INSTITUT LADY DAVIS DE RECHERCHES MÉDICALES / LADY DAVIS INSTITUTE FOR MEDICAL RESEARCH Cancer and Aging: Two faces of the same coin (4) Telomerase and Telomere Regulation Centre Bloomfield de recherche sur le vieillissement The Bloomfield Centre for Research in Aging Telomerase and Telomere Regulation TPP, Pot1 TERRA Pontin Reptin RNA maturation accumulation Posttranslational modification Processivity Podlevsky, J. and Chen, J.-J.L. 2012. It all comes together at the ends: Telomerase structure, function, and biogenesis. Mutation Research 730, 3-11. Transcriptional Regulation of hTERT Positive regulators: c-myc, Sp1, estrogen receptor, NF-KB (mTERT) Negative regulators: WT1 (Wilms tumor 1 suppressor), MZF-2 (myeloid-specific zinc finger protein implicated in cell cycle progression), p53 Chromatin and epigenetic regulation of the hTERT gene Cifuentes-Rojas, C. and Shippen, D.E. 2012. Telomerase regulation. Mutation Research 730, 20-27. hTERT promoter inactive in telomerase-negative cells Oh, S. et al. 1999. BBRC 263, 361-365. acetylated chloramphenicol chloramphenicol telomerase + telomerase - Cifuentes-Rojas, C. and Shippen, D.E. 2012. Telomerase regulation. Mutation Research 730, 20-27. Post-Translational Regulation of TERT CHIP (C terminus of Hsc70-interacting protein), a co-chaperone with E3 ubiquitin ligase Roles for pontin and reptin in telomerase RNP accumulation and assembly Baek, S.H. 2008. When ATPases pontin and reptin met telomerase. Cell 14, 459-450. ATPases with multiple binding partners and functions Pontin and reptin, like NAF1, aid in RNP assembly but do not remain associated with the mature telomerase enzyme TCAB1: driving telomerase to Cajal bodies and telomeres Venteicher, A.S. et al. 2009. A human telomerase holoenzyme protein required for cajal body localization and telomere synthesis. Science 323, 644-648. TERRA, telomeric RNA http://www.sciencedirect.com/cache/MiamiImageURL/1-s2.0-S0014579310005909-gr1_lrg.jpg/0?wchp=dGLzVl S-zSkWz Feuerhahn, S. et al. 2010. FEBS Lett. TERRA biogenesis, turnover and implications for function. 584, 3812-3818. Telomerase processivity and telomere length regulation Cifuentes-Rojas, C. and Shippen, D.E. 2012. Telomerase regulation. Mutation Research 730, 20-27. Unique characteristics of telomerase and telomeres as potential anti-cancer targets Copy of IMG_0227 Shusen Zhu and Sanjida Khondacker Human and mouse telomerase spliced variants May Shawi, Johanna Mancini, Shusen Zhu, Ricky Kwan Human and mouse telomerase-associated proteins Yasmin D’Souza Contribution of telomerase processivity to telomere function Josephine Chu Role of unique ‘insertion in fingers motif’ in telomere function SANJIDA.jpg Copy of IMG_0227 Me.jpg The Role of Alternatively-Spliced INS3 and INS4 hTERT mRNAs in Telomerase Function Copy of IMG_0227 Genomic organization and alternatively spliced sites of the hTERT gene From: Saeboe-Larssen, S., E. Fossberg, and G. Gaudernack. 2006. BMC Mol. Biol. 7 :26-35. Role of alternatively spliced TERT mRNA variants? The potential for complex splicing patterns may reflect a specific aspect of telomerase regulation in proliferation, differentiation and apoptosis Notably, some splice variants are expressed in primary and cancer cells that lack detectable telomerase activity In human development, the specific expression of hTERT splice variants that are predicted to encode catalytically-defective telomerases, correlates with telomere shortening and suggests that these transcripts may have important physiological roles Ectopic expression of the a-deletion variant that lacks conserved catalytic residues results in dominant-negative inhibition of telomerase, telomere shortening, and cell death Spliced TERT variants have also been reported in rice, chicken, mouse and rat Arabidopsis POT1A reported to interact