vertebrate telomeres are essential nucleoprotein structures that  protect chromosome ends from promiscuous dna repair activities and nucleolytic degradation ( for review , see de lange ,  2005 ) .
telomeric dna is composed of repetitive double - stranded ttaggg sequences that extend into a 3 single - stranded overhang on the g - rich strand ( moyzis et  al . , 1988 ; wellinger et  al . ,
telomeric repeats can be extended by telomerase , a reverse transcriptase that utilizes an associated rna moiety ( terc ) as a template to add de novo telomeric sequences to the 3 end of the g - rich strand of the telomere ( greider and blackburn , 1985 ; shippen - lentz and blackburn , 1990 ) .
telomere function is also critically dependent on a complex formed by six telomere - associated proteins , known as shelterin , which comprises trf1 ( telomere repeat binding factor 1 ) , trf2  ( telomere repeat binding factor 2 ) , pot1 ( protection of telomeres  1 ) , tin2 ( trf1-interacting nuclear factor 2 ) , rap1 ( repressor activator protein 1 ) , and tpp1 ( tinf2-interacting protein 2 ) that function to safeguard chromosome ends from the dna damage response ( ddr ) and to control telomere maintenance by telomerase ( for review , see palm and de lange , 2008 ) .
shelterin associates with telomeres by virtue of intrinsic duplex telomeric repeat binding conferred by trf1 and trf2 and binding of the single - stranded telomeric 3 overhang by  pot1 ( baumann and cech , 2001 ) . while trf1 promotes telomere replication and functions as a negative regulator of telomere length ( sfeir et  al . , 2009 )
, trf2 plays a crucial role in telomere end protection ( van steensel et  al . , 1998 ) .
consequently , loss of trf1 compromises telomere replication , giving rise to telomere fragility , whereas loss of trf2 results in loss of the 3 overhang and extensive chromosome end - to - end fusions ( celli and de lange , 2005 ; sfeir et  al . ,
electron microscopy and stochastic optical reconstruction microscopy have revealed that telomeres can adopt lasso - like configurations called t - loops ( doksani et  al . , 2013 ;
griffith et  al . , 1999 ) , which are believed to form as a result of strand invasion of the 3 single - stranded telomeric dna into upstream duplex ttaggg repeats ( griffith et  al . , 1999
as the 3 end of the telomere is buried within the t - loop , this structure has been proposed to safeguard the chromosome ends against nucleolytic attack or promiscuous dna repair activities ( for review , see osullivan and karlseder , 2010 ) .
the amino - terminal basic domain of the shelterin component trf2 is important for stabilizing t - loops and acts to protect the structure from unscheduled nucleolytic processing ( wang et  al . , 2004 ) .
trf2 is also essential for t - loop formation in  vivo ( doksani et  al . , 2013 ) , which likely contributes to its key role in end protection .
mechanisms must also exist to disassemble t - loops to allow telomerase access to the 3 end and/or to avoid collisions with the replisome during s phase .
rtel1 ( regulator of telomere length 1 ) ( ding et  al . , 2004 ) is an essential helicase with intrinsic d - loop - disrupting activity that is co - opted to telomeres to dismantle t - loops ( uringa et  al . , 2012 ) .
in the absence of rtel1 , t - loops are inappropriately resolved by the slx1-slx4 nuclease complex , leading to catastrophic telomere length changes and loss of the telomere as double - stranded telomere circles ( t - circles or tcs ) ( vannier et  al . , 2012 ) .
rtel1 also executes a second function to counteract the formation of telomeric guanine quadruplex ( g4 ) dna structures during telomere replication , which are a major source of telomere fragility ( vannier et  al . ,
direct binding of rtel1 to proliferating cell nuclear antigen ( pcna ) is critical for preventing telomere fragility but is dispensable for t - loop disassembly ( vannier et  al . , 2013 ) .
the importance of rtel1 in human disease has been established with the identification of variants that confer increased risk of human brain tumors ( shete et  al . , 2009 ; wrensch et  al . ,
mutations in rtel1 also give rise to hoyeraal - hreidarsson syndrome ( hhs ) , a severe form of the telomereopathy dyskeratosis congenita ( dkc ; for review , see vannier et  al . , 2014 ) .
hhs is characterized by its similarity to dkc , but patients also present with additional complications including bone marrow failure , microcephaly , and immunodeficiency ( vulliamy et  al . , 2006 ) .
so far , 18 distinct rtel1 mutations have been identified in hhs ( for review , see vannier et  al .
