Z.
        Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
        Mol. Cell. 2012; 47 (22885005): 839-850
        • Ohishi T.
        • Hirota T.
        • Tsuruo T.
        • Seimiya H.
        TRF1 mediates mitotic abnormalities induced by Aurora-A overexpression.
        Cancer Res. 2010; 70 (20160025): 2041-2052
        • Ohishi T.
        • Muramatsu Y.
        • Yoshida H.
        • Seimiya H.
        TRF1 ensures the centromeric function of Aurora-B and proper chromosome segregation.
        Mol. Cell Biol. 2014; 34 (24752893): 2464-2478
        • Long J.
        • Huang C.
        • Chen Y.
        • Zhang Y.
        • Shi S.
        • Wu L.
        • Liu Y.
        • Liu C.
        • Wu J.
        • Lei M.
        Telomeric TERB1-TRF1 interaction is crucial for male meiosis.
        Nat. Struct. Mol. Biol. 2017; 24 (29083416): 1073-1080
        • Biroccio A.
        • Cherfils-Vicini J.
        • Augereau A.
        • Pinte S.
        • Bauwens S.
        • Ye J.
        • Simonet T.
        • Horard B.
        • Jamet K.
        • Cervera L.
        • Mendez-Bermudez A.
        • Poncet D.
        • Grataroli R.
        • de Rodenbeeke C.T.
        • Salvati E.
        • et al.
        TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.
        Nat. Cell Biol. 2013; 15 (23792691): 818-828
        • El Maï M.
        • Wagner K.-D.
        • Michiels J.-F.
        • Ambrosetti D.
        • Borderie A.
        • Destree S.
        • Renault V.
        • Djerbi N.
        • Giraud-Panis M.-J.
        • Gilson E.
        • Wagner N.
        The telomeric protein TRF2 regulates angiogenesis by binding and activating the PDGFRβ promoter.
        Cell Rep. 2014; 9 (25437559): 1047-1060
        • Hussain T.
        • Saha D.
        • Purohit G.
        • Kar A.
        • Kishore Mukherjee A.
        • Sharma S.
        • Sengupta S.
        • Dhapola P.
        • Maji B.
        • Vedagopuram S.
        • Horikoshi N.T.
        • Horikoshi N.
        • Pandita R.K.
        • Bhattacharya S.
        • Bajaj A.
        • et al.
        Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
        Sci. Rep. 2017; 7 (28912501): 11541
        • Su C.-H.
        • Cheng C.
        • Tzeng T.-Y.
        • Lin I.-H.
        • Hsu M.-T.
        An H2A histone isotype, H2ac, associates with telomere and maintains telomere integrity.
        PLoS ONE. 2016; 11 (27228173): e0156378
        • Konishi A.
        • Izumi T.
        • Shimizu S.
        TRF2 protein interacts with core histones to stabilize chromosome ends.
        J. Biol. Chem. 2016; 291 (27514743): 20798-20810
        • Kaur P.
        • Wu D.
        • Lin J.
        • Countryman P.
        • Bradford K.C.
        • Erie D.A.
        • Riehn R.
        • Opresko P.L.
        • Wang H.
        Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.
        Sci. Rep. 2016; 6 (26856421): 20513
        • Rawal P.
        • Kummarasetti V.B.R.
        • Ravindran J.
        • Kumar N.
        • Halder K.
        • Sharma R.
        • Mukerji M.
        • Das S.K.
        • Chowdhury S.
        Genome-wide prediction of G4 DNA as regulatory motifs: role in Escherichia coli global regulation.
        Genome Res. 2006; 16 (16651665): 644-655
        • Huppert J.L.
        • Balasubramanian S.
        G-quadruplexes in promoters throughout the human genome.
        Nucleic Acids Res. 2007; 35 (17169996): 406-413
        • Verma A.
        • Halder K.
        • Halder R.
        • Yadav V.K.
        • Rawal P.
        • Thakur R.K.
        • Mohd F.
        • Sharma A.
        • Chowdhury S.
        Genome-wide computational and expression analyses reveal G-quadruplex DNA motifs as conserved cis-regulatory elements in human and related species.
        J. Med. Chem. 2008; 51: 5641-5649
        • Yadav V.K.