with an N-terminal variant of telomerase The G-quadruplex ligand A549 induces telomerase downregulation by modulating hTERT alternative splicing Reviewed in Sykorova, E. and Fajkus, J. 2009. Biol. Cell 101, 375-392) Alternative splicing of TERT in other organisms Sykorova, E. and Fajkus, J. 2009. Biol. Cell 101, 375-392 2 Expression of INS3 spliced hTERT mRNA in some telomerase positive cancer cell lines analyzed SK-N-SH: neuroblastoma, K562: leukemia, Huh-7: hepatoma, MCF-7:mammary adenocarcinoma, HeLa: cervical adenocarcinoma, RKO: colon carcinoma, LIM1215: colon carcinoma, Ovcar-3: ovarian adenocarcinoma, HMEC: SV40 transformed dermal microvascular endothelial cells, HA5: SV40 transformed embryonic kidney cells, GM847: SV40 transformed skin fibroblasts (ALT cells), WI38:primary lung fibroblasts. 4 3 Expression of INS4 spliced hTERT mRNA in most telomerase-positive cancer cell lines analyzed 5 The in vitro INS3- and INS4-reconstituted enzymes do not express telomerase activity, likely due to the importance of the hTERT C-terminus for activity Confirmation of spliced mRNAs using polyA purified RNA from K562 leukemia cell line TRAP_081009 Stable expression of INS3 and INS4 variants in Huh7 hepatocarcinoma cell lines inhibits telomerase activity and slows cell growth At PD 30 Ins 3 and Ins 4 stably expressing cell lines exhibit shorter telomeres compared to Huh7 parental cells and hTERT control, but telomeres do not progressively shorten with increasing population doubling No significant difference in cell cycle profile or percentage apoptosis in cells stably expressing INS3 or INS4 variant compared to parental cells or cells expressing hTERT PD 60-80 TRAP18022010 Loss of Ins3 transgene expression results in loss of telomerase inhibition at late population doublings Conclusion Ins3- and Ins4-reconstituted telomerases are inactive in vitro Stable expression of Ins3 and Ins4 variants in telomerase positive Huh7 leads to telomerase inhibition, slowed growth and shorter telomere lengths, but no significant changes in cell cycle profile Telomerase inhibition is not maintained at late population doublings, likely due to loss of expression of the Ins3 transgene expression Antisense oligonucleotide-mediated modulation of Ins3 and Ins4 splicing Ins3-1 Ins3-2 Ins4-1 Ins4-2 Used 2’-O-methyl-RNA phosphorothioate oligonucleotides -Investigated in the context of genetic diseases to prevent aberrant splicing of genes -With cancer-related genes has been exploited to redirect the splicing of an anti-apoptotic to a pro-apoptotic Bcl-x variant -One report of telomerase and cell growth inhibition upon oligomer-mediated modulation of hTERT alternative splicing in prostate cancer cells (Brambilla, C. et al. 2004. CMLS 61, 1764-1774) Increased expression of Ins3 and Ins4 spliced variants and decreased expression of full length hTERT mRNA upon antisense oligonucleotide treatment Quantitation of endogenous Ins3 levels by Real-time PCR Quantitation of endogenous Ins4 levels by Real-time PCR 200409TRAP Increased expression of Ins3 and Ins4 spliced variants and decreased expression of full length hTERT mRNA reduces telomerase activity Growth inhibition upon antisense oligonucleotide treatment Antisense oligonucleotide treatment decreased cell proliferation as measured by colony formation ability Antisense oligomer-mediated expression of Ins3 and Ins4 spliced hTERT mRNAs: Inhibited endogenous hTERT expression in Huh7 cells Inhibited endogenous telomerase activity in Huh7 cells Inhibited the growth, viability and proliferation of Huh7 cells Perspective Short term effects are most likely telomere-shortening independent and may be due to the loss of telomere protection Investigate if apoptosis is induced Verify off target effects using hTERT-negative cell line Conclusion Identification and