Identification of human TERT elements necessary for telomerase recruitment to telomeres
Linear human chromosomes end in G-rich telomeric repeat DNA with a single-stranded overhang ( Blackburn , 2005 ) . Chromosome ends pose two challenges to cells: they resemble DNA damage sites , and they shorten progressively each cell cycle due to the end-replication problem ( Levy et al . , 1992 ) . The six-protein shelterin complex specifically binds telomeric DNA and prevents the DNA damage machinery from recognizing telomeres ( Palm & de Lange , 2008 ) . Additionally , shelterin recruits telomerase , an RNA-containing reverse transcriptase that adds telomeric repeats to the 3ʹ-end of the single-stranded overhang to maintain telomere length ( Nandakumar and Cech , 2013 ) . Loss-of-function mutations in telomerase and shelterin components cause human diseases like dyskeratosis congenita , aplastic anemia , and pulmonary fibrosis ( Armanios and Blackburn , 2012 ) . In addition , like stem cells and germ cells , 90% of cancers depend on telomerase activity to maintain the potential to proliferate indefinitely ( Stewart and Weinberg , 2006; Shay and Wright , 2011 ) . Telomerase is a ribonucleoprotein ( RNP ) complex . Its catalytic core is comprised of hTERT and the telomerase RNA component ( hTR ) , which contains the template used for telomeric DNA synthesis ( Cech , 2004 ) . In the holoenzyme , hTR also associates with dyskerin for stability and with TCAB1 for telomerase maturation and trafficking to Cajal bodies ( Mitchell et al . , 1999; Venteicher et al . , 2009 ) . The hTERT protein contains four domains , the telomerase N-terminal domain ( TEN-domain ) , the telomerase RNA-binding domain ( TRBD ) , the reverse transcriptase domain ( RT-domain ) , and the C-terminal extension ( CTE ) or putative thumb domain of the reverse transcriptase . The TRBD- and RT-domains of hTERT bind hTR and position the template of hTR in the RT-domain active site to synthesize telomeric repeats ( Cech , 2004 ) . The TEN-domain participates in the catalysis of telomeric repeat addition ( Jacobs et al . , 2006; Jurczyluk et al . , 2011; Robart and Collins , 2011; Wu and Collins , 2014 ) . Additionally , both the TEN-domain and CTE contain DAT ( dissociates activities of telomerase ) regions , N-DAT and C-DAT , respectively ( Armbruster et al . , 2001; Banik et al . , 2002 ) . Mutations in either DAT-region disrupt the ability of telomerase to immortalize cells , but they retain catalytic activity ( Armbruster et al . , 2001; Banik et al . , 2002 ) . Based on experiments with N-DAT mutants , the TEN-domain has been proposed to mediate telomerase recruitment to telomeres ( Armbruster et al . , 2003 , 2004 ) . Telomerase is recruited to telomeres in S-phase , while it remains in Cajal bodies for the remainder of the cell cycle ( Jady et al . , 2006; Tomlinson et al . , 2006 ) . Telomerase localization to Cajal bodies requires the interaction of hTR with TCAB1 ( Venteicher et al . , 2009 ) . TCAB1 is not required for enzymatic activity but for function in vivo ( Venteicher et al . , 2009 ) , indicating that recruitment to Cajal bodies is a key step in telomerase trafficking or maturation . Recruitment of telomerase to the telomere requires the shelterin component TPP1 ( Xin et al . , 2007; Abreu et al . , 2010 ) . TPP1 is recruited to the shelterin complex via an interaction with TIN2 ( Abreu et al . , 2010 ) . TPP1 also tightly binds to POT1 ( Wang et al . , 2007 ) , which specifically binds the telomeric single-stranded DNA overhang ( Baumann and Cech , 2001; Lei et al . , 2004 ) . Biochemical studies have demonstrated that the interaction between the POT1-TPP1 complex and telomerase stimulates its repeat addition processivity ( RAP ) , that is , increases the number of consecutive repeats that telomerase synthesizes in a single round of telomeric DNA addition ( Wang et al . , 2007 ) . Recently , the TEL-patch , a group of mostly acidic amino acids on the oligosaccharide binding ( OB ) -fold domain of TPP1 was shown to be necessary for telomerase recruitment in vivo and RAP stimulation in vitro ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) . Furthermore , cell biological assays demonstrated that the OB-fold domain of TPP1 is sufficient to recruit telomerase to a non-telomeric chromosomal locus ( Zhong et al . , 2012 ) . Although TPP1 is critical for recruitment of telomerase to the telomere , it has been unclear whether TPP1 makes a direct interaction with telomerase or if their interaction is bridged by another component . Conversely , the site on telomerase that associates with the telomere is not well defined . The TEN-domain of hTERT is a potential candidate to contain key residues necessary for the interaction with TPP1 . A mutation in the TEN-domain of hTERT , G100V , is defective in stimulation of telomerase processivity in vitro ( Zaug et al . , 2010 ) and fails to localize to telomeres in vivo ( Zhong et al . , 2012 ) . However , G100V telomerase has substantially reduced enzymatic activity ( Zaug et al . , 2010 ) ; thus , it is difficult to assess whether its failure to function in vivo is due to an activity or recruitment defect . Furthermore , the interaction between TPP1 and telomerase may require a bridging factor . In Schizosaccharomyces pombe , Ccq1 bridges the interaction between Tpz1 , the TPP1 homologue , and telomerase ( Miyoshi et al . , 2008; Moser et al . , 2011 ) . A human homologue of Ccq1 has not been identified ( Nandakumar and Cech , 2013 ) . Thus , it is unclear if telomerase directly interacts with TPP1 in human cells . In this study , we identify separation-of-function mutations in the TEN-domain of hTERT . The mutants retain high catalytic activity , but their interaction with POT1-TPP1 is compromised in vitro . Furthermore , the TEN-domain mutants fail to localize to telomeres and cannot maintain telomere length in vivo . Importantly , a compensatory mutation in the TEL-patch of TPP1 rescues a mutation in the TEN-domain of hTERT both in vitro and in vivo , indicating that the interaction between telomerase and the OB-fold domain of TPP1 is direct . These observations open the door to more directed approaches for targeting the TPP1-telomerase interface as a therapeutic strategy for human diseases .