, 2014 ) , but how these mutations impact on the known functions of rtel1 is not known . here
, we define the mechanism by which rtel1 is recruited to telomeres to promote t - loop disassembly , which we show is dependent on an s phase - specific interaction with the shelterin component trf2 .
we demonstrate that the trf2 interaction site in rtel1 maps to an uncharacterized c4c4 metal - binding motif , which is commonly mutated in hhs patients .
we establish that the p.r1264h mutation within the c4c4 motif , which has a carrier frequency of 1 in 100 in the ashkenazi jewish population ( fedick et  al . , 2014 ) , compromises the rtel1-trf2 interaction , leading to catastrophic t - loop resolution accompanied by rapid  changes in telomere length , telomere loss , and tc formation .
conversely , we identify a single amino acid substitution ( p.i124d ) within the trfh dimerization domain of trf2 that specifically abolishes rtel1 binding and prevents its recruitment to telomeres . remarkably , the trf2 p.i124d mutation gives rise to rapid changes in telomere length , telomere loss , and tc formation and thereby phenocopies the p.r1264h mutation in rtel1 .
our results reveal that t - loops are highly dynamic and regulated structures whose assembly and disassembly is coordinated during the cell cycle .
rtel1 serves a dual function in telomere integrity ; it suppresses telomere fragility by unwinding telomeric g4 dna and also disassembles t - loops to prevent catastrophic loss of the telomere ( vannier et  al . , 2012 ) .
recently , we have shown that rtel1 s ability to suppress telomere fragility strictly depends on an interaction with pcna .
unexpectedly , however , the rtel1-pcna interaction was found to be dispensable for t - loop unwinding ( vannier et  al . , 2013 ) , suggesting the existence of a separate mechanism for recruiting rtel1 to telomeres .
we reasoned that rtel1 could be recruited to telomeres via an interaction with one of the shelterin components , which are the major constituents of vertebrate telomeres . to test this hypothesis
, we first modified a human bacterial artificial chromosome ( bac ) carrying the rtel1 genomic locus by inserting a flap tag ( containing gfp and flag ) at the n terminus ( nflap ) of the rtel1 coding sequence using homologous recombination ( poser et  al . , 2008 ) .
extracts from hek293 cells stably expressing nflap - tagged rtel1 at near endogenous levels were subjected to immunoprecipitation for 5 of the 6 endogenous shelterin components and then blotted for rtel1 . although known partner shelterin components were present in  immunoprecipitates of endogenous trf1 , tpp1 , or pot1 , nflap - tagged rtel1 was not detectable ( figure  s1a ) . however , blotting of immunoprecipitates of endogenous trf2 , and to a lesser extent rap1 , revealed an association with nflap - tagged rtel1 ( figure  1a ) .
importantly , these interactions were resistant to benzonase treatment , indicating that the interactions between trf2/rap1 and rtel1 are independent of dna / rna bridging .
furthermore , trf2 immunoprecipitated rtel1 from lysates that were mock depleted with igg but not from extracts immunodepleted for nflap - tagged rtel1 with a gfp antibody ( figure  1b ) , thus demonstrating the specificity of the interaction .
trf2 was coprecipitated from hek293 cells with nflap - tagged rtel1 , but not with irrelevant control protein gfp - alc1 ( figure  1c ) .
silencing of rap1 with lentivirus - mediated rna interference ( shrna ) did not significantly affect binding of rtel1 to trf2 ( data not shown ) , which raised the possibility that rtel1 interacts indirectly with rap1 and directly with trf2 . to confirm direct binding of rtel1 to trf2 in  vitro , we performed gst pull - down assays with purified full - length and truncated versions of human trf2 and affinity purified nflap - rtel1 .
western blotting analysis revealed that nflap - rtel1 associates with full - length trf2 protein ( gst - trf2 ) and trf2 lacking the n - terminal  basic domain ( gst - trf2b ) , but not with the basic n - terminal domain ( gst-b ) or gst alone ( figure  1d ) . taken together , these data indicate that rtel1 directly interacts with trf2 . since rtel1 only transiently associates with telomeres ( uringa et  al . , 2012 ) , we next examined whether the association of rtel1 with trf2 varied throughout the cell cycle . to this end
we synchronized hek293 nflap - rtel1 cells and rtel1v5 mouse embryonic fibroblasts ( mefs ) , stably expressing myc - tagged trf2 , by double thymidine block or by incubation with thymidine followed by nocodazole treatment ( figures s1b and s1c ) .
although a low level of association between rtel1 and trf2 was detected throughout the cell cycle , the rtel1-trf2 interaction is significantly increased at the g1-to - s transition and peaked in s phase ( figures 1e and s1d ) .