characterization of alternatively-spliced mTERT mRNAs Copy of IMG_0227 SANJIDA.jpg mTERT Alternatively-Spliced Variants mTERT ASPS •Exons = white boxes with numbering inside. •Arrowheads above full-length mTERT mRNA = the regions of each PCR product covered. •mTERT ASPSs have been assigned descriptive names. Five of the suspected variants were confirmed by sequencing. CB17: mouse fibroblast cell line NIH3T3: Mouse embryonic fibroblast FM3A: mouse mammary carcinoma * Strausberg et al., Unpublished Work * Similar variant in hTERT 081008-3 Generated PCR products of alternatively spliced mRNA using splicing variant-specific primers and polyA purified RNA from NIH3T3 cells mTERT Alternatively-Spliced Variants Variant Primers used for PCR 1 Primers used for PCR 2 Expected Size of Product (base pairs) Ins a InsaF 1069R InsaF 500R 291 Del k DelkF 2092R DelkF 1689R 682 Del b 806F DelR2 DelbF 2092R 558 Del d DeldF 3056R DeldF DelR2 427 Del e DeleF Ins4-R3 DeleF Ins3-R2 459 Insertion Variant, Insi1 •Found in intron 1 •Ins i1 is in a region which corresponds to the hTERT RID1 motif. • The telomerase reverse transcriptase (TERT) is divided into three regions, the TERT essential N-terminal (NTE) domain, the reverse-transcriptase (RT) motifs and the TERT C-terminal extension (CTE). Adapted from (Autexier and Lue, 2006.) Deletion Variant, Dele12 •Deletion falls in a region which corresponds to the hTERT CTE •Deleting a sequence in such an essential part of the mTERT may modify it slightly or it might render it completely inactive. mTERT insertion but not the deletion variant reconstitutes telomerase activity WT mTERT Insertion Variant WT mTERT (µl) Deletion Variant (µl) 0 0 0 1 1 1.5 1 2 1 0 1 1 (b) Telomerase activity of mixed wildtype mTERT/insertion variant suggests the insertion variant has no inhibitory effects on wildtype mTERT 0 0 1 0.5 1 1 1 1.5 1 2 0 1 1 0 WT mTERT Deletion Variant 0 0 1 0.5 1 1 1 1.5 1 2 0 1 Telomerase activity of mixed wildtype mTERT/deletion variant the deletion variant seems to be inhibiting telomerase activity and may have dominant negative effects on telomerase activity E:\DNA Binding\DEC2,2011-DNAbindingWTmTERT,INS,DEL.bmp E:\DNA Binding\DEC2,2011-DNAbindingWTmTERT,INS,DEL.bmp WT mTERT Insertion Deletion WT mTERT Insertion Deletion 1 2 1 2 1 2 3 4 3 4 3 4 1)5’-biotinylated primer 2)5’-biotinylated antisense primer 3)Non-biotinylated primer 4)No primer Insertion and deletion variants do not appear to have DNA-binding defects in vitro Bound hTR Precipitated TERT Protein Both variants exhibit RNA-binding defects in vitro •A major accessory domain containing the RNA-interaction domain 1 (RID1) •RID1 interacts with the hTR pseudoknot-template domain and hTERT's RT motifs and putative thumb and was shown to be essential for processivity, but not DNA synthesis. RID1 Motif and RNA Binding Autexier and Lue, 2006 •These preliminary results suggest that both the insertion and deletion variants have decreased binding affinities. •For the former, the decrease in binding may be due to the fact that the 102-nucleotide insert falls in a region which corresponds to the human RID1 motif. Stable expression of the deletion variant in the CB17 mouse fibroblast cell line slows cell growth Alternatively spliced variants are stably expressed with increasing population doublings E M L E M L E M L E M L Ins 2 Ins 8 Del 1 Del 9 •Confirm levels by qPCR •Confirm protein expression by Western analysis F:\CELL TRAP\feb1,2012-CELLTRAPDEL9-E,M,L.bmp Early Middle Late (-) Internal Control Dilutions 100 ng 40 ng 20 ng 10ng 4 ng 2 ng Stable expression of the deletion variant (clone 9) leads to inhibition of telomerase with increasing population doublings Stable expression of the deletion variant (clone 9) leads to inhibition of telomerase with increasing population doublings Conclusions 1.Five mTERT alternatively-spliced variants were identified by RT-PCR and sequencing. 