Conserved acidic amino acids on the surface of the TPP1 OB-domain ( TEL-patch ) are necessary for the recruitment of telomerase to telomeres ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) . We hypothesized that a corresponding region of basic amino acids would exist on the surface of hTERT , which directly associates with the TEL-patch via charge–charge and other interactions to recruit telomerase to telomeres ( Figure 1A ) . 10 . 7554/eLife . 03563 . 003Figure 1 . Identification of mutants in the TEN-domain of hTERT that affect the interaction with POT1-TPP1 in vitro but not enzymatic activity . ( A ) Domain structures of hTERT and TPP1 proteins . Question mark , proposed interaction between the TEL-patch on TPP1 and the N-DAT region of the TEN-domain of hTERT . In TPP1 , the OB-domain ( OB ) is followed by the POT1-binding domain ( PBD ) , and finally the TIN2-binding domain ( TIN2 BD ) . For hTERT , the Telomerase Essential N-terminal ( TEN ) domain is adjacent to the RNA binding domain ( RBD ) , which precedes the reverse transcriptase domain ( RT ) and finally the C-terminal extension ( CTE ) . ( B ) An alignment of the N-DAT region of the TEN-domain of TERT proteins from selected species . Basic residues conserved in greater than five of the seven species are highlighted blue . The conserved residue G100 is highlighted in orange above the alignment . ( C ) Western blot , probed for hTERT , showing the immuno-purification of telomerase over-expressed in HEK 293T cells . To monitor relative quantities of hTERT , equal fractions of lysate , flow through ( FT ) , IgG bead capture ( capture ) , and cleaved eluate ( elution ) , were analyzed by SDS-PAGE . ( D ) Western blot of the relative quantities of wild-type and mutant hTERTs after immuno-purification of the telomerases . ( E ) Direct telomerase activity assays in the absence and presence of the POT1-TPP1 heterodimer ( PT ) for wild-type ( WT ) and mutant telomerases . LC , loading control . +4 , oligonucleotide marker corresponding to the addition of the first four nucleotides to primer . Numbers on left , telomeric repeats added . ( F–H ) Bar graphs representing the quantification of activity , RAP , and RAP stimulation ( decay method ) by wild-type POT1-TPP1 . Values are normalized to WT telomerase , and to WT telomerase with WT POT1-TPP1 for RAP stimulation ( n = 3 , Mean ± SD ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 00310 . 7554/eLife . 03563 . 004Figure 1—figure supplement 1 . Additional TEN-domain mutants tested in this study . ( A ) Direct telomerase assays of additional hTERT TEN-domain mutants ( all with WT hTR ) without ( − ) and with ( + ) WT POT1-TPP1 . LC and +4 marker as in Figure 1E . Quantification in Table 1 . ( B ) Western blot of the relative quantities of wild-type and mutant hTERTs following immuno-purification . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 00410 . 7554/eLife . 03563 . 005Figure 1—figure supplement 2 . Comparison of methods for quantifying RAP stimulation by POT1-TPP1 . ( A ) Example of linear regression of data from one replicate that was used to calculate RAP stimulation by POT1-TPP1 in Figure 1H , using the decay method . All four assays included wild-type POT1-TPP1 . R2 values and slope of regression are shown . ( B ) Comparison of RAP stimulation by POT1-TPP1 for data calculated using the decay ( shown in Figure 1H ) and fraction methods as described in ‘Materials and methods’ . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 005 To identify candidate residues on the TEN-domain of hTERT that might interact with the TPP1 TEL-patch , we performed a multiple sequence alignment of TERT from higher eukaryotes ( Figure 1B ) . The alignment revealed a number of conserved basic amino acids; we tested these , as well as some non-conserved basic amino acids in close proximity in the primary sequence . We generated a panel of hTERT mutants , replacing individual or combinations of basic amino acids on hTERT with the acidic amino acid aspartate . Mutant hTERTs and hTR were over-expressed in HEK293T cells and the telomerases were immuno-purified ( Figure 1C ) . The hTERT variants were expressed at levels similar to those of wild-type telomerase ( Figure 1D ) . To identify separation-of-function hTERT mutants that do not interact with POT1-TPP1 but retain near wild-type telomerase activity in vitro , we carried out direct telomerase extension assays . ( In these assays , dATP , dTTP , and 32P-dGTP are added to the 3ʹ end of a DNA oligonucleotide by immuno-purified telomerase and products are separated on a polyacrylamide sequencing gel and visualized by autoradiography . The enzymatic activity of each telomerase is determined by quantifying the total amount of 32P-dGTP incorporated into reaction products , and RAP is measured by analyzing the distribution of product lengths . ) To measure the physical interaction between TPP1 and telomerase , we carried out direct telomerase assays with and without a previously described minimal POT1-TPP1 complex bound to substrate oligonucleotides ( Wang et al . , 2007 ) and calculated ‘RAP stimulation by PT’ ( equation given in ‘Materials and methods’ ) . RAP stimulation by PT depends on the association of telomerase with POT1-TPP1 and therefore provides a biochemical readout for this interaction . In the hTERT mutant screen , some telomerases had defects in either activity ( R72E , R143E , R142E;R143E ) , RAP ( R87E;R91E;K94E , R120E ) or had no effect ( R142E ) ( Figure 1—figure supplement 1 , Table 1 ) . However , hTERT K78E retained wild-type activity and processivity ( 92% and 100% respectively ) , but RAP stimulation by POT1-TPP1 was reduced to 68% that of wild-type telomerase ( Figure 1E–H; Figure 1—figure supplement 2 ) . Additionally , the R132D mutant was reported to have wild-type activity but failed to localize to telomeres in vivo ( Stern et al . , 2012 ) . We generated a R132E mutant hTERT , which retained moderate activity ( 73% ) but was defective in RAP stimulation by POT1-TPP1 ( 24% of wild-type hTERT , Figure 1E–H; Figure 1—figure supplement 2 ) . Furthermore , the K78E;R132E double mutant retained 63% activity but further reduced RAP stimulation by POT1-TPP1 to 18% of wild-type hTERT ( Figure 1E–H ) . Two different methods of quantitating RAP gave comparable results ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 03563 . 006Table 1 . Telomerase TEN domain mutant activity , processivity , and RAP stimulation by wild-type TPP1DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 006TEN domain mutantActivity % of WT*Processivity % of WTRAP stimulation by PT† % of WTR72E48 ± 399 ± 191 ± 2K78A19 ± 2101 ± 788 ± 2K78E92 ± 1100 ± 168 ± 5R87E;R91E;K94E74 ± 675 ± 0 . 361 ± 4R120E999655K78E;R120E82 ± 1887 ± 338 ± 2R132E73 ± 292 ± 324 ± 2R132E;K78E63 ± 1094 ± 818 ± 1R142E97 ± 7111 ± 10103 ± 4R143E16 ± 1281 ± 3N . D . ‡R142E;R143E4 ± 1N . D . ‡N . D . ‡V144M48 ± 695 ± 544 ± 7*Percentage of wild-type telomerase activity , processivity , or RAP stimulation . Activity values normalized to hTERT levels , loading control , ± standard deviation for 2 or more replicates . †Repeat addition processivity ( RAP ) stimulation upon addition of WT POT1-TPP1 , values relative to WT telomerase with WT POT1-TPP1 ( i . e . RAP stimulation by PT % of WT = ( ( WT PT RAP stimulation of telomerase mutant ) / ( WT PT RAP stimulation WT telomerase ) ) *100 ) . ‡Not determined ( N . D . ) due to low telomerase activity . An alignment of the human TEN-domain with the Tetrahymena thermophila TEN-domain in combination with a secondary structure prediction shows that K78 and R132 are in close proximity to each other , on a surface distinct from the proposed DNA-binding region of the TEN-domain ( Figure 2 , Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 03563 . 