        • Abraham J.K.
        • Mani P.
        • Kulshrestha R.
        • Chowdhury S.
        QuadBase: genome-wide database of G4 DNA–occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes.
        Nucleic Acids Res. 2008; 36 (17962308): D381-D385
        • Dhapola P.
        • Chowdhury S.
        QuadBase2: web server for multiplexed guanine quadruplex mining and visualization.
        Nucleic Acids Res. 2016; 44 (27185890): W277-W283
        • Yadav V.
        • Hemansi
        • Kim N.
        • Tuteja N.
        • Yadav P.
        G quadruplex in plants: a ubiquitous regulatory element and its biological relevance.
        Front. Plant Sci. 2017; 8 (28725233): 1163
        • Saha D.
        • Singh A.
        • Hussain T.
        • Srivastava V.
        • Sengupta S.
        • Kar A.
        • Dhapola P.
        • Dhople V.
        • Ummanni R.
        • Chowdhury S.
        Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.
        J. Biol. Chem. 2017; 292 (28717007): 15205-15215
        • Guilbaud G.
        • Murat P.
        • Recolin B.
        • Campbell B.C.
        • Maiter A.
        • Sale J.E.
        • Balasubramanian S.
        Local epigenetic reprogramming induced by G-quadruplex ligands.
        Nat. Chem. 2017; 9 (29064488): 1110-1117
        • Mukherjee A.K.
        • Sharma S.
        • Chowdhury S.
        Non-duplex G-quadruplex structures emerge as mediators of epigenetic modifications.
        Trends Genet. 2019; 35 (30527765): 129-144
        • Wang X.
        • Kam Z.
        • Carlton P.M.
        • Xu L.
        • Sedat J.W.
        • Blackburn E.H.
        Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy.
        Epigenetics Chromatin. 2008; 1 (19014413): 4
        • Burla R.
        • La Torre M.
        • Saggio I.
        Mammalian telomeres and their partnership with lamins.
        Nucleus. 2016; 7 (27116558): 187-202
        • Gonzalo S.
        • Eissenberg J.C.
        Tying up loose ends: telomeres, genomic instability and lamins.
        Curr. Opin. Genet. Dev. 2016; 37 (27010504): 109-118
        • Wood A.M.
        • Danielsen J.M.R.
        • Lucas C.A.
        • Rice E.L.
        • Scalzo D.
        • Shimi T.
        • Goldman R.D.
        • Smith E.D.
        • Le Beau M.M.
        • Kosak S.T.
        TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends.
        Nat. Commun. 2014; 5 (25399868): 5467
        • Smith E.D.
        • Garza-Gongora A.G.
        • MacQuarrie K.L.
        • Kosak S.T.
        Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.
        Differentiation. 2018; 102 (29979997): 19-26
        • Mathur S.
        • Glogowska A.
        • McAvoy E.
        • Righolt C.
        • Rutherford J.
        • Willing C.
        • Banik U.
        • Ruthirakuhan M.
        • Mai S.
        • Garcia A.
        Three-dimensional quantitative imaging of telomeres in buccal cells identifies mild, moderate, and severe Alzheimer's disease patients.
        J. Alzheimers Dis. 2014; 39 (24121960): 35-48
        • Garcia A.
        • Mathur S.
        • Kalaw M.C.
        • McAvoy E.
        • Anderson J.
        • Luedke A.
        • Itorralba J.
        • Mai S.
        Quantitative 3D telomeric imaging of buccal cells reveals Alzheimer's disease-specific signatures.
        J. Alzheimers Dis. 2017; 58 (28387668): 139-145
        • Tichy E.D.
        • Sidibe D.K.
        • Tierney M.T.
        • Stec M.J.
        • Sharifi-Sanjani M.
        • Hosalkar H.
        • Mubarak S.
        • Johnson F.B.
        • Sacco A.
        • Mourkioti F.
        Single stem cell imaging and analysis reveals telomere length differences in diseased human and mouse skeletal muscles.
        Stem Cell Reports. 2017; 9 (28890163): 1328-1341
        • Gardner J.P.
        • Kimura M.
        • Chai W.
        • Durrani J.F.
        • Tchakmakjian L.
        • Cao X.
        • Lu X.
