Abstract
Chromosomal and telomeric reprogramming was assessed in intraspecies hybrids obtained by fusion of embryonic stem (ES) cells and mouse embryonic fibroblasts. Evaluation of the ploidy of ES-somatic hybrids revealed that 21 of 59 clones had a tetraploid DNA profile while the remaining clones showed deviations from the expected profile of fusion between two diploid cells. Microsatellite polymerase chain reaction analysis of four of these clones demonstrated no random loss of somatic chromosome pairs in the ES-somatic cell hybrids. Pluripotential of ES-somatic hybrids was assessed by gene expression analysis, antibody staining for Oct4 and SSEA-1 and teratoma formation containing derivatives of the three germ layers. Reprogramming of telomeric maintenance was observed with ES-somatic hybrids showing high telomerase activity and increased telomere lengths. However, we detected no significant increase in the expression of the three critical telomerase subunits: telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), and dyskerin. This indicates that activation of telomerase and telomere maintenance is not reliant on changes in gene expression of TERT, TERC, and dyskerin following ES-somatic cell fusion or sister chromatid recombination and may arise through elimination of negative regulation of telomerase activity. This is the first demonstration of telomere lengthening following cell fusion and offers a new model for studying and identifying new regulators of telomere maintenance.
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References
Betts D, Bordignon V, Hill J, Winger Q, Westhusin M, Smith L, King W (2001) Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. PNAS 98(3):1077–1082
Blackburn EH (1991) Telomeres. Trends Biochem Sci 16(10):378–381
Cuthbert AP, Bond J, Trott DA, Gill S, Broni J, Marriott A, Khoudoli G, Parkinson EK, Cooper CS, Newbold RF (1999) Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells. J Natl Cancer Inst 91(1):37–45
Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309(5739):1369–1373
de Lange T (2005) Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19(18):2100–2110
Ducrest AL, Amacker M, Mathieu YD, Cuthbert AP, Trott DA, Newbold RF, Nabholz M, Lingner J (2001) Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity. Cancer Res 61(20):7594–7602
Hanada H, Takeda K, Tagami T, Nirasawa K, Akagi S, Adachi N, Takahashi S, Izaike Y, Iwamoto M, Fuchimoto D, Miyashita N, Kubo M, Onishi A, King WA (2005) Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer. Mol Reprod Dev 71(1):36–44
Jeon HY, Hyun SH, Lee GS, Kim HS, Kim S, Jeong YW, Kang SK, Lee BC, Han JY, Ahn C, Hwang WS (2005) The analysis of telomere length and telomerase activity in cloned pigs and cows. Mol Reprod Dev 71(3):315–320
Kishigami S, Wakayama S, Hosoi Y, Iritani A, Wakayama T (2008) Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome. Exp Cell Res 314(9):1945–1950
Kugoh H, Shigenami K, Funaki K, Barrett JC, Oshimura M (2003) Human chromosome 5 carries a putative telomerase repressor gene. Genes Chromosomes Cancer 36(1):37–47
Lansdorp PM (2008) Telomeres, stem cells, and hematology. Blood 111(4):1759–1766
Li H, Zhao LL, Funder JW, Liu JP (1997) Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells. J Biol Chem 272:16729–16732
Liu L, Bailey SM, Okuka M, Munoz P, Li C, Zhou L, Wu C, Czerwiec E, Sandler L, Seyfang A, Blasco MA, Keefe DL (2007) Telomere lengthening early in development. Nat Cell Biol 9(12):1436–1441
Marion RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, Serrano M, Blasco MA (2009) Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4(2):141–154
Matveeva NM, Shilov AG, Kaftanovskaya EM, Maximovsky LP, Zhelezova AI, Golubitsa AN, Bayborodin SI, Fokina MM, Serov OL (1998) In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes. Mol Reprod Dev 50(2):128–138
Matveeva NM, Pristyazhnyuk IE, Temirova SA, Menzorov AG, Vasilkova A, Shilov AG, Smith A, Serov OL (2005) Unequal segregation of parental chromosomes in embryonic stem cell hybrids. Mol Reprod Dev 71(3):305–314
Morrish TA, Greider CW (2009) Short telomeres initiate telomere recombination in primary and tumor cells. PLoS Genet 5(1):e1000357