similarly , we observed that the rtel1-trf2 interaction is significantly enhanced in s phase rtel1v5 mouse cells as determined by proximity ligation assay pla ( figures 1f and s1e ) and is unaffected by inhibiting dna replication ( figure  s2 ) .
these results establish that the rtel1-trf2 interaction occurs predominantly during s phase and does not require active dna replication .
to determine the minimal trf2 interaction site within rtel1 , we conducted a peptide array binding experiment , as previously described ( ward et  al . , 2010 ) .
we reasoned that the trf2 interaction site was unlikely to map to the n - terminal helicase motif and so focused our attention on the c terminus of rtel1 , which contains three recognizable motifs : a pip box ( pcna binding ) , a harmonin n - like domain ( faure et  al . , 2014 ) , and a c4c4 motif ( unknown function ) .
binding of gst - trf2 to a 20-mer peptide - scanning array encompassing the c - terminal region of rtel1 identified a single trf2-binding peptide that overlapped with the uncharacterized c4c4 motif ( figure  2a ) . to confirm that the c4c4 motif of rtel1 is sufficient to confer binding to trf2 and to determine if this interaction requires metal coordination , we generated recombinant gst and gst - c4c4 motif fusions and performed pull - downs of endogenous trf2 from hek293 cell extracts in the absence ( edta ) or presence ( + edta ) of edta . as shown in figure  2b ( lane 2 ) , the lower isoform of endogenous trf2 is specifically pulled down from whole - cell extract by gst - c4c4 , but not with gst alone ( figure  2b ; lane 1 ) .
addition of edta to the pull - down , which is predicted to abolish metal coordination of the c4c4 motif , resulted in the complete loss of trf2 binding ( figure  2b ; lane 4 ) .
these results reveal that the c4c4 motif of rtel1 is sufficient for binding to trf2 , and this requires metal coordination .
to assess the biological importance of the c4c4 domain for rtel1 activities , we introduced c4c4 mutations by site - directed mutagenesis into the human and mouse versions of rtel1 ( see  map in figure  2c ) .
the rtel1 ( human)/rtel1 ( mouse ) mutation corresponds to the hhs disease - causing mutation ( ballew et  al . , 2013 ) and is located between the second and third metal - coordinating pair of cysteines ( kellenberger et  al . , 2005 ) .
we also generated two cysteine - to - alanine substitutions ( rtel1 in human or rtel1 in mouse ) that are predicted to disrupt the fourth pair of cysteines coordinating a second metal ion ( kellenberger et  al .
first , we evaluated whether the mutations within the c4c4 motif affect the rtel1-trf2 interaction . to this end
u2os cells stably expressing empty vector , wild - type ( wt ) myc - c4c4 ( an  equivalent to the human rtel112341292 region ) , or two c4c4 mutants , myc - c4c4 and myc - c4c4 , were analyzed by immunoblotting with anti - myc antibody .
the decreased steady - state abundance of the mutated human c4c4 motif suggests that the mutations might impact on its stability ( figure  s3a ) . to account for the reduced expression , we compensated myc - c4c4 protein levels of both mutants in immunoprecipitation experiments .
western blotting analysis revealed that endogenous trf2 was efficiently immunoprecipitated with wt myc - c4c4 peptide but not with either mutant ( figure  s3b ) .
we also introduced the c4c4 motif mutations into the full - length human rtel1 and found that endogenous trf2 interacted exclusively with wt flag - rtel1 , whereas mutations within the c4c4 motif abolished rtel1 s ability to bind trf2 ( figure  2d ) .
these results were also confirmed by an in  vitro gst pull - down experiment , in which we observed robust interaction of gst - trf2 with full - length flag - tagged wt rtel1 , but not with rtel1 or double - mutant rtel1 ( figure  2e ) .
finally , we examined the interaction of trf2 with rtel1 in normal bj human fibroblasts and in patient - derived msk-41 cells harboring inherited homozygous rtel1 hhs disease - causing mutations ( ballew et  al . , 2013 ) .
as shown in figure  2f , the endogenous rtel1-trf2 interaction is undetectable in msk-41 cells ( lane 1 ) but is readily detectable in normal bj human fibroblasts ( figure  2f , lane 2 ) .
an interaction between endogenous trf2 and rap1 , which served as a positive control , was observed by pla assay in bj fibroblasts , retinal - pigmented epithelial rpe1 cells , and in the patient - derived msk-41 fibroblasts ( figure  2 g ) .