2.The splicing patterns are different from rat and human (except for Dele6, which has also been identified in the human). 3.The variants were confirmed by RT-PCR using purified polyA+ mRNA from NIH 3T3. 4.In the in-vitro TRAP assay, the insertion variant reconstitutes telomerase activity while the deletion variant does not. 5.In vitro, while the insertion variant seems to have no inhibitory effects on wildtype mTERT, the deletion variant seems to be inhibiting telomerase activity and may have dominant negative effects on telomerase activity. 8.The insertion and deletion variants are present in different mouse cell lines and specific mouse tissues, shown through RT-PCR using variant-specific primers. 9.Deletion variant stable cells (clone 9) exhibit a noticeable growth defect and inhibition of telomerase activity with increasing population doublings Perspectives 1.The potential negative-regulatory or dominant-negative function of the alternative-spliced deletion variants will continue to be assayed when expressed in telomerase-positive cell line, CB17 by assessing telomere length or function at various passages 2.Modulation of splicing in NIH 3T3 cells using anti-sense oligonucleotides and its consequences in gene expression, and telomerase activity and cell proliferation 3.Can alternatively-spliced mTERT variants function to reduce end-to-end fusions typically observed in late passage mTERT-/- ES cells by assessing signal-free ends Identification of a Box C/D SnoRNP component as a human telomerase-associated protein Telomerase-associated Proteins Shay et al, 1999 Cohen et al, 2007 : dimer of hTERT, dyskerin and hTR (total of 1300kDa); Complex in the range of 500-1000 kDa (Schapp et al., 1998) Reptin/Pontin TERT 148 kDa TAP-tagged hTERT reconstitutes an active enzyme in vitro Created stably expressingTAP-tagged hTERT expressing Flp-In 293 cell line Mass Spectrometry identifies NOP17 as a potential interacting protein NOP17 :a telomerase interacting protein IP: FLAG Depletion of NOP17 reduces telomerase activity NOP17 overexpression prevents the knockdown of telomerase activity NOP17 forms a complex with pontin Role for Nop17 in telomerase assembly? = Pih1=Nop17 Rvb1/2=pontin/reptin pontin/reptin hTERT Dyskerin/NOP10/NHP2/GAR1 Conclusions and Perspective •NOP17 is a potential interacting protein of telomerase •Immunoprecipitation against calmodulin binding peptide tagged hTERT confirms coimmunoprecipitates NOP17 •Immunoprecipitation of FLAG-NOP17 co-immunoprecipitates telomerase activity •siRNA against NOP17 reduces telomerase activity •Overexpression of NOP17 prevents knockdown of telomerase activity •Assess if stable knockdown of NOP17 causes telomere shortening and reduced accumulation of hTR •Assess if hTR, and various snoRNAs (H/ACA and C/D) co-immunoprecipitate with NOP17 • • Characterization of unique properties of telomerase that regulate telomere maintenance Unique features of telomerase such as its repeat addition mechanism of DNA polymerization could be excellent specific targets What are the determinants of processivity? In vitro studies implicate TR, TERT, telomere and telomerase-interacting proteins (TPP1, Pot1), proofreading activities? Does processivity in vitro correlate with telomere maintenance? Emerging pharmacological and genetic evidence, primarily in yeast, suggests that telomerase processivity is a significant determinant of telomere length However, human telomerase is much more processive than yeast telomerase, and a fundamental unknown aspect of human telomerase biology is the contribution of enzyme processivity to telomere length maintenance and human cellular immortalization Residues predicted to be important for processivity hTERT Mutant Previous Mutation (TERT species) Phenotype of TERT mutant Effect on telomere length Reference V791Y LYID589AAAA (Est2p) Decreased processivity Telomere shortening Lue et al, 2003 W930F FCA720WCG (Est2p) Increased processivity Telomere length increase Peng et al, 2001 L866Y L813Y (tTERT) Increased processivity N/A Bryan et al, 2000 V791Y Wild-type W930F In vitro-reconstituted hTERT RT domain mutants -W930F and -V791Y are less active than wild-type 1/50; 1/65; 1/100; 1/200; 1/250; 1/400 Processivity of in vitro-reconstituted hTERT-W930F and -V791Y enzymes are severely compromised • Feb92007A.JPG oct122007A.JPG hTERT mutant T2AG3 repeats -W930F 3 -V791Y 4 Mutant telomerases reconstituted in cells with limited lifespan methods.jpg hTERT-/- hTR +/+ Fibroblasts expressing SV40 early region Late passage, near crisis V791Y hTERT W930F Growth Activity Protein expression Telomere length Despite similar activities of in vitro-reconstituted enzymes, HA5 cells expressing hTERT-V791Y are unable to survive in culture, unlike HA5 cells expressing hTERT–W930F Days Days Growth curve of mortal Ha5-hTERT mutant clones in culture B Actin hTERT 127Kd HA5 cells expressing hTERT-V791Y undergo apoptosis hTERT-V791Y and hTERT-W930F expressed in cells can reconstitute active enzymes Wild-type F1 V791YD V791YE PD8.4 PD5.6 PD5.6 PD8.4 PD18 (-) TRAP1redomarch302011.bmp TRAP3march272011.bmp Wild-type F1 PD 111 W930F F2 PD 12 W930F F2 PD 87 W930F F2 PD 207 The processivity of mutant telomerase enzymes expressed in cells parallels the processivity of in vitro-reconstituted enzymes • Overexpress both hTERT and hTR components in 293T cells, collect extract and perform direct primer extension assay • Critical telomere length •It is disputed whether it is one critically short telomere or a subset of critically short telomeres that directs the cell towards senescence/cell death • Telomerase Telomerase Critically short telomere: “SIGNAL FREE END” (SFE) Kamranvar and Masucci, 2011 HA5 cells expressing hTERT-V791Y contain higher number of telomeric signal free ends than cells expressing hTERT–W930F 3000 telomere ends 1000 telomere ends Possible identification of hTERT residues that regulate substrate utilization and/or recruitment to telomeres – –Telomerase enzymes with similar activities and processivities function differently in telomere maintenance –Activities and processivities of in vitro-reconstituted enzymes and enzymes expressed in cells are similar –hTERT-W930F and -V791Y both accumulate SFEs however only -W930F is able to rescue those SFEs •We speculate that hTERT-W930F may more efficiently bind or elongate shorter substrates than hTERT-V791Y, or be more efficiently recruited to telomeres, allowing it to immortalize late passage HA5 cells with short telomeres Conclusion In vitro-expressed hTERT-L866Y reconstitutes similar levels of activity than wild-type enzyme L866Y Wild-type In vitro-expressed hTERT-L866Y displays increased processivity compared to wild-type enzyme Feb92007A.JPG Feb92007A.JPG Feb92007A.JPG Used first 4 repeats Say did it two ways Growth rate of HA5-hTERT-L866Y is similar to HA5 expressing wild-type hTERT despite increased processivity hTERT-L866Y enzyme extracted from cells is highly processive conventional_extractmarch102011.bmp Processivity of L866Y hTERT-L866Y-expressing clones display heterogenous telomere lengths L866YM3 L866YF1 Wild-type M2 TRF_L866YM3_W930FF2_april2009_converted.tif 10 8 6 3 2 1.5 1.2 9 12 45 102 141 kb 10 8 6 3 2 1.5 1.2 TRF_WTM2_deltas_Ha5.jpg 24 48 126 165 180 198 30 Digest with RsaI and HinfI PFGE Probe with radiolabelled (C3TA2)4 15 39 111 144 162 189 TRF_L866YF102-04-09.tif 30 12 4.3 3.6 3.1 hTERT-L866Y-expressing clones initially display an increase in telomere length followed by telomere length heterogeneity L866YM3 Wild-type M2 TRF_L866YM3_W930FF2_april2009_converted.tif 