007Figure 2 . Conserved basic residues K78 and R132 are found in close proximity on the surface of the TEN-domain . Cartoon representation of the TEN-domain crystal structure from Tetrahymena thermophila ( PDB: 2B2A ) . Amino acid numbers are from Tetrahymena ( human counterparts in parentheses ) . Anchor site ( DNA binding ) residues are highlighted in blue . The positions of the residues corresponding to human K78 and R132 are highlighted in red , and F143 corresponding to human V144 is highlighted in green . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 00710 . 7554/eLife . 03563 . 008Figure 2—figure supplement 1 . Alignment and secondary structure prediction for human TEN-domain . Secondary structure prediction for hTERT TEN domain and alignment to selected eukaryotic TERT TEN domains . Human secondary structure prediction made with JPRED3 ( Cole et al . , 2008 ) , while T . thermophila secondary structure based on crystal structure ( PDB: 2B2A ) . The T . thermophila and S . cerevisiae TEN-domains were aligned to the human TEN-domain using Psi-blast , followed by manual alignment of the remaining species . Similar amino acids are shaded by color . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 008 These results demonstrate that K78 and R132 within the TEN-domain of hTERT make a much larger contribution to RAP stimulation by POT1-TPP1 than to enzymatic activity in vitro . RAP stimulation defects in K78E and R132E mutants are most likely due to the failure of telomerase to interact with TPP1 . Thus , this surface of hTERT is a candidate TPP1-interacting element . If mutations of K78 and R132 in hTERT affect the interaction of telomerase with TPP1 , they should disrupt telomerase localization to telomeres in vivo . To test whether the mutant telomerases are recruited to telomeres , we transiently over-expressed mCherry-tagged hTERT variants and hTR in HeLa cells and determined the subcellular localization of telomerase by immuno-fluorescence ( IF ) . Under all conditions hTERT and hTR were expressed at similar levels , indicating that mutations in the TEN-domain did not affect hTERT or hTR stability ( Figure 3A–B ) . As previously described ( Zhong et al . , 2012 ) , wild-type telomerase localized to telomeres and promoted the formation of neo-Cajal bodies at most telomeres , as shown by the co-localization of TRF2 , hTERT , and coilin foci ( Figure 3C ) . In contrast , all three hTERT mutants tested ( K78E , R132E , K78E;R132E ) localized to bona fide Cajal bodies , forming ∼1–3 large foci per cell that co-localized with coilin but did not localize to telomeres marked by TRF2 ( Figure 3C ) . These results demonstrate that TEN-domain mutants that do not interact with POT1-TPP1 in vitro also fail to localize to telomeres in vivo . Furthermore , because Cajal body localization of hTERT requires its association with hTR , our observations indicate that telomerase assembly is unaffected by mutations in the TEN-domain . 10 . 7554/eLife . 03563 . 009Figure 3 . TEN-domain mutations disrupt telomere localization of telomerase . ( A ) Western blots of lysates of HeLa cells transfected with expression plasmids for various hTERT alleles and hTR , probed with an antibody against hTERT . Actin was used as a loading control . ( B ) Northern blots of RNA isolated from HeLa cells transfected with expression plasmids for various hTERT alleles and hTR , using probes for hTR . In vitro transcribed hTR ( 500 pg ) was used as positive control . Blots were probed for RNase P RNA as loading control . ( C ) Immuno-fluorescence ( IF ) analysis of HeLa cells transiently transfected with mCherry-hTERT and hTR plasmids . Cells were fixed and probed with antibodies against mCherry , coilin , and TRF2 to visualize telomerase , Cajal bodies and telomeres , respectively . Images were deconvolved . Numbers indicate the fraction of cells analyzed showing the displayed phenotype ( scale bar = 5 μm ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 009 To determine the effects of the mutant telomerases on telomere maintenance , we generated cell lines stably expressing mCherry-tagged hTERT variants by retroviral transduction . Overexpression of hTERT alone leads to a moderate increase in telomerase activity per cell , more closely resembling endogenous telomerase levels than those obtained by overexpression of both hTERT and hTR ( Cristofari et al . , 2007; Xi and Cech , 2014 ) . Thus , while the absolute expression level of mCherry-hTERT varied between the stable cell lines ( Figure 4A ) , telomerase immuno-purified from the cell lines had comparable activities per cell , and these activity levels were threefold to fourfold higher than levels in untransfected control cells ( Figure 4B ) . This result confirmed that mutations in the TEN-domain did not strongly reduce telomerase activity . The exogenous hTERT was overexpressed relative to endogenous hTERT , which was not detectable by western blot ( Figure 4A ) . Therefore , the majority of endogenous hTR should be assembled into telomerase RNPs containing the mutant hTERT protein ( Figure 4A ) . 10 . 7554/eLife . 03563 . 010Figure 4 . TEN-domain mutants that do not localize to telomeres fail to elongate telomeres in vivo . ( A ) Western blot probed for hTERT and for Actin as a loading control , showing hTERT expression in lysates of parental HeLa cells and cell lines stably expressing mCherry-hTERT variants . hTR was not ectopically expressed . ( B ) Direct enzyme assay of telomerase immuno-purified from lysates , generated using equal number of cells , from parental HeLa or cell lines stably expressing mCherry-hTERT variants . Activity per cell relative to WT hTERT overexpressing cells ( n = 4 , Mean ± SD , p < 0 . 05 ) . ( C ) Telomeric restriction fragment Southern blot of cell lines stably expressing mCherry-hTERT variants over the time course of 8 weeks . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 010 To analyze the effect of mutant hTERT expression on telomere length , DNA isolated from stable cell lines over the course of 8 weeks after viral transduction was subjected to telomeric restriction fragment ( TRF ) analysis by Southern blot . The telomere length of the parental HeLa cell line was stable over the course of the experiment at ∼6 . 6 kb ( Figure 4C ) . Expression of wild-type hTERT caused a progressive increase in telomere length to ∼14 . 8 kb ( Figure 4C ) . In contrast , TEN domain mutants K78E and R132E failed to elongate telomeres , despite the fact that telomerase enzymatic activity per cell was higher than in the parental HeLa cell line ( Figure 4B , C ) . In addition the TEN domain mutant K78E;R132E also failed to elongate telomeres , but due to its reduced activity ( Figure 1F ) , we cannot conclude that this is exclusively due to a localization defect . These observations reinforce the conclusion that telomerase with K78E or R132E mutations in the TEN-domain fails to elongate telomeres due to an inability to localize to telomeres , not an activity defect . Taken together , our experiments demonstrate that mutations in the TEN-domain of hTERT recapitulate the effects of mutations in the TEL-patch of TPP1 ( i . e . , loss of telomerase recruitment to telomeres in vivo , and reduced POT1-TPP1 RAP stimulation in vitro ) . If the stimulation of telomerase RAP and telomerase recruitment to telomeres are the manifestation of a direct interaction between charged residues in the TEN-domain of hTERT and the OB-fold of TPP1 , a charge-swap mutation in the OB-fold of TPP1 could rescue the TEN-domain mutation ( Figure 5A ) . On the other hand , if a bridging factor were necessary for the interaction of TPP1 with hTERT , individually deleterious mutations in both TPP1 and the TEN-domain should have an additive negative impact on the TEN-TPP1 interaction when combined ( Figure 5A ) . 