Report Flap Endonuclease 1 Contributes to Telomere Stability

Department of Biomedical EngineeringBoston University School of MedicineBoston UniversityBoston, Massachusetts 02118SummaryTelomerestabilityplaysanimportantroleinthepreservationof genomic stability and is maintained through the coordi-nated actions of telomere-specific proteins and DNA repairand replication proteins [1, 2]. Flap endonuclease 1 (FEN1)is a protein that plays a role in lagging-strand DNA replica-tion, base excision repair, homologous recombination, andreinitiationofstalledreplicationforks[3,4].Here,wedemon-strate that FEN1 depletion leads to telomere dysfunctioncharacterized by the presence of gH2AX and sister telomereloss. Expression of catalytically active telomerase, the re-verse transcriptase that adds telomeric repeats to chromo-some ends, was sufficient to rescue telomere dysfunctionuponFEN1depletion.Strikingly,FEN1depletionexclusivelyabrogatestelomeresreplicatedbylagging-strandDNArepli-cation. Genetic rescue experiments utilizing FEN1 mutantproteins that retained the ability to localize to telomeric re-peats revealed that FEN1’s nuclease activity and ability tointeractwiththeWernerprotein(WRN)andtelomere-bindingprotein (TRF2) were required for FEN1 activity at the telo-mere. Given FEN1’s role in lagging-strand DNA replicationand reinitiation of stalled replication forks, we propose thatFEN1 contributes to telomere stability by ensuring efficienttelomere replication.Results and DiscussionHigh-fidelityreplicationoftelomeresiscriticaltomaintaintelo-mere stability and is confounded by both the end replicationproblem and repetitive G-rich nature of telomeric DNA [5]. Re-petitive DNA sequences such as those found in the telomerepresent a challenging template for the replication machinerydue to a propensity to form secondary structures that canlead to stalled replication forks [6, 7]. Due to the importanceand difficulty of high-fidelity replication through the telomere,recent studies have focused on the roleDNA replication/repairproteins play in telomere stability [8–11]. Rad27, the FEN1homolog, is one such replication and repair protein that playsarole atSaccharomycescerevisiaetelomeres[8,12].Here, wedemonstrate that FEN1 plays a critical role in mammalian telo-mere stability.PreviousworkdemonstratedthatFEN1localizedtothetelo-mereinacell-cycle-dependentmanner[13].Weconfirmedthisobservation by chromatin immunoprecipitation (ChIP) fromcells (1) synchronized with thymidine and aphidicolin (Fig-ure S1 available online) and (2) enriched in different phasesof the cell cycle by centrifugal elutriation (Figure S2). In agree-ment with previous work, we found that FEN1 localized to thetelomere in the S and G2/M phases of the cell cycle. PurifiedFEN1 has been shown to interact directly with TRF2 throughboth the basic and myb domains of TRF2 [14]. Utilizingantibodies specific for endogenous FEN1 and TRF2, wedemonstrate that these proteins interact in vivo (Figure S3).FEN1’s presence at the telomere and its interaction withTRF2 raised the intriguing possibility that it played a role intelomerebiology.Toaddressthisdirectly,lentiviral-expressedRNA interference (RNAi) hairpins targeting FEN1 (shFEN) or ascrambled hairpin (negative control, shSCR) were introducedinto BJ fibroblasts (Figure 1A). Upon transduction, FEN1 pro-tein expression was virtually undetectable compared to con-trol cells (Figure 1B). To determine whether FEN1 depletion re-sulted in telomere dysfunction, we analyzed telomeres for thepresence of gH2AX (an indicator of DNA damage) by ChIP.Lysates from cells expressing shSCR or shFEN were subjectto immunoprecipitation using an antibody to gH2AX, followedby quantitation of isolated telomeric and genomic DNA (ALU).We found that, upon FEN1 depletion, immunoprecipitationof gH2AX resulted in a significant increase in the amount ofisolated telomeric DNA compared to control cells (1.39-foldgreater than control; p < 0.05; Figures 1C and 1D). In contrast,no significant increase was observed in gH2AX associatedwith ALU DNA (1.09-fold; p = 0.59), indicating that there isincreased DNA damage upon FEN1 depletion at telomericsequences compared to the genome at large. A similar in-crease in gH2AX-associated telomeric and genomic DNAwas observed when cells were treated with the ribonucleotidereductase inhibitor hydroxyurea (data not shown). Together,these results indicate that FEN1 depletion results in telomeredysfunction similar to that observed upon replication stressfollowing hydroxyurea treatment.We next assessed the telomeres directly upon FEN1 deple-tion. FEN1 was depleted in BJ fibroblasts expressing the SV40early region (BJL) (the presence of the early region facilitatedisolation of metaphase chromosomes) (Figure 2A). FollowingFEN1 depletion, we utilized fluorescence in situ hybridization(FISH)tovisualizetelomeres.WefoundthatFEN1depletionre-sulted in increased sister telomere loss (STL) (Figures 2B and2C).On average,9.4%of thechromosomes isolated fromcon-trolcellsdisplayedSTLs(Figure2C).UponFEN1depletion,thepercentageofchromosomesdisplayingSTLsincreasednearly2-fold (16.8%, p < 0.0001; Figure 2C), indicating that FEN1depletion impacted telomere stability.Depletion of FEN1 leads to sister telomere loss (Figure 2),resulting in recognition of telomeres by the DNA damagemachinery (Figure 1). Several papers have demonstrated that