in contrast , the rtel1-trf2 interaction was only detectable by pla assay in bj fibroblasts and rpe1 cells but not in the msk-41 fibroblasts ( figure  2h ) .
these results establish that the interaction of rtel1 and trf2 is compromised by the rtel1 p.r1264h mutation in hhs . to investigate the impact of c4c4 motif mutations on the telomere functions of rtel1 , we complemented conditional rtel1 mefs with retroviruses expressing either empty vector , full - length wt v5-rtel1 , single - mutant v5-rtel1 , or double - mutant v5-rtel1 .
transient expression of cre recombinase in the transduced rtel1 mefs resulted in the expected loss of the floxed rtel1 alleles and concomitant elimination of the endogenous rtel1 protein within 96  hr , leaving the v5-rtel1 wt or mutants as the only source of rtel1 protein ( figures 3a and 3b ) .
conditional deletion of rtel1 in mefs leads to rapid accumulation of tcs as a consequence of processing of persistent t - loops by the slx1/4 nuclease ( vannier et  al . ,
cells expressing empty vector and mefs complemented either with v5-rtel1 or v5-rtel1 exhibited very high levels of tcs after eliminating the rtel1 floxed alleles .
in contrast , rtel1-deficient cells expressing wt v5-rtel1 exhibited low levels of tcs , comparable to control virus - infected cells ( figure  3c ) .
next , we examined the levels of telomere fragility and telomere loss in complemented rtel1 mefs by fluorescent in  situ hybridization ( fish ) using telomere - specific probes .
as expected , inactivation of rtel1 in rtel1 mefs , 96  hr after cre infection , resulted in high levels of fragile telomeres , which manifest as  multiple spatially distinct telomere fish signals ( figure  3d ) . 
however , inactivation of rtel1 in rtel1 mefs complemented with either wt v5-rtel1 , v5-rtel1 , or v5-rtel1 did not result in significant induction of  telomere fragility when compared to cells complemented with an empty vector control ( figure  3d ) .
strikingly , inactivation of  rtel1 in rtel1 mefs expressing v5-rtel1 or v5-rtel1 resulted in a significant increase in telomere loss , corresponding to 6.2   2.8 and 5.8   3.3 telomeres that lacked a telomere fish signal on one or both sister chromatid ends , which is comparable to the levels observed in rtel1 null cells ( 7.5   3.2 ) and significantly different from wt v5-rtel1-complemented cells that present 2.7   1.4 ends lacking detectable telomeric signals in wt v5-rtel1-complemented cells ( figure  3e ) .
these results establish that a functional c4c4 motif in rtel1 is essential for suppressing tcs and telomere loss , but is dispensable for preventing telomere fragility .
we next evaluated how loss of the rtel1-trf2 interaction might affect the ddr at telomeres . to this end
we quantified the number of dysfunctional telomeres by scoring for 53bp1  colocalization with telomeres .
conditional deletion of rtel1  in  rtel1 mefs expressing v5-rtel1 or v5-rtel1 resulted in enhanced telomere - dysfunction - induced foci ( tifs ) , corresponding to 7.2%   0.5% and 8.3%   1.3% foci per nucleus compared to 2.5%   1.6% tifs per nucleus in the cells expressing wt v5-rtel1 ( figures 3f and s4 ) .
thus , disrupting the trf2 binding site on rtel1 causes a mild ddr that resembles the phenotype of rtel1 deletion . to determine whether loss of the rtel1-trf2 interaction induces telomere recombination , we employed chromosome orientation ( co)-fish to monitor the frequency of telomere sister chromatid exchange ( t - sces ) .
inactivation of rtel1 in rtel1 mefs expressing v5-rtel1 or v5-rtel1 resulted in a 3-fold increase in the number of t - sces when compared to wt v5-rtel1-complemented cells ( figure  3 g ) .
collectively , these results lead us to propose that recruitment of rtel1 to telomeres by trf2 is important for t - loop disassembly and prevents catastrophic loss of the telomere , but is dispensable for efficient telomere replication .
given that a functional c4c4 motif and rtel1-trf2 interaction is required for t - loop disassembly but is dispensable for preventing telomere fragility , we wished to determine the impact of c4c4 motif mutations on genome - wide replication dynamics using the molecular combing method ( michalet et  al . , 1997 ) .
as previously described ( vannier et  al . , 2013 ) , the progression of  sister replication forks , 96  hr after inactivation of rtel1 , in  rtel1 mefs is significantly reduced relative to wild - type cells and is associated with increased fork asymmetry and elevated origin usage .