        • Li G.
        • Peppas A.P.
        • Skurnick J.
        • Wright W.E.
        • Shay J.W.
        • Aviv A.
        Telomere dynamics in macaques and humans.
        J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2007; 62 (17452729): 367-374
        • Sunpaweravong S.
        • Sunpaweravong P.
        • Sathitruangsak C.
        • Mai S.
        Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.
        Dis. Esophagus. 2016; 29 (25625311): 307-313
        • Caria P.
        • Dettori T.
        • Frau D.V.
        • Lichtenzstejn D.
        • Pani F.
        • Vanni R.
        • Mai S.
        Characterizing the three-dimensional organization of telomeres in papillary thyroid carcinoma cells.
        J. Cell. Physiol. 2019; 234 (30328617): 5175-5185
        • Takahashi K.
        • Yamanaka S.
        Induced pluripotent stem cells in medicine and biology.
        Development. 2013; 140 (23715538): 2457-2461
        • Liu L.
        Linking telomere regulation to stem cell pluripotency.
        Trends Genet. 2017; 33 (27889084): 16-33
        • Marión R.M.
        • López de Silanes I.
        • Mosteiro L.
        • Gamache B.
        • Abad M.
        • Guerra C.
        • Megías D.
        • Serrano M.
        • Blasco M.A.
        Common telomere changes during in vivo reprogramming and early stages of tumorigenesis.
        Stem Cell Reports. 2017; 8 (28162998): 460-475
        • Agarwal S.
        • Loh Y.-H.
        • McLoughlin E.M.
        • Huang J.
        • Park I.-H.
        • Miller J.D.
        • Huo H.
        • Okuka M.
        • dos Reis R.M.
        • Loewer S.
        • Ng H.-H.
        • Keefe D.L.
        • Goldman F.D.
        • Klingelhutz A.J.
        • Liu L.
        • et al.
        Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
        Nature. 2010; 464 (20164838): 292-296
        • Kamada M.
        • Mitsui Y.
        • Matsuo T.
        • Takahashi T.
        Reversible transformation and de-differentiation of human cells derived from induced pluripotent stem cell teratomas.
        Hum. Cell. 2016; 29 (26069211): 1-9
        • Wood L.D.
        • Heaphy C.M.
        • Daniel H.D.-J.
        • Naini B.V.
        • Lassman C.R.
        • Arroyo M.R.
        • Kamel I.R.
        • Cosgrove D.P.
        • Boitnott J.K.
        • Meeker A.K.
        • Torbenson M.S.
        Chromophobe hepatocellular carcinoma with abrupt anaplasia: a proposal for a new subtype of hepatocellular carcinoma with unique morphological and molecular features.
        Mod. Pathol. 2013; 26 (23640129): 1586-1593
        • Lee J.-C.
        • Jeng Y.-M.
        • Liau J.-Y.
        • Tsai J.-H.
        • Hsu H.-H.
        • Yang C.-Y.
        Alternative lengthening of telomeres and loss of ATRX are frequent events in pleomorphic and dedifferentiated liposarcomas.
        Mod. Pathol. 2015; 28 (26022452): 1064-1073
        • Rodriguez F.J.
        • Brosnan-Cashman J.A.
        • Allen S.J.
        • Vizcaino M.A.
        • Giannini C.
        • Camelo-Piragua S.
        • Webb M.
        • Matsushita M.
        • Wadhwani N.
        • Tabbarah A.
        • Hamideh D.
        • Jiang L.
        • Chen L.
        • Arvanitis L.D.
        • Alnajar H.H.
        • et al.
        Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia.
        Brain Pathol. 2019; 29 (30192422): 126-140
        • Hariharan N.
        • Quijada P.
        • Mohsin S.
        • Joyo A.
        • Samse K.
        • Monsanto M.
        • De La Torre A.
        • Avitabile D.
        • Ormachea L.
        • McGregor M.J.
        • Tsai E.J.
        • Sussman M.A.
        Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging.
        J. Am. Coll. Cardiol. 2015; 65 (25593054): 133-147
        • Diao D.
        • Wang H.
        • Li T.
        • Shi Z.
        • Jin X.
        • Sperka T.
        • Zhu X.
        • Zhang M.