10 . 7554/eLife . 03563 . 011Figure 5 . A compensatory mutation in the TEL-patch of TPP1 rescues RAP stimulation of TEN-domain mutant telomerase in vitro . ( A ) Schematic of the charge-swap experiment to test the interaction between specific amino acids on the TEL-patch on TPP1 and the N-DAT region of the TEN-domain of hTERT . Predicted experimental outcomes are illustrated for two competing models: a direct TPP1 telomerase interaction and an interaction bridged by a yet-unidentified factor ( BF ? ) . ( B ) Coomassie-stained gel of WT , E169K , and E215K TPP1 co-purified with wild-type POT1 . ( C ) Overlays of the Superdex 200 16/60 sizing column chromatograms for wild-type and mutant TPP1-POT1 complexes . ( D ) Direct telomerase activity assay to test the rescue of various telomerase containing WT , K78E , R132E , and K78E;R132E mutant hTERTs in the absence of POT1-TPP1 ( No PT ) or WT , E169K , and E215K TPP1 in complex with wild-type POT1 . LC and +4 marker as in Figure 1E . ( E ) Quantification ( decay method ) of the RAP stimulation by POT1-TPP1 ( PT ) relative to the stimulation of wild-type telomerase with wild-type POT1-TPP1 ( n = 3 , Mean ± SD , *p < 0 . 05 , **p < 0 . 01 , Student's t test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 01110 . 7554/eLife . 03563 . 012Figure 5—figure supplement 1 . The charge-swap is statistically significant by alternative quantitation methods . RAP stimulation by PT calculated using the fraction method to quantitate data presented in Figure 5D ( n = 3 , Mean ± SD , *p < 0 . 05 , **p < 0 . 01 , Student's t test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 01210 . 7554/eLife . 03563 . 013Figure 5—figure supplement 2 . E215K TPP1 rescues RAP stimulation of K78E mutant telomerase produced in RRLs . ( A ) Western-blot for hTERT produced in RRLs . Also shown is a serial dilution of overexpressed telomerase immuno-purified from HEK293T cells . 1X signifies amount of telomerase used in direct extension assays . ( B ) Direct telomerase activity assay to test the rescue of WT and K78E telomerases produced in RRLs , in the absence of POT1-TPP1 ( No PT ) or WT , E169K , and E215K TPP1 in complex with wild-type POT1 . LC as in Figure 1E . Rescue is apparent by looking at the long extension products ( >12 repeats ) , which decrease in intensity from lane 2 to lane 4 for WT hTERT , but then increase from lane 2 to lane 4 for K78E hTERT . ( C ) Quantification ( decay method ) of the RAP stimulation by POT1-TPP1 ( PT ) relative to the stimulation of wild-type telomerase with wild-type POT1-TPP1 ( n = 3 , Mean ± SD , *p < 0 . 05 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 01310 . 7554/eLife . 03563 . 014Figure 5—figure supplement 3 . The IPF allele V144M is deficient in RAP stimulation by TPP1 . ( A ) Direct telomerase assays of wild type and V144M hTERT telomerases in the presence of WT , E169K , or E215K POT1-TPP1 . LC and +4 marker as in Figure 1E . ( B ) Western blot of the relative quantities of wild-type and V144M hTERTs following immuno-purification . ( Note: activity is proportional to hTERT , but RAP is independent of the amount of hTERT in these assays . ) ( C ) Quantification of the RAP stimulation by POT1-TPP1 ( PT ) relative to the stimulation of wild-type telomerase with wild-type POT1-TPP1 ( n = 6 , Mean ± SD , **p < 0 . 01 , Student's t test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 014 To test this hypothesis , we generated a number of mutant TPP1N proteins , changing individual conserved negatively charged residues in the TEL-patch to the basic amino acid lysine . Mutant TPP1 proteins were co-purified with POT1 to apparent homogeneity ( Figure 5B ) . All mutant POT1-TPP1 complexes had identical elution profiles from the sizing column ( Figure 5C ) . The presence of a single elution peak indicated that all TEL-patch mutant TPP1 proteins were not globally misfolded and formed heterodimers with POT1 ( Figure 5C ) . To test the ability of the mutant TPP1 proteins to bind TEN-domain mutants , we used direct telomerase enzyme assays to measure RAP stimulation by POT1-TPP1 . Strikingly , TPP1 E215K fully rescued the processivity defect of hTERT K78E; processivity increased from 68% to 98% relative to wild-type telomerase and wild-type TPP1 ( Figure 5D , E ) . Importantly , the non-cognate combinations of ( i ) wild-type telomerase and E215K TPP1 and ( ii ) K78E telomerase with wild-type TPP1 showed a similar reduction of RAP stimulation , to 78% and 68% of the full level , respectively ( Figure 5D , E ) . TPP1 E169K gave dramatic reductions in RAP and did not stimulate any of the tested telomerases above 24% of wild-type ( Figure 5D , E ) . hTERT mutant R132E and the double mutant K78E;R132E were stimulated at levels less than 23% of wild-type by all TPP1 proteins tested ( Figure 5D , E ) . An alternative method for quantification of RAP stimulation by POT1-TPP1 gave similar results ( Figure 5—figure supplement 1 ) . Furthermore , to rule out the possibility of a bridging factor that co-purifies with telomerase from HEK293 T cells , we carried out the charge-swap experiment with telomerase purified from rabbit reticulocyte lysates ( RRLs ) . The charge-swap rescue was statistically significant when using telomerase from RRLs , although the absolute PT stimulation of RRL telomerase was diminished ( Figure 5—figure supplement 2 ) . To further illustrate the specificity of the charge-rescue , a disease allele V144M found in some cases of idiopathic pulmonary fibrosis ( IPF ) ( Tsakiri et al . , 2007 ) was tested for interaction with POT1-TPP1 . V144M telomerase retained moderate telomerase activity and high processivity ( 48% and 95% of wild-type telomerase , respectively ) but POT1-TPP1 RAP stimulation was significantly decreased to 61% when compared to wild-type telomerase ( Table 1 , Figure 5—figure supplement 3 ) . Furthermore , TEL patch mutants E169K and E215K each resulted in an additive loss of RAP stimulation when tested with disease mutant V144M , to 28% and 37% respectively ( Figure 5—figure supplement 3 ) . Thus , the specific and compensatory nature of the hTERT K78E and TPP1 E215K mutant combination strongly suggests that K78 on the TEN-domain of hTERT directly interacts with E215 on the TEL-patch of TPP1 to stimulate RAP in vitro . To analyze mutant hTERT-TPP1-OB-fold interactions in vivo , we utilized a Lac-repressor ( LacI ) assay previously described ( Zhong et al . , 2012 ) . The TPP1-OB-fold domain was inserted between GFP and the LacI protein ( Figure 6A ) . GFP-TPP1-OB-LacI was expressed alongside mCherry-tagged hTERT and hTR in the U2OS 2-6-3 cell line , which contains a single Lac-operator DNA array on chromosome 1 ( Janicki et al . , 2004 ) , thereby tethering the OB-fold domain of TPP1 to a non-telomeric chromosomal locus ( Figure 6A ) . The interaction between telomerase and the TPP1-OB-fold domain was assessed by co-localization of GFP- and mCherry foci in cell nuclei . Wild-type telomerase co-localized with the TPP1-OB-fold domain in ∼90% of nuclei . In contrast , in cells expressing K78E , R132E , or K78E;R132E telomerase , the fraction of TPP1-OB-fold domain foci showing telomerase signal was reduced to ∼55% , ∼30% , and 0% , respectively ( Figure 6B , C , additional examples in Figure 6—figure supplement 1 ) . The reduction in co-localization demonstrated that mutation of the hTERT TEN-domain interferes with the interaction between telomerase and the TPP1-OB-fold domain in vivo . 