in contrast , rtel1 mefs complemented with v5-rtel1 or v5-rtel1 exhibited comparable replication fork dynamics to that of v5-rtel1 wt cells ( figures 4a and 4b ) .
these results predict that mutations in the c4c4 motif should not affect the rtel1-pcna interaction , which we first examined using in  situ pla . as expected , loss of rtel1 abolished the  rtel1-pcna interaction detectable by pla in rtel1 mefs complemented with empty vector ( figures 4c and 4d ) . in  contrast , rtel1-depleted cells complemented with v5-rtel1 and v5-rtel1 c4c4 mutations retained the rtel1-pcna interaction , which corresponded to 13.4   2.9 and 11.6   2.0 pla foci per nucleus when compared to 11.5   2.1 foci per nucleus in rtel1 mefs expressing wt v5-rtel1 ( figures 4c and 4d ) .
similar results were obtained in coimmunoprecipitation experiments from the same cells ( figure  4e ) .
we suggest that the c4c4 motif is dispensable for the rtel1-pcna interaction and does not contribute to the global and telomere replication functions of rtel1 in cells . to identify the region of trf2 that interacts with rtel1 , we synthesized peptide arrays of overlapping 20-mer peptides ( offset by a single amino acid ) that comprised 150 amino acids of trf2 , equivalent to human trf243183 .
we found that gst - tagged full - length human rtel1 but not gst alone interacted with a peptide corresponding to human trf2106125 ( figure  s5a ) , which is located within the trfh dimerization domain at the helix1-helix2 boundary ( figure  5a ) . to confirm this result , we purified untagged human trf2trfh , a 6xhis - sumo - c4c4 motif ( human rtel112341292 ) , and 6xhis - sumo control .
6xhis - sumo - c4c4 but not control bound to trf2trfh at both low and high salt concentrations as confirmed by coomassie staining of the eluted material and resolution on sds - page gels ( figure  5b ) .
consistent with our previous result that the binding of trf2 to the c4c4 motif is dependent on metal coordination ( figure  2b ) , we found that the interaction of trf2trfh with 6xhis - sumo - c4c4 is greatly reduced by the addition of edta ( figure  s5b ) . to identify key residues in trf2 that are necessary for the trf2-rtel1 interaction , we generated a substitution peptide array in which the human trf2106125 peptide was systematically substituted ( one amino acid substitution at a time ) with each of the 20 natural amino acids . probing this array with 6xhis - sumo - c4c4 revealed that several amino acid substitutions at isoleucine 121 severely reduced the interaction ( figure  5c ) .
in contrast , a purified 6xhis - sumo control peptide did not bind to any spot within the trf2106125 substitution peptide array , confirming specificity of the interaction ( data not shown ) . to test
if the trf2 p.i121 mutation compromises the rtel1 interaction in  vivo , we introduced the corresponding mutation ( p.i124d ) into full - length mouse trf2 , which was used to stably transduce hek293 flap - tagged rtel1 cells and rtelv5 mefs with retroviruses along with controls ( figure  s6 ) .
strikingly , we found that the p.i124d mutation completely abolished binding of trf2 to flag - rtel1 or v5-rtel1 ( figure  5d ) .
thus , isoleucine 124 within the trf2trfh domain is critical for the trf2-rtel1 interaction in  vivo . to examine how the p.i124d mutation affects trf2 activities at  telomeres , we subjected whole - cell extracts of hek293 flap - tagged rtel1 cells stably expressing an empty vector , wt myc - trf2 , or myc - trf2 to immunoprecipitation with anti - myc or control igg .
while the trf2 mutation specifically abolished the trf2-rtel1 interaction ( figure  5d ) , it did not measurably affect its ability to interact with the shelterin proteins trf1 and rap1 ( figure  5e ) . since trf2 is not in close proximity to the tin2 binding site , which is separate from the trfh domain , and maps to residues 352365 ( chen et  al .
