Feedback control of telomere length D Shore and A Bianchi MRX Tel1 P Primase Est1 Pol1 Pol12 Telomerase Est2 TLC1 Figure 3 Summary of some of the proposed steps in TG tract length-dependent regulation of telomerase action. The Rap1–Rif complex generates a number-dependent negative regulatory signal that inhibits telomerase recruitment. Rif2 (grey sphere) competes with Tel1 for binding to the Xrs2 component of MRX, thus inhibiting Tel1 binding. Rap1 appears to block MRX–Tel1 binding through an uncharacterized mechanism. The molecular basis of Rif1 inhibition on telomerase action is unknown. Tel1 in turn phosphorylates Cdc13, creating a positive signal for telomerase recruitment through a direct interaction with its Est1 subunit. The DNA polymerase a/primase complex interacts with the CST complex, thus blocking telomerase recruitment. Regulation and timing of this step are not understood at present. Note that Est1, or other components of telomerase, may be activated through as yet unknown mechanisms, perhaps involving Tel1 phosphorylation (see text for discussion). interpretations of these results indicates that telomerase recruitment to telomere ends in yeast is regulated by telomere length. Distinguishing conclusively between these different possibilities appears to be beyond the reach of present ChIP techniques, which cannot monitor conformational or posi- tional changes for proteins at a given chromatin site and are therefore unsuitable for assessing activation models. An additional important ﬁnding to emerge from these studies was the observation that Tel1 protein is signiﬁcantly enriched at a shortened telomere (Sabourin et al, 2007; Bianchi and Shore, 2007b), a conclusion also supported by ChIP studies in cells lacking telomerase activity (Hector et al, 2007). As pointed out above, the Tel1 kinase is required for normal telomerase association with telomeres, is essential for telomerase action, together with the related Mec1 kinase (Ritchie et al, 1999), and may phosphorylate the Cdc13 RD in vivo to promote Est1 binding and thus telomerase recruit- ment (Tseng et al, 2006). This latter ﬁnding is particularly signiﬁcant in light of the ChIP ﬁndings with Tel1, as it is very easy to imagine that increased Tel1 association with short telomeres will lead to increased Cdc13 phosphorylation, and in turn, increased telomerase recruitment. It should be noted that Cdk1-dependent phosphorylation of Cdc13 also pro- motes Est1 binding (Li et al, 2009). However, based on genetic data, this effect seems less important than that of Tel1, and it is unclear whether it is regulated by TG tract length. At this point, it seems necessary to understand why Tel1 recruitment (which, like at a DSB, depends on the C-terminus of Xrs2 (Sabourin et al, 2007)) is more efﬁcient at short compared with longer TG tracts. Recent work sheds some light on this question. To begin with, Marcand and co-work- ers present evidence that Rif2 has a direct function in telomere capping, and suggest that this could result from a direct inhibition of MRX and/or Tel1 binding (Marcand et al, 2008). In a more recent report, Sugimoto and co-workers show, using a ‘telomere healing’ (de novo formation) assay, that both Rif1 and Rif2 directly block Tel1 (but not MRX) association at longer TG tracts, with Rif2 showing a stronger effect (Hirano et al, 2009). Signiﬁcantly, this study also provides biochemical evidence that Rif2 acts by competing 2316 The EMBO Journal VOL 28 | NO 16 | 2009 with Tel1 for binding to a surface on the C-terminus of Xrs2. The molecular basis for Rif1 action remains unknown, and the authors are careful to point out that a Rap1-dependent but Rif-independent mechanism for telomere length control is likely to exist, consistent with earlier ﬁndings (Negrini et al, 2007; Marcand et al, 2008). A working model for TG tract length regulation of telomerase, based on the ﬁndings dis- cussed above, is summarized in cartoon form in Figure 3. A direct effect of Tel1 on Cdc13, and in turn telomerase recruitment, appears not to be the only mechanism promot- ing elongation of short telomeres. In their Cre-LoxP-based telomere shortening experiments, Bianchi and Shore (2007a) made the unexpected ﬁnding that shortened telomeres, un- like normal-length controls, replicate early in S phase due to the earlier ﬁring of subtelomeric DNA replication origins. They showed that this effect accelerates the elongation of a critically short telomere, perhaps providing an advantage to cells in the event of replication fork collapse at the telomere (Miller et al, 2006) or rapid telomere deletion (Li and Lustig, 1996). The mechanism by which telomere length feeds back to control nearby replication origins is not known at present, but seems unlikely to be fully explained by the effects of yeast Ku or SIR proteins on subtelomeric gene silencing and replication origin ﬁring (Stevenson and Gottschling, 1999; Cosgrove et al, 2002). Intriguingly, a local effect of DSBs on the ﬁring of nearby origins has recently been uncovered, which might be related to the phenomenon observed at shortened telomeres (Doksani et al, 2009). Although it is unclear why early replication accelerates telomere elonga- tion, one possibility is that it increases the time that the telomere is available for telomerase binding, particularly during a period in early S phase when association of the inhibitor Rif1 is lowest (Bianchi and Shore, 2007a). A number of loose ends As suggested at various points above, several aspects of telomere replication, even in the well-studied yeast system, remain poorly understood. Thus, apart from the ﬁrst hints of a mechanism by which Rif2 blocks Tel1 recruitment, we still & 2009 European Molecular Biology Organization