10 . 7554/eLife . 03563 . 015Figure 6 . A compensatory mutation in the TEL-patch of TPP1 rescues TEN-domain mutant telomerase binding to the TPP1 OB-domain in vivo . ( A ) Model showing the experimental design . Fusion of the OB-domain of TPP1 to the lac repressor ( LacI ) recruits the OB-domain to a single non-telomeric chromosomal locus ( LacO array ) , allowing the interaction between telomerase and the TPP1 OB-domain to be assessed by co-localization of GFP ( TPP1 OB ) and mCherry ( hTERT ) in cell nuclei . ( B ) Fluorescence images showing the localization of GFP-TPP1-OB-LacI and mCherry-hTERT fusion proteins in cell nuclei stained by DAPI . Cells were fixed , permeabilized , and stained with DAPI . The intrinsic GFP- and mCherry-fluorescence was used to detect GFP-TPP1-OB-LacI and mCherry-hTERT ( scale bar = 5 μm ) . ( C ) Quantification of the experiments shown in ( B ) , showing the fraction of nuclei with co-localization of GFP- and mCherry-foci ( n = 3 , 117–176 nuclei total , Mean ± SD , *p < 0 . 05 , **p < 0 . 01 , Student's t test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 01510 . 7554/eLife . 03563 . 016Figure 6—figure supplement 1 . Additional examples of U2OS 2-6-3 cells expressing GFP-TPP1 OB-LacI and mCherry-telomerase . Fluorescence images showing the localization of GFP-TPP1-OB-LacI and mCherry-hTERT fusion proteins in cell nuclei stained by DAPI . Cells were fixed , permeabilized , and stained with DAPI . The intrinsic GFP- and mCherry-fluorescence was used to detect GFP-TPP1-OB-LacI and mCherry-hTERT ( scale bar = 5 μm ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 016 To determine whether mutations introduced in the OB-fold domain of TPP1 can compensate for the mutations introduced in hTERT in vivo , we carried out the LacI-assay with E215K TPP1-OB . As shown in Figure 6B , C , the co-localization of wild-type telomerase with the E215K TPP1-OB-fold domain was greatly reduced; instead , wild-type telomerase localized to many foci distinct from the LacI site , presumably telomeres . In contrast , introduction of the E215K mutation significantly increased the co-localization of K78E telomerase with the TPP1-OB-fold domain ( p < 0 . 05 ) . Thus the E215K mutation in the TPP1-OB-fold compensates for the presence of the K78E mutation in hTERT in vivo . Importantly , E215K TPP1 had no effect on the localization of R132E and K78E;R132E telomerases , demonstrating that E215K specifically compensates for the presence of the K78E mutation in hTERT ( Figure 6B , C ) . If another protein were bridging telomerase and TPP1 , any combination of two defective mutations should be even more defective . Thus , finding a compensatory double mutant demonstrates that telomerase is recruited to telomeres by a direct interaction between the TEN-domain of telomerase and the TPP1-OB-fold domain in part by a contact formed between K78 in the TEN-domain and E215 in the TPP1-OB-fold domain . To address whether TPP1 E215K could rescue recruitment of hTERT K78E to telomeres , we transduced cell lines stably expressing WT and K78E mCherry-hTERT with retrovirus expressing wild-type ( WT ) , E169K , and E215K TPP1-FLAG alleles . The resulting cell lines expressed both TPP1-FLAG and mCherry-hTERT proteins ( Figure 7A–B ) . Importantly , retroviral transduction was carried out after substantial telomere erosion had occurred due to the expression of K78E hTERT ( Figure 4C , transduction carried out after 8 weeks ) . 10 . 7554/eLife . 03563 . 017Figure 7 . TPP1 E215K rescues telomere maintenance in cells expressing hTERT K78E . ( A ) Western blot probed for TPP1-FLAG and for Actin as a loading control , showing TPP1 expression in lysates of parental HeLa cells and cell lines stably expressing TPP1-FLAG and mCherry-hTERT variants . ( B ) Western blot probed for hTERT and for Actin as a loading control , showing hTERT expression in lysates of cell lines stably expressing TPP1-FLAG and mCherry-hTERT variants . ( C ) Telomeric restriction fragment Southern blot of cell lines stably expressing K78E mCherry-hTERT and TPP1-FLAG variants over the time course of 6 weeks . DOI: http://dx . doi . org/10 . 7554/eLife . 03563 . 017 To test whether the expression of E215K TPP1 rescued telomere maintenance in cells expressing K78E hTERT , we carried out TRF analysis by Southern blot ( Figure 7C ) . Telomere length in the cell line expressing only K78E hTERT was constant at ∼3 . 4 kb during the 6-week experiment . Expression of WT TPP1 led to an increase of telomere length to ∼6 . 2 kb . Importantly , expression of E215K TPP1 triggered a more pronounced increase in telomere length to ∼7 . 7 kb over the same time period , while E169K gave lead to a subtle increase to ∼4 . 9 kb . These observations demonstrate that expression of E215K TPP1 in cells expressing K78E hTERT rescues telomere length maintenance , most likely by driving telomerase recruitment to telomeres .
During the S-phase of the cell cycle , telomerase moves from Cajal bodies to telomeres to counteract the progressive shortening of the telomere that occurs during DNA replication ( Jady et al . , 2006; Tomlinson et al . , 2006; Cristofari et al . , 2007; Stern et al . , 2012 ) . The regulatory mechanisms underlying this process are not well understood , in part because the necessary molecular interfaces are not fully defined . On the telomere side , TPP1 is well established as being necessary for the recruitment of telomerase ( Abreu et al . , 2010; Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) . Localization of telomerase to an artificial non-telomeric focus by the TPP1 OB-domain ( Zhong et al . , 2012 ) suggests that the TPP1 OB-domain is the minimal sufficient telomerase recruitment module of the shelterin complex . The TEL-patch provides a molecular surface that mediates telomerase binding , as single mutations in the TEL-patch result in loss of telomere recruitment and of RAP stimulation of telomerase by TPP1 ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) . While the telomeric half of the interface required for telomerase recruitment to telomeres