Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.Telomere integrity is essential for chromosome stability, and the maintenance of telomeric DNA is required for long-term proliferation of eukaryotic cells. Telomeres are maintained by telomerase, a reverse transcriptase that adds telomeric repeats to chromosome ends (14; reviewed in reference 31). In most normal human somatic tissue telomerase is repressed, and as a result, telomeres shorten (17, 18). Critically short telomeres lose their ability to protect chromosome ends, resulting in chromosomal degradation and fusion. In contrast to normal somatic human cells, immortalized cells (including cancer cells) and germ cells express telomerase (21, 34) and maintain their telomeres. In these cells telomere maintenance is regulated by a homeostatic mechanism (reviewed in reference 29). Thus, in the mammalian germ line telomeres show a speciesspecific telomere length setting which is constant over the generations (23). Regulation is also apparent in many human tumor cell lines, where despite the presence of high levels of telomerase telomeres do not grow, but rather, they are stably maintained within a given size range (9, 10).Mammalian telomeres consist of long tandem arrays of TTAGGG repeats bound by the DNA-binding proteins, TRF1 and TRF2 (4, 5, 7; reviewed in reference 8). The TRFs are related in their primary structure; both contain carboxy-terminal Myb-type DNA-binding motifs and internal, conserved domains required for homodimerization (2, 5). The proteins do not form heterodimers (5). A dis...

clinicaltrials.gov Identifier: NCT00206141.

Our findings suggest a dynamic cross talk between TRF1 and TRF2 and provide a molecular mechanism for telomere length homeostasis by TRF2 in the absence of TRF1.

clinicaltrials.gov Identifier: NCT00119652.

Mammalian telomeres are coated by the sequence-specific, DNA-binding protein, TRF1, a negative regulator of telomere length. Previous results showed that ADP-ribosylation of TRF1 by tankyrase 1 released TRF1 from telomeres and promoted telomere elongation. We now show that loss of TRF1 from telomeres results in ubiquitination and degradation of TRF1 by the proteasome and that degradation is required to keep TRF1 off telomeres. Ubiquitination of TRF1 is regulated by its telomerebinding status; only the telomere-unbound form of TRF1 is ubiquitinated. Our findings suggest a novel mechanism of sequential posttranslational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres. Received January 22, 2003; revised version accepted March 28, 2003. In most human tumor cells, telomere length is maintained by telomerase, a specialized reverse transcriptase that adds telomere repeats to chromosome ends (Greider and Blackburn 1985). Telomere length is tightly controlled in tumor cells, where despite high levels of telomerase, telomeres are maintained at a constant length setting (Counter et al. 1992). Cells employ a complex set of positive and negative regulatory mechanisms to modulate telomere length, including regulating access of telomerase to chromosome termini (Evans and Lundblad 2000).Mammalian telomeres consist of tandem arrays of TTAGGG repeats bound by the sequence-specific, double-stranded, DNA-binding proteins TRF1 (Chong et al. 1995) and TRF2 (Bilaud et al. 1997;Broccoli et al. 1997). TRF2 is required to protect chromosome ends (van Steensel et al. 1998;de Lange 2002), possibly through its ability to assemble t-loops, higher-order structures at telomeres (Griffith et al. 1999). In addition, TRF2 can influence telomere length through a telomeraseindependent mechanism (Ancelin et al. 2002;Karlseder et al. 2002). TRF1, on the other hand, is a negative regulator of telomerase-mediated telomere length, acting in cis at chromosome ends to repress telomere elongation (van Steensel and de Lange 1997;Ancelin et al. 2002).Tankyrase 1 is a telomeric poly(ADP-ribose) polymerase (PARP) that binds and modifies TRF1 (Smith et al. 1998). Like other PARP family members (Smith 2001), tankyrase 1 uses NAD + as a substrate to catalyze formation of ADP-ribose polymers onto specific protein acceptors, including itself and TRF1 (Smith et al. 1998;Rippmann et al. 2002). ADP-ribosylation of TRF1 by tankyrase 1 inhibits TRF1 binding to telomeric DNA in vitro (Smith et al. 1998 These findings suggest that tankyrase 1-mediated removal of TRF1 from telomeres by ADP-ribosylation could allow access of telomerase to chromosome termini.Here, we show that tankyrase 1 induces proteasomemediated degradation of TRF1. We demonstrate that TRF1 is ubiquitinated in vivo and in vitro. We present evidence to suggest that it is not ADP-ribosylation per se, but rather, release of TRF1 from telomeres that serves as a signal for ubiquitination and subsequent degradation. Results and Discussion Tankyrase...