, 2008 , 2011 ) , it is unlikely that this mutation affects interaction with tin2 .
next , we examined whether the trf2 mutant affects telomere binding of itself or other shelterin components by chromatin immunoprecipitation ( chip ) . complemented trf2 cells were crosslinked with formaldehyde and dna slot blots were hybridized with a ttaggg probe to determine relative levels of protein - associated telomeric dna ( figure  5f ) . as expected , cre - mediated deletion of trf2 in cells expressing empty vector resulted in a severe reduction in telomeric binding of trf2 accompanied by a 5-fold reduction of trf1 and rap1 associated with telomeres ( figure  5 g ) .
importantly , trf2 cells complemented with myc - trf2 or myc - trf2 retained wt or mutant trf2 as well as endogenous shelterin proteins at telomeres ( figure  5 g ) .
these results suggest that the trf2 mutant does not compromise the ability of trf2 to associate with telomeres and does not measurably impact on telomeric binding by trf1 and rap1 . to gain further insight into the impact of the trf2 mutant at telomeres , we conducted tc amplification assays and telomere fish experiments to examine telomere stability and telomere length changes in these cells .
we first examined trf2 mefs complemented with empty vector , myc - trf2 , or myc - trf2 for the presence of tcs .
strikingly , inactivation of trf2 in trf2 mefs complemented with myc - trf2 resulted in a 3-fold induction of tcs when compared to cells complemented with wt myc - trf2 ( figure  6c ) .
intriguingly , this result is comparable to the tc ratio observed between wt and c4c4 motif mutants expressed in rtel1 mefs ( figure  3c ) . as previously described ( celli and de lange , 2005 ) , cre - mediated deletion of trf2 in trf2 mefs complemented with empty vector results in chromosome end - to - end fusions with telomeric dna signals evident at the junction of each fusion ( figure  6d ) .
in contrast , trf2 mefs complemented with either wt myc - trf2 or myc - trf2 did not present with telomere fusions ( figure  6d ) . since loss of tin2 gives rise to telomere fusions ( takai et  al . , 2011 )
, these data further suggest that the trf2 mutation does not impact on trf2-tin2 binding .
while we found no evidence that the trf2 mutation is associated with telomere fusions or fragility ( figures 6d and 6e ) , we did observe a high incidence of telomere loss following inactivation of trf2 in trf2 mefs complemented with myc - trf2 .
telomere fish analysis revealed that disruption of the rtel1-trf2 interaction , associated with the trf2 mutation , resulted in the loss of a telomere signal on 13   7.7 chromosome ends per metaphase .
this contrasted with the loss of 5.1   2.8 telomeres per metaphase in cells expressing wt myc - trf2 ( figure  6f ) . to address whether the trf2 mutation gives rise to telomere length heterogeneity , we conducted quantitative fluorescence in  situ hybridization ( q - fish ) to measure the intensity of the telomere signal from images of fish - stained metaphases .
analysis of trf2 mefs expressing myc - trf2 , performed 96  hr after cre introduction , revealed increased telomere length heterogeneity when compared to cells with wt myc - trf2 ( figure  6 g ) .
interestingly , these changes in the mean telomere length , which included a statistically significant increase in both short and long telomeres , resembled rtel1 deficiency , and more specifically the defect associated with mutations in the c4c4 motif ( vannier et  al . , 2012 ) . to examine the impact of the trf2 mutation on the ddr , we quantified the number of 53bp1 foci that colocalize with telomeric dna .
tif analysis revealed that conditional deletion of trf2 in trf2 mefs expressing empty vector control resulted in 71% of telomeres with tifs . in contrast , deletion of trf2 in trf2 mefs complemented with either wt myc - trf2 or myc - trf2 mutant diminished the percentage of tif foci at telomeres by 10.5-fold and 6.1-fold , respectively ( figures 6h and s7 ) .
these results suggest that loss of the trf2-rtel1 interaction , which compromises the recruitment of rtel1 to telomeres , elicits a mild telomere - associated ddr , which is comparable in severity to the ddr observed at telomeres in rtel1-deficient cells or associated with the rtel1 mutation . finally , we performed co - fish analysis to examine the frequency of recombination at telomeres induced by the loss of the trf2-rtel1 interaction .
inactivation of trf2 in trf2 mefs complemented with myc - trf2 resulted in a greater than 5-fold induction in the frequency of t - sces with preferential addition of lagging telomere dna when compared with cells expressing wt myc - trf2 ( figure  6i ) .
these results indicate that the recruitment of rtel1 to telomeres via its interaction with trf2 protects against telomeric recombination .
overall , these results establish that trf2 recruits rtel1 to telomeres to facilitate t - loop disassembly .
telomeres are dependent on two distinct activities of rtel1 to maintain the integrity of chromosome ends : rtel1 disassembles t - loops and also counteracts the formation of g4-dna secondary structures to prevent telomere loss and telomere fragility , respectively ( vannier et  al . , 2012 ) .
our previous study established that rtel1 interacts with the replisome through a pip - box - dependent interaction with pcna , which is important for the replication functions of rtel1 ( vannier et  al . , 2013 ) .