is well understood , the molecular determinants on telomerase required for telomere localization were thus far ill defined . Several lines of evidence , including naturally occurring disease-associated alleles of hTERT ( V144M ) ( Tsakiri et al . , 2007 ) and synthetic mutations ( G100V , R132D , N-DAT ) , have implicated the TEN-domain of hTERT in telomerase recruitment to telomeres ( Armbruster et al . , 2001; Zaug et al . , 2010; Stern et al . , 2012 ) . Unfortunately , due to the involvement of the TEN-domain in catalysis and RAP , many of these mutations also alter the enzymatic activity of telomerase . Thus , TEN-domain mutants can have pleotropic effects and may not specifically disrupt the telomere–telomerase interface ( Zaug et al . , 2010 , 2013 ) . Our results demonstrate that a region in the TEN-domain including K78 , R132 , and V144 is required for telomerase recruitment in vivo and POT1-TPP1-mediated RAP stimulation in vitro , albeit to varying degrees . The differences in RAP stimulation may reflect the importance of the respective residue in an interaction with TPP1 . The recruitment defects and failure to maintain telomeres in vivo occur in the context of wild-type levels of activity , suggesting that the mutants described in this study are separation-of-function alleles that dissociate enzymatic activity from recruitment . Importantly , the recruitment defects of K78E and R132E telomerases recapitulate those of TEL-patch mutant TPP1 proteins ( Zhong et al . , 2012; Nandakumar et al . , 2012 ) . The interaction between the TEL-patch of TPP1 and the TEN-domain of hTERT provides a molecular mechanism to explain the failure of N-DAT mutations to immortalize cells . Our observation that charge-swap mutations on the TEN-domain of hTERT and the TEL-patch of TPP1 restore telomerase recruitment indicates that these elements interact directly; for other examples see Jucovic and Hartley ( 1996 ) ; Tansey and Herr ( 1997 ) ; Pennock et al . ( 2001 ) . If instead there were an ‘adapter’ protein bridging the two elements , the combination of two individually deleterious mutations would lead to an additive loss of function ( Figure 5A ) . We note that hTERT K78E and TPP1 E215K lead to similar reductions in RAP stimulation when combined with wild-type TPP1 and wild-type hTERT , respectively . This observation adds additional support to the model that hTERT K78E and TPP1 E215K eliminate reciprocal residues of the same molecular interaction . In contrast , the V144M mutation in hTERT and the E215K mutation in the TEL-patch of TPP1 display an additive reduction in RAP stimulation , indicating that they affect separate components of the telomerase–TPP1 interface . In other organisms , however , the association of telomerase with the telomere occurs through different mechanisms . For example , a bridging factor Ccq1 appears to be necessary for recruitment of telomerase to the telomeric protein Tpz1 ( TPP1 homolog ) in S . pombe ( Miyoshi et al . , 2008; Moser et al . , 2011 ) . A homologue of Ccq1 has not been identified in humans ( Nandakumar and Cech , 2013 ) . Another variation occurs in Saccharomyces cerevisiae , where Est3 shares structural conservation with TPP1 ( Yu et al . , 2008; Rao et al . , 2014 ) . Est3 is part of the telomerase holoenzyme instead of the telomeric cap ( Hughes et al . , 2000 ) . Est3 is thought to make direct contacts with the TEN-domain of Est2 , the yeast TERT ( Friedman et al . , 2003; Talley et al . , 2011; Yen et al . , 2011 ) . However , Est3 appears to stimulate telomerase activity rather than act as a recruitment factor ( Talley et al . , 2011 ) . Recruitment is instead mediated by the interaction of the Est1 subunit of the telomerase holoenzyme and Cdc13 at the telomere ( Evans and Lundblad , 1999 ) , and charge-swap mutations indicate that this interaction is direct ( Pennock et al . , 2001 ) . Thus , in budding yeast the interaction between a TPP1-like OB-domain protein ( Est3 ) and the TEN-domain of TERT ( Est2 ) is conserved , but the recruitment mechanism differs significantly from that in humans . In addition to the interaction between TPP1 and hTERT in humans , other factors contribute to the productive recruitment of telomerase to the telomere . Depletion of TIN2 from shelterin ( Abreu et al . , 2010 ) and perturbation of the TCAB1-hTR interaction ( Stern et al . , 2012 ) both result in a reduction of telomerase association with the telomere . In spite of the aforementioned defects , these proteins likely play important but indirect roles in telomeric recruitment . TCAB1 is necessary for telomerase maturation and trafficking to Cajal bodies ( Venteicher et al . , 2009 ) , while TIN2 is required for TPP1 localization to telomeres ( Abreu et al . , 2010 ) . Previous work also suggested that the C-DAT region of the CTE of hTERT contributed to telomerase recruitment ( Banik et al . , 2002; Zhong et al . , 2012 ) . However , synthetic C-DAT mutants have severe activity defects ( Huard et al . , 2003; Jurczyluk et al . , 2011 ) . In addition , disease-associated mutations in and near C-DAT have varying effects on activity and cellular localization of telomerase . E1117X has ∼10% of wild-type activity and appears to be sequestered to Cajal bodies in vivo ( Tsakiri et al . , 2007; Zhong et al . , 2012 ) ; in contrast , F1127L has reasonable activity ( ∼70% ) and localizes to telomeres , and it is stimulated by POT1-TPP1 ( Zhong et al . , 2012; Zaug et al . , 2013 ) . Thus , CTE mutants do not have clear recruitment defects dissociated from activity loss , despite their importance based on disease association . In contrast , the observation that a mutant TPP1-OB-domain increases the localization of a mutant hTERT to a non-telomeric locus in vivo , while it strongly decreases the association with wild-type hTERT , demonstrates that a direct TEN-domain-TEL-patch interaction is necessary and may be sufficient for telomerase recruitment to telomeres in humans . The identification of a direct protein–protein interaction surface between telomerase and the telomere is key to identifying modulators of telomerase recruitment as potential therapeutic agents . Multiple disease mutations associated with IPF—P33S , L55Q , Pro112ProfsX16 , and V144M—cluster in TEN-domain of hTERT . Mutation carriers have significantly shorter telomeres than non-carrier relatives ( Armanios et al . , 2007; Tsakiri et al . , 2007 ) . These mutations have variable impacts on telomerase enzymatic activity ( Armanios et al . , 2007; Tsakiri et al . , 2007; Zaug et al . , 2013 ) . Importantly , V144M is defective in TPP1-mediated RAP stimulation in vitro ( this study ) and fails to localize to telomeres in vivo ( Zhong et al . , 2012 ) . Given the propensity of both synthetic and IPF-associated mutations in the TEN-domain of hTERT to disrupt telomere localization , it is likely that some cases of IPF are directly caused by a decrease in telomerase recruitment to telomeres . Agonists of the TEL-patch-TEN-domain interaction that compensated for these mutations and attenuated telomere shortening associated with the disease might be pharmaceutically important . In contrast to the loss of telomerase function associated with IPF , reactivation of telomerase is a hallmark of most human cancers . Inhibitors that disrupt telomerase recruitment to telomeres would provide an additional approach to target telomerase activity in cancer cells . In support of this idea , disruption of the TEL-patch diminishes cell growth and triggers apoptosis in HeLa cells , and this effect is exacerbated in combination with a small molecule inhibitor of telomerase activity ( Nakashima et al . , 2013 ) . Our finding that the TEN-domain of hTERT interacts directly with the TEL-patch of TPP1 to bring telomerase to the telomere defines a key interface and provides a direct target for the design of novel therapeutic inhibitors of telomerase action .
