Journal of Cell Science ernlpoeiosadmedulloblastoma cerebellar and mouse progenitors proliferating neuronal highly in foci RNA (TERRA) repeat-containing telomeric of Formation Article Research ehns sbpse nnra rlfrtn el and cells protective proliferating this normal but in cells, senescing bypassed normal is cell and to in mechanism leads (Sahin function arrest capping replicative cycle and of Loss arrest 2010). cycle Depinho, can that cell signal damage to DNA end- lead a attrition, elicit In by nucleolytic shorten 2010). short critically telomeres al., and activity, and et Ye of factors, 2010; absence Cooper, the machinery, and associated (Jain replication enzymes its an processing In by DNA and 2008). maintained Lange, involving telomerase is de mechanism length and repeat Palm elaborate 2004; telomere al., The cells, (de et proliferating regulation 2004). Liu length 2005a; are Lange, and Lange, end-protection telosome, de telomere or for 2002; important termed Lange, cap , to stranded (de telomere-associated structures ends double nucleoprotein 3 higher-order stranded Mammalian length complex single form 2004). variable a can in Cech, of ending repeats 2006; consists TTAGGG al., DNA linear of et telomere ends (Blackburn the genome stability and at maintenance chromosome structures for required DNA repetitive are Telomeres Introduction telomere of form early novel highly a in body reflect nuclear novel levels words: a TERRA form Key may elevated aggregates that RNA TERRA suggest the and findings cells. evolution, These cell mammalian colocalize cancer cancers. not proliferating and did human stress foci progenitor replication of TERRA during granule tissue. variety regulation purified responsive in a in for expression expression in TERRA TERRA model also elevated enhance to could mouse contribute could aggregates signals SHH TERRA a normal proliferation cerebellum, neurons. foci with that developing adjacent Using progenitor TERRA suggesting normal not of of vitro, the stress. formation zones but In in the proliferating replication cells. cells tumor, revealed tumor highly FISH in or proliferating RNA in detected rapidly blotting. nuclear detected damage of northern was be large and regions DNA RNA (FISH) forms nuclear hybridization TERRA the be and of situ in signaling, cells, in can (TERFs) markers fluorescence (SHH) remains cancer and RNA hedgehog development and characterized both RNA cell Sonic normal using previously cells nuclear proliferating cancer chronic from highly heterogeneous and by in normal driven a distinct accumulates in medulloblastoma as are TERRA TERRA persists that can of which telomeres that show regulation foci, eukaryotic we (TERRA) and Most Here, function RNA senescence. understood. cellular precise repeat-containing poorly of the telomeric control However, and a regulated. integrity generate developmentally genome of to maintenance transcribed the be in roles crucial play Telomeres Summary 10.1242/jcs.108118 doi: 4383–4394 125, ß Science Cell of Journal 2012 May 1 Accepted ( correspondence for *Authors 4 3 2 1 Deng Zhong ioe Xu Xiaowei eateto ahlg n aoaoyMdcn,Uiest fPnslai colo eiie hldlha A114 USA 19104, PA Philadelphia, Medicine, of School USA Pennsylvania 19104, of PA University Philadelphia, Medicine, Medicine, USA Laboratory of 19104, and School PA Pathology Pennsylvania Philadelphia, of USA of Institute, Department 19104, University Wistar PA Neurosurgery, The Philadelphia, of Program, Institute, Department Oncogenesis Wistar Cellular The and Program, Molecular Regulation and Expression 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. c 2Xfc,poylctcluei PL rCjlbde nmuetmrtsu.W lopoieeiec htTRAis TERRA that evidence provide also We tissue. tumor mouse in bodies Cajal or (PML) leukemia promyelocytic foci, H2AX eoeerpa-otiignncdn N,TRA ernlpoeios acr eeelm eulbatm,Snchedgehog Sonic Medulloblastoma, Cerebellum, Cancer, progenitors, Neuronal TERRA, RNA, non-coding repeat-containing Telomere 4 1 alM Lieberman M. Paul , hoWang Zhuo , [email protected] 1 hoe Xiang Chaomei , ; [email protected] 1, n ai Dahmane Nadia and * 9 vragthat overhang 2,3 la Molczan Aliah , ) 2,3, ihpoieaiecpct n otiue onuclear to contributes and capacity correlates expression proliferative TERRA Blasco, that 2007; and suggesting and with al., human Schoeftner cells, both 2009; in iPS et elevated al., are mouse levels (Azzalin et TERRA addition, Deng state In 2009; 2008). al., epigenetic cellular by et telomere status, regulated Caslini and developmental be and Schoeftner can including 2010; stress, expression al., mechanisms, RNA et several TERRA (Redon 2008). vitro Blasco, in telomerase inhibit activity and (TERT) telomere enzyme telomerase of subunit regulate catalytic the al., by et to (Flynn capping 1 Pot Lo and 2011; proteins binding 2009) DNA strand al., single telomere shelterin et the (Deng with RNA formation TERRA heterochromatin 2012). interact Cooper, and antisense Greenwood can and 2012; sense al., sequence telomere et fission both adjacent (Bah and In for tract repeat 2008). telomere detected the Blasco, of be strands and can Schoeftner transcripts 2007; yeast, al., chromosomes et eukaryotic of (Azzalin repeats telomere terminal the through 2005; 2005b). (Blasco, Lange, cancers De 2007; human Blasco, in deregulated further potentially * ER sahtrgnoslnt o-oigRAtranscribed RNA non-coding length heterogeneous