Ohmura H, Tahara H, Suzuki M, Ide T, Shimizu M, Yoshida MA, Tahara E, Shay JW, Barrett JC, Oshimura M (1995) Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res 86(10):899–904
Osterhage JL, Friedman KL (2009) Chromosome end maintenance by telomerase. J Biol Chem. doi:101074/jbcR900011200
Palm W, de Lange T (2008) How shelterin protects mammalian telomeres. Annu Rev Genet 42:301–334
Perrem K, Colgin LM, Neumann AA, Yeager TR, Reddel RR (2001) Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol Cell Biol 21(12):3862–3875
Pralong D, Mrozik K, Occhiodoro F, Wijesundara N, Sumer H, Van Boxtel AL, Trounson A, Verma PJ (2005) A novel method for somatic cell nuclear transfer to mouse embryonic stem cells. Cloning Stem Cells 7(4):265–271
Sagawa Y, Nishi H, Isaka K, Fujito A, Takayama M (2001) The correlation of TERT expression with c-myc expression in cervical cancer. Cancer Lett 168(1):45–50
Shiels PG, Kind AJ, Campbell KH, Waddington D, Wilmut I, Colman A, Schnieke AE (1999) Analysis of telomere lengths in cloned sheep. Nature 399(6734):316–317
Sumer H, Jones KL, Liu J, Rollo BN, van Boxtel AL, Pralong D, Verma PJ (2009) Transcriptional changes in somatic cells recovered from ES-somatic heterokaryons. Stem Cells Dev. doi:101089/scd20080361
Stadtfeld M, Maherali N, Breault DT, Hochedlinger K (2008) Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2(3):230–240
Tada M, Tada T, Lefebvre L, Barton SC, Surani MA (1997) Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO J 16:6510–6520
Tada M, Takahama Y, Abe K, Nakatsuji N, Tada T (2001) Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol 11:1553–1558
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:1–12
Tanaka H, Horikawa I, Kugoh H, Shimizu M, Barrett JC, Oshimura M (1999) Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer. Mol Carcinog 25(4):249–255
Tycowski KT, Shu MD, Kukoyi A, Steitz JA (2009) A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol Cell 34(1):47–57
Tian XC, Xu J, Yang X (2000) Normal telomere lengths found in cloned cattle. Nat Genet 26(3):272–273
Vasilkova AA, Kizilova HA, Puzakov MV, Shilov AG, Zhelezova AI, Golubitsa AN, Battulin NR, Vedernikov VE, Menzorov AG, Matveeva NM, Serov OL (2007) Dominant manifestation of pluripotency in embryonic stem cell hybrids with various numbers of somatic chromosomes. Mol Reprod Dev 74(8):941–951
Venteicher AS, Abreu EB, Meng Z, McCann KE, Terns RM, Veenstra TD, Terns MP, Artandi SE (2009) A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 323(5914):644–648
Wang F, Podell ER, Zaug AJ, Yang Y, Baciu P, Cech TR, Lei M (2007) The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature 445(7127):506–510
Wu KJ, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, Dalla-Favera R (1999) Direct activation of TERT transcription by c-MYC. Nat Genet 21(2):220–224
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukin II, Thomson AJ (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920
Acknowledgments
This project was supported by funding from the Australian Stem Cell Centre, National Health and Medical Research Council of Australia, and Cancer Council of Victoria, Australia. HS receives a NH&MRC Biomedical Training Fellowship supported by an Establishment Gift from the Clive and Vera Ramaciotti Foundation.
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Supplemental Table 1
Chromosome contribution of ES-somatic cell hybrids as determined by microsatellite analysis (GIF 86 kb)
Supplemental Fig. 1
DNA content of ES-somatic hybrids. Ploidy profiles as determined by FACs analysis and compared to known diploid and tetraploid controls (shaded). Examples of a near-diploid profile, b near-tetraploid, c hypertetraploid, and d mixed profiles, all shown in solid shading. e Summary of distribution of ploidy ES-somatic hybrids (GIF 22 kb)
Supplemental Fig. 2
Telomerase activity following reprogramming. Telomerase activity as determined by TRAP assay for the various cell lines showing three serial dilutions for each sample (GIF 253 kb)
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Sumer, H., Nicholls, C., Pinto, A.R. et al. Chromosomal and telomeric reprogramming following ES-somatic cell fusion. Chromosoma 119, 167–176 (2010). https://doi.org/10.1007/s00412-009-0245-1
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DOI: https://doi.org/10.1007/s00412-009-0245-1