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Htert Associates with Human Telomeres and Enhances Genomic Stability and DNA Repair

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Htert Associates with Human Telomeres and Enhances Genomic Stability and DNA Repair Oncogene (2003) 22, 131–146 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc hTERT associates with human telomeres and enhances genomic stability and DNA repair Girdhar G Sharma1,2, Arun Gupta1,2, Huichen Wang3, Harry Scherthan4, Sonu Dhar1,2, Varsha Gandhi5, George Iliakis3,7, Jerry W Shay6, Charles SH Young2 and Tej K Pandita1,2,* 1Radiation and Cancer Biology Division, Washington University School of Medicine, St Louis, MO 63108, USA; 2College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA; 3Thomas Jefferson University, Philadelphia, PA 19107, USA; 4Max-Plank-Institute for Molecular Genetics, Ihnestraße 73, D-14195 Berlin, Germany; 5University of Texas, MDACC, Houston, TX 70330, USA; 6University of Texas Southwestern Medical Center, Dallas, TX 75390, USA Ectopic expression of telomerase in telomerase-silent cells a function of age in cells derived from normal human is sufficient to overcome senescence and to extend cellular blood, colonic mucosa and skin (Hastie et al., 1990; lifespan. We show here that the catalytic subunit of human Allsopp et al., 1992; Levy et al., 1992; Vaziri et al., telomerase (hTERT) crosslinks telomeres. This interac- 1994). It has been suggested as a result of this shortening tion is blocked by the telomere repeat binding factor 1, but that transcriptional profile of critical genes at the ends not by a dominant negative form of this protein. It is also of the chromosomes may be altered (Levy et al., abolished by destruction of the RNA component of 1992; Olovnikov, 1992) or undergo a change in gene telomerase as well as by mutations in the hTERT protein. expression (Wright and Shay, 1992), leading to cell Ectopic expression of hTERT leads to transcriptional growth arrest. Biochemical and genetic studies have alterations of a subset of genes and changes in the established an association between telomere mainte- interaction of the telomeres with the nuclear matrix. This nance and extended lifespan mediated through the is associated with reduction of spontaneous chromosome expression of TERT. Telomerase is a reverse transcrip- damage in G1 cells, enhancement of the kinetics of DNA tase that synthesizes telomeric DNA thereby compen- repair and an increase in NTP levels. The effect on DNA sating for telomere loss that occurs with each replication repair is likely indirect as TERT does not directly affect cycle. DNA end rejoining in vitro or meiotic recombination in Ectopic expression of hTERT prevents replicative vivo. The observed effects of hTERT occurred rapidly senescence in several cell types including fibroblasts and before any significant lengthening of telomeres was epithelial cells (Bodnar et al., 1998; Vaziri and Bench- observed. Our findings establish an intimate relationship imol, 1998; Ouellette et al., 2000; Wood et al., 2001). It between hTERT–telomere interactions and alteration in may also exert an antiapoptotic action at an early stage transcription of a subset of genes that may lead to of the cell death process prior to mitochondrial increased genomic stability and enhanced repair of genetic dysfunction and caspase activation (Fu et al., 2000). It damage. These novel functions of telomerase are distinct has been proposed that telomere shortening during from its known effect on telomere length and have human replicative aging generates antiproliferative potentially important biological consequences. signals that mediate p53-dependent G1 arrest as is Oncogene (2003) 22, 131–146. doi:10.1038/sj.onc.1206063 observed in senescent cells (Vaziri et al., 1997). In support of this idea, Wong et al. (2000) reported that Keywords: hTERT; telomeres; transcription; DNA re- telomere dysfunction in mTerc null mice impairs DNA pair; ionizing radiation repair and subsequently leads to cell growth arrest; and Goytisolo et al. (2000) reported radiosensitivity of the late generation telomerase knockout mice. Choi et al. Introduction (1997) demonstrated that telomerase expression sup- presses senescence-associated genes in Werner syndrome The mammalian telomeres are composed of TTAGGG cells. However, it is not known how the TERT protein arrays bound by a complex of specialized proteins that influences gene expression and subsequently extends the protect chromosome ends from exonucleolytic attack, lifespan of a cell. Here we show for the first time that fusion and incomplete replication. Telomeres shorten as hTERT interacts with the telomeres and influences the interaction of telomeres with the nuclear matrix. It leads to transcriptional alteration along with increased *Correspondence: TK Pandita, 4511 Forest Park, Suite 411, St Louis, MO 