        • Yang F.
        • Cong Y.
        • Shen L.
        • Zhan Q.
        • Yan J.
        • Song Z.
        • Ju Z.
        Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan.
        EMBO Rep. 2018; 19 (30126922): e45494
        • Choumerianou D.M.
        • Martimianaki G.
        • Stiakaki E.
        • Kalmanti L.
        • Kalmanti M.
        • Dimitriou H.
        Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults.
        Cytotherapy. 2010; 12 (20662612): 881-887
        • Dan J.
        • Rousseau P.
        • Hardikar S.
        • Veland N.
        • Wong J.
        • Autexier C.
        • Chen T.
        Zscan4 inhibits maintenance DNA methylation to facilitate telomere elongation in mouse embryonic stem cells.
        Cell Rep. 2017; 20 (28834755): 1936-1949
        • Schneider R.P.
        • Garrobo I.
        • Foronda M.
        • Palacios J.A.
        • Marión R.M.
        • Flores I.
        • Ortega S.
        • Blasco M.A.
        TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells.
        Nat. Commun. 2013; 4 (23735977): 1946
        • Varela E.
        • Schneider R.P.
        • Ortega S.
        • Blasco M.A.
        Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21873233): 15207-15212
        • Alder J.K.
        • Barkauskas C.E.
        • Limjunyawong N.
        • Stanley S.E.
        • Kembou F.
        • Tuder R.M.
        • Hogan B.L.M.
        • Mitzner W.
        • Armanios M.
        Telomere dysfunction causes alveolar stem cell failure.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25840590): 5099-5104
        • Orun O.
        • Tiber P.M.
        • Serakinci N.
        Partial knockdown of TRF2 increase radiosensitivity of human mesenchymal stem cells.
        Int. J. Biol. Macromol. 2016; 90 (26598048): 53-58
        • Serakinci N.
        • Mega Tiber P.
        • Orun O.
        Chromatin modifications of hTERT gene in hTERT-immortalized human mesenchymal stem cells upon exposure to radiation.
        Eur. J. Med. Genet. 2018; 61 (29288791): 288-293
        • Lagunas A.M.
        • Wu J.
        • Crowe D.L.
        Telomere DNA damage signaling regulates cancer stem cell evolution, epithelial mesenchymal transition, and metastasis.
        Oncotarget. 2017; 8 (29113290): 80139-80155
        • Wu M.
        • Lin Z.
        • Li X.
        • Xin X.
        • An J.
        • Zheng Q.
        • Yang Y.
        • Lu D.
        HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2.
        Sci. Rep. 2016; 6 (27782152): 36045
        • Zhang P.
        • Pazin M.J.
        • Schwartz C.M.
        • Becker K.G.
        • Wersto R.P.
        • Dilley C.M.
        • Mattson M.P.
        Nontelomeric TRF2-REST interaction modulates neuronal gene silencing and fate of tumor and stem cells.
        Curr. Biol. 2008; 18 (18818083): 1489-1494
        • Zhang P.
        • Casaday-Potts R.
        • Precht P.
        • Jiang H.
        • Liu Y.
        • Pazin M.J.
        • Mattson M.P.
        Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21903926): 16434-16439
        • Bai Y.
        • Lathia J.D.
        • Zhang P.
        • Flavahan W.
        • Rich J.N.
        • Mattson M.P.
        Molecular targeting of TRF2 suppresses the growth and tumorigenesis of glioblastoma stem cells.
        Glia. 2014; 62 (24909307): 1687-1698
        • Saha A.
        • Roy S.
        • Kar M.
        • Roy S.
        • Thakur S.
        • Padhi S.
        • Akhter Y.
        • Banerjee B.
        Role of telomeric TRF2 in orosphere formation and CSC phenotype maintenance through efficient DNA repair pathway and its correlation with recurrence in OSCC.
        Stem Cell Rev. Rep. 2018; 14 (29872959): 871-887
        • Tejera A.M.
        • Stagno d'Alcontres M.
        • Thanasoula M.
        • Marion R.M.
        • Martinez P.
        • Liao C.
        • Flores J.M.
        • Tarsounas M.
        • Blasco M.A.
        TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.
        Dev. Cell. 2010; 18 (20493811): 775-789