a is TERRA 2 Vale , ´ e eSlnse l,21) ER a loitrc with interact also can TERRA 2010). al., et Silanes de pez reBaubet ´rie 2,3 oeConejo-Garcia Jose , 2 , 4383 Journal of Cell Science enfursec nest eaiet omlclsi adjacent in cells normal to relative intensity fluorescence mean oaayeteepeso fTRAi os oe fhuman of model mouse a employed in we medulloblastoma TERRA cancer, for of model expression the mouse analyze a To in foci form in TERRA elevated organs. is Results diverse mouse in TERRA originating normal cancers that human in during show of found types we cells various be also Finally, progenitor can development. foci proliferating TERRA shortened. highly has from DNA exhibit distinct repeat and are granule (TERFs) TERRA foci proliferating this of These c In foci. cancer level TERRA 2002). and of high al., formation normal express et progenitors that Altaba by neuron show i (reviewed Ruiz we 1997) 2001; al., work, important Scott, et al., an et and (Goodrich Johnson models Corcoran and 1996; mouse al., ligand et and (Hahn 1996) SHH human 1 1999; in the in medulloblastoma Patched Mutations to for Altaba, signaling. SHH 1999). receptor of i regulator Scott, negative a during Ruiz and is progenitors Wechsler-Reya (Ptch1) and neuronal 1999; (Dahmane these Wallace, development of for proliferation required normal is rapid (SHH) neuron hedgehog during proliferation Sonic the of granule period. Ellison, rates postnatal high and early (Ellison, very of the Gilbertson to subject cerebellum 2004; are deregulation GNPs Gilbertson, the 2008). 2010; from in Ellison, arise development 2002; (GNPs) pediatric to tumors human progenitors these of the thought all subset A of of is 2010). (Ellison, incidence tumors malignancy highest brain for malignant the model a TERRA mouse with is a examined cerebellum in Medulloblastoma and we biopsies development, medulloblastoma. cancer human cell in cancer expression and normal 2011). al., of et context understood. the poorly in Yehezkel remains TERRA cancer of 2009; function and expression al., the However, et (Marion reprogramming 4384 N IHeprmnsuigcmue mgn software imaging computer using foci multiple distinct that experiments of any indicated Quantification FISH detect S2B). to RNA Fig. failed material TERRA PNA (supplementary antisense FAM-conjugated a RNA Fig. for control, material to probe specificity supplementary additional signal corresponds an panels; As indeed the S1B). lower probe that 1B, (Fig. TERRA indicating expression the A, non-tumor with adjacent RNase detected the with in detected foci in not pre-treated were not TERFs discrete 1B). sections but (Fig. cerebellum forms same cells, the of tumor TERRA cells the that in revealed (TERFs) RNA-FISH telomere from A DNA. RNA 2011). non-denaturing telomere distinguish al., under selectively et to used Flynn conditions was 2009; of al., probe detection et PNA (Deng TAMRA-conjugated for RNA optimized unstable been and have hybridizationrare that situ methods in fluorescence using RNA cancer(FISH) employed and normal first mouse we in tissue, expression TERRA Fig. examine material supplementary To 1A; S1). (Fig. normal/non- markers and various histology from of distinguished on analysis based readily tissue be cerebellar can tumor material Tumor supplementary S1). 1A; (Fig. Fig. 1997) al., et (Goodrich pathway express and 2005), by proliferating marked of composed are GNPs tumors These 1997). al., et 2010; Goodrich (Ellison, medulloblastoma subtype SHH-positive human for 2XDAdmg oi u cu nclsweetetelomere the where cells in occur but foci, damage DNA H2AX oivsiaeterglto n ucino ER in TERRA of function and regulation the investigate To ora fCl cec 2 (18) 125 Science Cell of Journal , 0 ftmrclshv a have cells tumor of 80% Gli1 Math1 Ptch1 agtadmdao fteSHsignaling SHH the of mediator and target a , as nw as known (also +/ 2 ie ieyue eei model genetic used widely a mice, Atoh1 , nsitu in Oie tal., et (Oliver ) .-odgreater 7.5-fold Ptch1 expression a lead can ofre yRAFS sn N lgncetd probe further oligonucleotide DNA were a findings using FISH These (TAACCC) RNA 1C–E). by (Fig. confirmed tissue non-tumor eetbesga ntenra ato h aecerebellum same a no with the observed with was of expression cells TERRA part (TAACAC) for mutated tumor normal signal No in the 1F). 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Supplementary 12 after to release 11SDG5330017 for PMC number in [grant Deposited from Association grant Z.D.]. Heart Development number Scientist [grant American a Health the Brain and of P.M.L.); Institutes National to National the RO1CA140652 N.D., Society N.D.]; to Cancer Society to Tumor American RSG-08-045-01-DDC the Core number CA10815]; Center Cancer [grant P30 Institute number Wistar [grant the by Grant supported was work This and Keeney Funding Core. Fred Microscopy and Institute Wistar RNA, the Dartmouth tissue in at tumor cancer Hayden Pathology James the of ovarian for Department for Center the University Cancer thank the Abramson also Feldman, the We Michael samples. and Valdivieso, bank Federico tissue thank Tumor to like would We Acknowledgements as added was cerebella hours. R&D) mouse 12 ng/ml, for (600 P5 SHH media from 2010). the al., purified to et were (Fernandez previously (GNPs) described progenitors neuron Granule purification progenitor neuron Granule eLne T. 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