63108, USA; E-mail: [email protected] G1 chromosome stability, enhanced DNA repair and 7Present address: University of Essen, D-45122 Essen, Germany increased NTP pools. These effects seem to be Received 23 July 2002; revised 18 September 2002; accepted 24 independent of the effect of telomerase on telomere September 2002 length. Associating telomeres GG Sharma et al 132 Results ELISA assay (Sawant et al., 1999) (Figure 1b). Im- hTERT associates with the telomeres munoprecipitates from HFF+hTERT and HeLa cells had telomerase activity. However, no activity was found To investigate whether hTERT associates with telo- in the immunoprecipitates of HFF+Ad.DhTERT or meres, human foreskin fibroblasts (HFF) with control HFF cells. These results confirmed the specifi- (HFF+hTERT) and without (HFF) ectopic hTERT city of the hTERT antibody for detection of hTERT expression were examined. Stable human fibroblasts protein and functional telomerase complex. (HFF+hTERT) were generated by infection with an To determine the association of hTERT with telo- hTERT-expressing retrovirus as previously described meres, formaldehyde-crosslinked chromatin from HFF, (Wood et al., 2001). To determine the levels of HFF+hTERT and HeLa cells was immunoprecipitated ectopically expressing hTERT in HFF+hTERT cells, using anti-hTERT and anti-p21 antibodies. By using the Western blotting was performed to detect hTERT by chromatin-immunoprecipitation (ChIP) procedure using an anti-hTERT antibody. The antibody recog- (Levy et al., 1992; Braunstein et al., 1993), we found nized hTERT in protein extracts of HFF+hTERT and that hTERT crosslinks with bulk genomic DNA as well HeLa cells while no such band was detected in HFF cells as telomeric DNA (Figure 1c). To rule out the (Figure 1a). Further, it was investigated whether possibility that hTERT interaction with the telomeres hTERT antibody recognizes the mutant hTERT is because of nonspecific binding, we investigated (DhTERT). This was achieved by infecting adenovirus- whether a negative regulator of telomerase, TRF1, expressing hTERT with deletions in the reverse tran- influences hTERT binding to telomeres. Association of scriptase (RT) motif in HFF cells (HFF+Ad.Dh- hTERT with telomeres was examined in the cells that TERT). hTERT antibody recognized the mutant overexpress intact or truncated version of TRF1. This protein in HFF+Ad.DhTERT cells (Figure 1a). The was achieved by infecting HFF+hTERT cells with hTERT antibody also immunoprecipitates (IP) func- adenovirus-expressing TRF1. The levels of TRF1 tional telomerase complex as determined by a TRAP- protein were analyzed by Western blots. TRF1 increased Figure 1 Human telomeric DNA coimmunoprecipitated with hTERT antibody after in vivo crosslinking. (a) Western analysis of hTERT with anti-hTERT antibody. Note hTERT antibody recognized both the wild (HFF+hTERT and HeLa) as well as mutant [HFF+hTERT(m), i.e, HFF+Ad.DhTERT] hTERT protein and endogenous hTERT in HeLa cells, whereas no such band was observed in HFF cells. (b) Telomerase activity of telomerase complex immunoprecipitated by anti-hTERT antibody. Note that the telomerase activity was found in IPs with anti-hTERT antibody in HFF+hTERT and HeLa cells, whereas no such activity was observed in HFF and HFF+Ad.DhTERT cells. (c) In vivo crosslinking of HeLa, HFF+hTERT and HFF cells, followed by immunoprecipitation with anti-hTERT or anti-a-p21 antibody. The following probes were used for hybridization: telomeric DNA, total human genomic DNA and Alu DNA; no formaldehyde treatment (ÀF) or formaldehyde treatment for 1 h (+F). The same blots were used for hybridization with different probes after the stripping of the probe from the blot. Note that no telomeric signal is detected in DNA immunoprecipitated with a-p21 antibody, whereas only DNA coimmunoprecipitated by hTERT antibody after formaldehyde crosslinking showed telomeric signals. Immunoprecipitation by hTERT in HFF cells resulted in no telomeric signals Oncogene Associating telomeres GG Sharma et al 133 at least four-fold 48 h postinfection (Figure 2a). Inter- HFF+hTERT cells were treated with an antisense estingly, cells overexpressing wild-type TRF1 showed oligonucleotide directed against hTR linked to a 2–5A reduced interaction of hTERT with telomeres (50-phosphorylated 20–50-linked oligoadenylate) mole- (Figure 2b). The degree of interaction correlated cule (2–5A-anti-hTR) as described by Kondo et al. inversely with the level of TRF1 (Figure 2a, b). (1998). A 2–5A-antisense-hTR oligonucleotide is tar- However, truncated TRF1 (lacking the Myb DNA- geted against the predicted loop of hTR (nucleotides 76– binding domain) has no effect on the interaction of 94). HFF+hTERT cells treated with 4 mm 2–5A hTERT with telomeres (data not shown). These antisense oligonucleotide for 48 h showed loss of hTR observations suggest that hTERT directly
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