surprisingly , however , the rtel1-pcna interaction is dispensable for t - loop disassembly , which suggested the existence of a distinct mechanism for recruiting rtel1 to telomeres to promote t - loop unwinding . here
, we establish that rtel1 is transiently recruited to telomeres to promote t - loop unwinding through an s phase - specific interaction with the shelterin component trf2 ( see figure  7 for a model ) . failure to disassemble t - loops
has severe consequences for telomere integrity , which may reflect the need to avoid collisions between t - loops and the replication fork , allow telomerase access to the 3 end of the telomere , and/or counteract the formation of t - loops in trans between sister telomeres .
rtel1 s ability to interact directly with trf2 is mediated by a cysteine - rich c4c4 motif , which is predicted to bind two zinc ions to form a cross - braced structure ( kellenberger et  al .
the finding that addition of edta is sufficient to abolish the trf2-rtel1 interaction underscores the importance of c4c4 metal coordination for this association . of direct clinical relevance is the recent discovery of mutations within the c4c4 motif that are causal for the telomeropathy hhs , including rtel1 and rtel1 mutations ( ballew et  al .
, 2013 ; le guen et  al . , 2013 ) . strikingly , the carrier frequency of the rtel1 mutation within the ashkenazi orthodox jewish and general ashkenazi jewish populations is 1% and 0.45% , respectively , which has prompted the need for urgent screening to identify heterozygous carriers ( ballew et  al . , 2013 ; fedick et  al . , 2014
we demonstrate here that the rtel1-trf2 interaction is compromised by the p.r1264h mutation in hhs patient - derived cells and in rtel1 mutant mefs .
in contrast to the rtel1 pip - box mutation , which eliminates binding to pcna and specifically affects the replication functions of rtel1 , the c4c4 mutations have no measurable impact on normal replication dynamics , telomere fragility , or the interaction with pcna .
instead , the c4c4 mutation conferred rapid accumulation of tcs concomitant with telomere length changes and telomere loss , which we attribute to the inappropriate resolution of the t - loop by the slx1/4 nuclease complex ( figure  7 and vannier et  al . , 2012 ) .
thus , we propose that the pip - box and c4c4 motif convey distinct and genetically separable functions , which impact on the targeting of rtel1 to replication forks and telomeres , respectively .
our analysis of the rtel1 binding sites in trf2 identified a  single point mutation , trf2 , located in helix2 of the trf2trfh dimerization domain ( fairall et  al . , 2001 ) , which abolishes the rtel1-trf2 interaction .
importantly , trf2 is not in close proximity to the binding sites for tin2 or rap1 ( chen et  al .
, 2008 , 2011 ) and does not affect its interaction with trf1 or rap1 , nor its ability , or the ability of other shelterin components , to bind to telomeres . furthermore , unlike loss of trf2 , which gives rise to telomere de - protection , telomere end - to - end fusions , and a massive induction of the ddr at telomeres , the trf2 mutation gives rise to a phenotype that is remarkably similar to that observed for the rtel1 mutation . both trf2 and rtel1 mutant cells present with high levels of tcs , telomere length heterogeneity , telomere loss , increased telomere sces , and a moderate induction of the ddr at telomeres , which we have previously attributed to the inappropriate resolution of persistent t - loops by the slx1/4 nuclease complex ( vannier et  al . , 2012 ) .
thus , we propose that the phenotypes of the trf2 and rtel1 mutations reflect a failure to recruit rtel1 to telomeres during s phase , which is necessary to transiently disassemble t - loops .
given the phenotypic similarity of trf2 and rtel1 mutations , our results also raise the possibility that trf2 coordinates both the assembly and disassembly of t - loops during the cell cycle .
previous studies have established that the n - terminal basic domain of trf2 binds to and protects telomeric holliday junctions ( hj)/d - loop structures from cleavage by endonucleases , which may play a role in the formation and protection of t - loops ( poulet et  al . , 2009 ; wang et  al . , 2004 ) .
the n - terminal basic domain of trf2 could also be used to target rtel1 to the telomere hj / d - loop during s phase , which must be unwound to release the 3 telomere end to disassemble the t - loop .
it is intriguing to note that the rtel1 interaction site within the trfh domain of trf2 is adjacent to the n - terminal basic domain of trf2 that confers telomere hj / d - loop binding ( fouch et  al .
given their close proximity , it will be important to determine if the binding of rtel1 to trf2 induces an allosteric change in the adjacent basic domain , which could alter hj / d - loop association to facilitate unwinding by rtel1 to dismantle the t - loop . in future studies
it will be important to further define how the rtel1-trf2 interaction is controlled during the cell cycle and how this is limited to s phase cells .