10 8 6 3 2 1.5 1.2 9 12 45 102 141 kb 10 8 6 3 2 1.5 1.2 TRF_WTM2_deltas_Ha5.jpg 24 48 126 165 180 198 30 Negative regulation of telomere length by a telomeric DNA trimming mechanism •Overexpression of both telomerase components in telomerase positive cancer cells results in increased telomere length that eventually reaches a plateau, accompanied by ALT characteristics such as telomere length heterogeneity and extrachromosomal telomeric repeat (ECTR) DNA in the form of T circles (Pickett et al, 2009) •Not all characteristics of ALT were observed, including no telomere exchange events and no telomere dysfunction-induced foci •Are we observing telomere trimming in HA5 cells expressing L866Y? • • • • • Telomerase hTERT-L866Y Telomeres are longer at earlier PD but then shorten to a regulated, pre-determined length Although it has been suggested that APBs are directly implicated in telomere metabolism of ALT cells, their precise role and structure have remained elusive. Here we show that PML bodies in ALT cells associate with chromosome ends forming small, spatially well-defined clusters, containing on average 2–5 telomeres. Using an innovative approach that gently enlarges PML bodies in living cells while retaining their overall organization, we show that this physical enlargement of APBs spatially resolves the single telomeres in the cluster, but does not perturb the potential of the APB to recruit chromosome extremities. We show that telomere clustering in PML bodies is cell-cycle regulated and that unique telomeres within a cluster associate with recombination proteins. Enlargement of APBs induced the accumulation of telomere-telomere recombination intermediates visible on metaphase spreads and connecting heterologous chromosomes. The strand composition of these recombination intermediates indicated that this recombination is constrained to a narrow time window in the cell cycle following replication. These data provide strong evidence that PML bodies are not only a marker for ALT cells but play a direct role in telomere recombination, both by bringing together chromosome ends and by promoting telomere-telomere interactions between heterologous chromosomes. ECTR (T circle) formation 0.jpeg Wang et al, Cell, 2004 Presence of T-circles in late passage hTERT-L866Y-expressing cells suggest that trimming events are occurring L866YF166_april122011_T.bmp tcirclefeb72011.bmp Ha5-hTERT-L866YF1 PD198 GM847 ALT cell tcircleWTM269_feb222011.bmp Ha5-hTERT PD207 Regulation of telomere length and homeostasis by processivity • •We speculate that processivity is a regulator of telomere homeostasis •This would be the first report that trimming occurs physiologically in limited lifespan cells that require telomerase expression for cell survival versus in immortal telomerase-positive cells that overexpress both telomerase components • – –L866Y reconstituted telomerase exhibits increased processivity –Telomeres of hTERT-L866Y expressing cells are heterogeneous in length –T-circles indicative of telomere trimming is observed in late passage L866Y Conclusion Promyelocytic leukaemia nuclear bodies (PML-NBs) are one of^ numerous distinct subnuclear structures that are thought to^ compartmentalize the nucleus. They contain the PML protein and^ are associated with various nuclear functions, including transcriptional^ regulation, apoptosis and the maintenance of genome stability.^ Now, Stig Ove Bøe and co-authors report that PML-NBs^ are predetermined processing sites for damaged DNA Acknowledgements Collaborators and Colleagues Kurt Dejgaard Joachim Lingner José-Arturo Londoño-Vallejo Silvia Bacchetti CIHRlogo_e Lab members Shusen Zhu Yasmin D’Souza Marie-Eve Brault May Shawi Catherine Lauzon Sanjida Khondaker Johanna Mancini Josephine Chu Nahid Golabi Ricky Kwan ac1_bandeau