Telomerase was overexpressed in HEK293T cells and purified as previously described ( Sauerwald et al . , 2013 ) . Briefly , 1 ml whole cell lysates of 50–60 × 106 HEK293T cells overexpressing ZZ-TEV-3xFlag-hTERT variants and hTR were incubated with ∼350 µl IgG-Sepharose at 4°C for 3–4 hr . Following a wash with 50 ml wash buffer ( 20 mM HEPES-KOH pH 7 . 9 , 300 mM KCl , 2 mM MgCl2 , 1 mM EDTA , 1 mM DTT , 1 mM PMSF , 0 . 1% Triton X-100 , 10% glycerol ) , telomerase was eluted from the beads by cleavage with TEV-protease . The telomerase bound to IgG-Sepharose was incubated with ∼500 µl of elution buffer ( 20 mM HEPES-KOH pH 7 . 9 , 150 mM KCl , 2 mM MgCl2 , 1 mM EDTA , 1 mM DTT , 1 mM PMSF , 0 . 1% Triton X-100 , 10% glycerol ) supplemented with 5 µl of Act-TEV ( Life technologies , Carlsbad , CA ) and 5 µl RNAsin+ ( Promega , Madison , WI ) overnight at 4°C . Telomerase-containing eluate was aliquoted , snap frozen in liquid nitrogen , and stored at −80°C until use . To purify endogenous telomerase from HeLa cells and HeLa cell lines overexpressing mCherry-hTERT variants , telomerase was immuno-purified using a sheep polyclonal antibody ( a kind gift from S Cohen ) as previously described ( Cohen and Reddel , 2008 ) . Briefly , cell lysates of 1 × 106 cells were incubated with 40 µg of anti-hTERT antibody , which was captured using protein-G agarose . Telomerase was eluted using the peptide antigen used to raise the antibody . Eluates were used for direct telomerase extension assays . Telomerase was produced in RRLs as previously described ( Zaug et al . , 2013 ) using ProA-tagged hTERT and purified using the same protocol as telomerase from HEK293T cells described above . Western blots were carried out using 4–12% polyacrylamide Bis Tris Glycine gels ( Life Technolgies ) and antibodies against hTERT ( Ab32020; 1:1000; Abcam , UK ) , beta-Actin ( A5441; 1:5000; Sigma ) , and an HRP-conjugated FLAG-antibody ( A8592; 1:1000; Sigma ) . Secondary antibodies ( Jackson ImmunoResearch , West Grove , PA ) were used at 1:5000 . Detection was carried out using SuperSignal Western Pico Chemiluminescence substrate ( ThermoFisher Scientific , Waltham , MA ) . Cell pellets from insect cells overexpressing GST-POT1 and Escherichia coli cells overexpressing 6xHis-SUMO-TPP1N were lysed by sonication in lysis buffer ( PBS supplemented with 250 mM KCl , 1 protease inhibitor tablet [ThermoFisher Scientific] , 1 mM PMSF ) . Lysates were cleared by centrifugation at 40 , 000×g for 35 min at 4°C . Equal volumes of the insect-cell lysates expressing GST-POT1 and bacterial lysates expressing 6xHis-SUMO-TPP1N were combined and incubated with 1 ml of glutathione–sepharose resin for 1 hr at 4°C . After washing three times with 50 ml of GST-wash buffer ( PBS supplemented with 250 mM KCl , 1 mM DTT , and 1 mM PMSF ) , the POT1-TPP1N complex was eluted with 3 ml of GST-elution buffer ( 50 mM Tris pH 8 . 1 , 75 mM KCl , 10 mM glutathione ) . The elutions were supplemented with 0 . 5% w/w PreScission protease and SUMO-protease and incubated on ice for 0 . 5–1 hr , followed by size-exclusion chromatography on a Superdex 200 16/60 column ( GE Healthcare , UK ) in gel-filtration buffer ( 50 mM Tris pH 7 . 0 , 150 mM NaCl , 1 mM DTT ) . Size-exclusion fractions were pooled , concentrated , supplemented with 10% glycerol vol/vol , snap-frozen in liquid nitrogen and stored at −80°C until use . Direct telomerase activity assays were carried out as previously described ( Zaug et al . , 2013 ) , with slight modifications . The final 20 µl reactions contained 2 µl eluted telomerase extract , 500 nM primer , 500 nM POT1-TPP1 complex , and 150 mM KCl . Direct telomerase assays were quantified as previously described ( Zaug et al . , 2013 ) , except the summed counts incorporated into extension products were normalized to both the hTERT levels ( determined by western blot ) and the loading control and then normalized to WT telomerase . Processivity was measured using the decay method previously described ( Latrick and Cech , 2010 ) . Briefly , counts for repeats 1–20 were corrected for the number of dGTP nucleotides incorporated . The natural log of the counts left behind in each repeat was graphed vs repeat number and fit by linear regression . The slopes of the linear regressions were compared and normalized to WT telomerase . RAP stimulation by PT , for direct extension assays with POT-TPP1 , was measured in the same manner as processivity except values were normalized to WT telomerase with WT POT1-TPP1 ( i . e . , RAP stimulation by PT = processivity of mutant telomerase with TPP1/processivity of WT telomerase with WT POT1-TPP1 ) . The processivity in the absence of POT1-TPP1 was not included the calculation , because ( 1 ) it did not vary greatly among the mutants ( Table 1 ) and ( 2 ) it was so small relative to the processivity in the presence of POT1-TTP1 that dividing by this distorted the calculations and led to irreproducibility . An alternative fraction method of quantitation was also used to determine RAP stimulation by PT , calculated as the fraction of counts in bands 9 and above divided by the total sum of counts incorporated , normalized as described for the decay method . Telomerase from stable cell lines was immuno-purified from identical number of cells and was subjected to direct telomerase assays as previously described ( Cohen and Reddel , 2008 ) . Activity was determined by dividing the total counts by the loading control and normalized to the activity level of cells overexpressing WT hTERT . Telomerase purified from RRLs was assayed using 10 µl of eluate under identical conditions as telomerase from HEK293T cells . Northern blots were carried out as previously described , using 5 separate probes for hTR and 2 probes for RNase P ( Xi and Cech , 2014 ) . The mCherry-hTERT vector was generated by restriction cloning of the hTERT gene into a modified pBABE-puro vector ( a kind gift from Iain Cheeseman ) containing the N-terminal mCherry-tag using SalI/EcoRI . GFP-TPP1-OB-LacI was cloned by restriction cloning of the TPP1-OB-fold domain into a modified pBABE-blast vector ( a kind gift from Iain Cheeseman ) containing the GFP and LacI sequences using XhoI/EcoRI . All point mutations in hTERT and TPP1 were introduced using the Quickchange II mutagenesis kit ( Agilent , Santa Clara , CA ) . The presence of the mutations was verified by Sanger sequencing . All human cell lines were cultured at 37°C , 5% CO2 in growth medium ( Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum , 2 mM glutamax [Life Technologies] ) , 100 units/ml penicillin , and 100 µg/ml streptomycin . The growth medium for the U2OS 2-6-3 cell line ( a kind gift from David Spector ) was additionally supplemented with 100 µg/ml hygromycin B to maintain the LacO array . All transient transfections of human cells were carried out using Lipofectamine 2000 ( Life Technologies ) according to the instructions of the manufacturer . Retroviruses were generated by transfection of pBABE vectors encoding mCherry-hTERT variants and a puromycin resistance gene along with a vector encoding the coat protein VSV-G into the packaging cell line 293-GP . Virus-containing supernatants were harvested 72 hr post-transfection , filtered through a 0 . 