while active replication is not required for the rtel1-trf2 interaction , it is intriguing to note that trf2 is a likely cdk substrate and could be subject to cell cycle - dependent modification .
indeed , trf2 contains numerous potential cdk phosphorylation sites and can be phosphorylated by a cyclin a - cdk2 complex in  vitro ( chi et  al . ,
however , the importance of these putative cdk sites in trf2 and whether or not they contribute to the rtel1-trf2 interaction remains unclear .
in addition , trf2 is detected as two closely migrating bands ( bilaud et  al . , 1997 ) .
how and why rtel1 preferentially binds to the lower isoform is unclear , but may reflect differences in post - translational modifications ( ptms ) that could be determinants of the interaction .
finally , it is also important to consider that rtel1 may be subject to ptms during the cell cycle , but this remains ill defined and warrants future investigation .
an improved understanding of the cell cycle control of rtel1 and trf2 is likely to provide important insights into the assembly and disassembly of t - loops and its contribution to the end protection problem .
trf2 ( celli and de lange , 2005 ; a gift from titia de lange , the rockefeller university ) , rtel1v5 , and rtel1v5 ( vannier et  al . , 2013 )
mefs were cultured in  dulbecco s modified eagle s medium ( dmem ) supplemented with 15% fetal bovine serum ( invitrogen ) , l - glutamine , and penicillin - streptomycin .
bj - htert , rpe-1 , patient - derived msk-41 fibroblasts , and u2os osteosarcoma cells were kept in dmem with 10% fbs .
production of retro- and lentiviral supernatants was performed as described earlier ( sarek et  al . , 2010 ) .
rtel mefs were transduced with pbabe - puromycin retroviruses expressing an empty vector , mouse v5-tagged wt , or mutant rtel1 .
trf2 mefs were infected with plpc - puromycin retroviruses expressing control vector , mouse wt , or mutant myc - tagged trf2 .
the human u2os cells were transduced with plpc - puromycin retroviruses carrying myc - tagged wt or mutant alleles of the c4c4 domain .
hek293 cells were infected with phage - pgk - puromycin lentiviral constructs to express wt or mutants of human flag - tagged rtel1 .
transduced cells were selected with puromycin ( 3  g / ml ) for 26  days .
deletion of floxed alleles in rtel and trf2 mefs was carried out with ad - gfp - cre adenovirus ( vector biolabs ) , and cells were genotyped by pcr at 96  hr post - infection as described earlier ( celli and de lange , 2005 ; vannier et  al . , 2012 ) .
cells were plated on coverslips at density 5   10 in 24-well plates and left in culture conditions overnight .
the next day cells were pre - extracted in csk buffer ( 10  mm pipes [ ph 6.8 ] , 100  mm nacl , 300  mm sucrose , 3  mm magnesium chloride , 1  mm egta , and 0.5% triton x-100 ) fixed with 5% formaldehyde ( thermo scientific ) for 10  min , permeabilized with pbs containing 0.5% ( v / v ) np-40 for 5  min , and blocked for 30  min with goat serum ( 5% ) in  pbs .
pla was performed following the manufacturer s instructions using the duolink anti - mouse minus and anti - rabbit plus in situ pla probes and the duolink in situ detection reagents red ( olink bioscience ) .
images were acquired with a zeiss axio imager m1 microscope equipped with an orca - er camera ( hamamatsu ) and using the volocity 4.3.2 software ( improvision ) .
 summarythe helicase rtel1 promotes t - loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres . 
 an interaction between rtel1 and pcna is important to prevent telomere fragility , but how rtel1 engages with the telomere to promote t - loop unwinding is unclear . here 
 , we establish that the shelterin protein trf2 recruits rtel1 to telomeres in s phase , which is required to  prevent catastrophic t - loop processing by structure - specific nucleases . we show that the trf2-rtel1 interaction is mediated by a metal - coordinating c4c4 motif in rtel1 , which is compromised by the hoyeraal - hreidarsson syndrome ( hhs ) mutation , rtel1r1264h . 
 conversely , we define a trf2i124d substitution mutation within the trfh domain of trf2 , which eliminates rtel1 binding and phenocopies the rtel1r1264h mutation , giving rise to aberrant t - loop excision , telomere length heterogeneity , and loss of the telomere as a circle . 
 these results implicate trf2 in the recruitment of rtel1 to facilitate t - loop disassembly at telomeres in s phase . 
Introduction
Results
Discussion
Experimental Procedures