22-µm filter , and supplemented with 8 µg/ml polybrene . HeLa cells were incubated with virus-containing supernatant for 16 hr . After infection , the growth medium was replaced with fresh medium containing 1 µg/ml puromycin for mCherry-hTERT constructs and 1 µg/ml blasticidin for TPP-FLAG constructs to select for transduced cells . To ensure maintenance of the transgenes , cell lines were kept under selection with 1 µg/ml puromycin and 1 µg/ml blasticidin ( TPP1 rescue only ) for the duration of the experiments . IF experiments were carried out as previously described ( Nandakumar et al . , 2012 ) , using the following antibodies: mouse monoclonal anti-TRF2 ( IMG-124A; 1:500; Imgenex ) , rabbit polyclonal anti-coilin ( sc-32860; 1:100; Santa Cruz , Dallas , TX ) , rat monoclonal anti-mCherry ( M11217; 1:1000; Life Technologies ) , and rabbit polyclonal anti-RAP1 ( NB-100-292; 1:500; Novus Biologicals , Littleton , CO ) . Secondary antibodies ( Life Technologies , Abcam ) were pre-absorbed to prevent cross-reactivity between rat and mouse antibodies . Telomeric restriction fragment length analysis by Southern blot was carried out as previously described ( Nandakumar et al . , 2012 ) . Briefly , 1 . 5 µg of genomic DNA extracted from human cell lines was digested with RsaI/HinfI and separated by gel electrophoresis using a 0 . 8% 1xTBE agarose gel for a total of 1100 V-hours . DNA was transferred onto a Hybond N+ membrane ( GE Healthcare ) and telomeric restriction fragments were detected using a radiolabeled ( TTAGGG ) 4 probe . To determine mean telomere length , lane intensity profiles were extracted using ImageQuant TL and fit to a Gaussian using Kaleidagraph . The mean of the Gaussian distribution was used as telomere length of the respective sample . Plasmids encoding mCherry-hTERT , hTR , and GFP-TPP1-OB-LacI were co-transfected into U2OS 2-6-3 cells grown on coverslips as described above . Cells were fixed for 5 min in PBS supplemented with 0 . 4% formaldehyde followed by permeabilization with PBS supplemented with 0 . 2% ( vol/vol ) Triton X-100 for 5 min . Finally , coverslips were embedded in DAPI containing mounting media ( Vector Laboratories , Burlingame , CA ) . Imaging was carried out visualizing the intrinsic fluorescence of the mCherry- and GFP-fusion proteins without additional staining . All images were acquired on a Deltavision Core deconvolution microscope ( Applied Precision , GE Healthcare ) using a 60× 1 . 42NA PlanApo N ( Olympus , Japan ) or 100× UPLanSApo 1 . 4NA ( Olympus ) objective and a sCMOS camera . 20 Z-sections with 0 . 2 µm spacing were acquired for each image with identical exposure conditions within each experiment . For analysis , maximum intensity projections were generated and scaled identically . For presentation in figures , representative images were deconvolved ( where indicated ) , followed by generation of maximum intensity projections of 10 Z-sections , which were scaled identically for all experimental conditions .
Human chromosomes terminate in telomeres , repetitive DNA sequences bound by the shelterin complex . Shelterin protects chromosome ends , prevents recognition by the DNA damage machinery , and recruits telomerase . A patch of amino acids , termed the TEL-patch , on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres . In contrast , the site on telomerase that interacts with the TPP1 OB-fold is not well defined . In this study , we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase ( hTERT ) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase . Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct . Our findings define the interaction interface required for telomerase recruitment to telomeres , an important step towards developing modulators of this interaction as therapeutics for human disease .
In the nucleus of a cell , the DNA that contains the cell's genetic information is packaged into long structures called chromosomes . Every time a cell divides , its chromosomes are duplicated . However , the proteins that are responsible for copying the DNA cannot reach the very end of the DNA strand , causing the chromosomes to progressively shorten . To ensure that this does not cause genetic information to be lost , each chromosome ends in a repetitive stretch of DNA called a telomere . Though the end of the telomere is lost whenever the DNA is copied , an enzyme called telomerase replaces the sequence that has been lost and counteracts the shortening of the telomeres . Shelterin is a protein complex that binds to telomeres to protect them and also helps telomerase to work correctly . Shelterin contains a specific site that attaches to telomerase , but exactly how the human versions of these two molecules bind to each other is unknown . A possible interaction site had been identified on the telomerase , which , when mutated , stops the telomerase working properly . However , as this region is also involved in lengthening the telomeres after the chromosomes have duplicated , it is not certain that these problems result from the telomerase failing to bind to shelterin . The enzyme telomerase is unusual; it has both RNA and protein components . Like all other proteins , the telomerase protein is made up of strings of amino acids . Schmidt et al . discovered that replacing two specific amino acids in human telomerase prevents its binding to shelterin . Cells that produced the modified form of the telomerase had chromosomes with shortened telomeres . However , if the cells also produced modified versions of the shelterin complex that were designed to bind to the modified telomerase , telomere length was normal . This indicates that the telomerase interacts directly with shelterin , rather than through a ‘bridging’ molecule . Mutations in the genes coding for both shelterin and the telomerase enzyme cause a number of human diseases , and cancers rely on the activity of telomerases to grow . Knowing how shelterin and telomerase interact could therefore help to design drugs that may either restore or disrupt the interaction and therefore can be used to treat these diseases .