Feedback control of telomere length D Shore and A Bianchi molecule (Greider and Blackburn, 1987), an integral part of the holoenzyme, as a template for the addition of simple TG- rich repeats to the 30 ends of chromosomes. The use of the telomerase enzyme to solve the ‘end-replication problem’ is remarkably widespread amongst eukaryotes. Oddly enough, though, in Drosophila melanogaster, in which early studies were among the ﬁrst to reveal the unique properties of telomeres, a retrotransposon-based mechanism is used to replenish the chromosome ends. The identiﬁcation of mutants defective in telomerase com- ponents in yeast (Lundblad and Szostak, 1989; Singer and Gottschling, 1994; Lendvay et al, 1996), or knockout of the telomerase RNA gene in mouse (Blasco et al, 1997), proved that telomerase is indeed required to prevent the slow erosion of chromosome ends, and thus for cell and, ultimately, whole organism viability. Nevertheless, a small fraction of telomer- ase-minus yeast cells escape senescence and survive through the activation of recombination-based telomere maintenance mechanisms. A similar telomerase-independent survival me- chanism, referred to as the ALT (alternative lengthening of telomeres) pathway, is also observed in a small percentage of human tumours (reviewed in Cesare and Reddel, 2008). For further discussion of recombination-based mechanisms for regulation of telomere stability and telomere repeat tract length, the reader is referred to recent reviews (Lustig, 2003; Bhattacharyya and Lustig, 2006). Recognizing the telomere length regulation problem: a case of localized feedback? The short repeated sequences found at telomeres (irregular TG1 3 repeats in yeast, regular T2AG3 ones in vertebrates; generally referred to hereafter as TG repeats) vary consider- ably in length between organisms (B300 bp in yeast, 5–10 kb in humans) but are centred about a ﬁxed average length characteristic of a given species. This implies that the telo- merase enzyme is instructed in some way as to when it is appropriate to add TG repeats to a given chromosome end. Formally, one could imagine an alternative hypothesis in which exonucleolytic processing of telomeres would be acti- vated in proportion to TG tract length. However, subsequent experiments (Marcand et al, 1999) would clearly show that the telomerase pathway, and not the end attrition that occurs in its absence, is regulated according to TG tract length. Murray et al (1988) recognized this problem early on, and, based on their studies of de novo telomere formation in yeast, they proposed that ‘(t)he constant average length of yeast telomeres implies a feedback mechanism that senses the length of telomeric DNA and reduces the extent of non- template-directed DNA synthesis when the telomeric DNA exceeds a certain length.’ This proposal of a feedback me- chanism regulating telomerase that can sense the length of telomeric DNA eventually gained experimental support. In budding yeast, the duplex portion of the irregular TG1 3 repeats at telomeres is bound directly by a tandem Myb domain containing protein called Rap1 (Shore, 1994). In the course of studying the effect of Rap1 on telomeric gene silencing, Marcand et al (1997) noticed that tethering of hybrid proteins containing the Rap1 C-terminus adjacent to a single-telomeric TG tract would reduce the length of that telomere, in a manner roughly proportional to the number of targeted Rap1 C-termini, without affecting other telomeres in 2310 The EMBO Journal VOL 28 | NO 16 | 2009 the same cell. They interpreted this result in terms of a negative feedback mechanism for telomerase regulation in- volving the Rap1 C-terminus (see Figure 1). At the same time, studies from the de Lange laboratory led to a similar propo- sal, namely that in human cells the telomere repeat binding protein TRF1 controls telomere length in cis by inhibiting telomerase action at individual telomeres (van Steensel and de Lange, 1997). Thus, overexpression of TRF1, which might be expected to increase its binding at telomeres, leads to telomere shortening, whereas expression of a dominant- negative allele that interferes with DNA binding leads to telomere elongation. Even before the emergence of this feedback (‘counting’) model for telomere length regulation, the ability to carry out sophisticated genetic analysis in yeast had already provided a clear foundation for the model as well as some hints regard- ing underlying mechanisms. Indeed, genetic experiments had already suggested that Rap1 might be a negative regulator of telomere elongation: point mutations in the Rap1 C-terminus had been shown to lead to telomere elongation and to a new equilibrium set point (Sussel and Shore, 1991), and complete deletion of a C-terminal domain of the protein was found to cause severe telomere elongation and an absence of any apparent regulation (Kyrion et al, 1992). These studies had thus already shown that Rap1 might have a key function in the proposed counting mechanism leading to feedback reg- ulation of telomerase action. However, other work suggested that Rap1 has a more complex function in telomere regula- tion. Rap1 is encoded by an essential gene in yeast, probably due to its involvement in gene activation in other contexts (Shore, 1994), and the ﬁrst temperature-sensitive (ts) lethal mutations in RAP1 displayed a telomere shortening pheno- type at semi-permissive temperatures (Lustig et al, 1990). Further study of the Rap1 C-terminus revealed two addi- tional proteins with direct roles in telomere length regulation. The ﬁrst hint that the Rap1 C-terminus might interact with other proteins to negatively regulate telomerase action came from experiments showing that overexpression of this do- main, in the absence of the centrally located DNA-binding domain, causes telomere elongation (Conrad et al, 1990). These experiments were interpreted to mean that Rap1 inter- acts with one (or more) negative regulators of telomere elongation, present in limiting amounts in the cell, which can be titrated away from telomeres by overexpression of the Rap1 C-terminal domain. The identity of two such negative regulators, Rif1 (Rap1-interacting factor 1) (Hardy et al, 1992) and Rif2 (Wotton and Shore, 1997), soon came to light through two-hybrid screens carried out with the Rap1 C-terminus as bait. Deletion of either RIF1 or RIF2 causes telomere elongation, B600 bp in the case of rif1-D and less (B150 bp) for rif2-D. Thus, both single mutants would seem to retain some form of feedback regulation on telomerase action, albeit with an alteration in the TG tract length set point at equilibrium. Signiﬁcantly, though, cells in which both RIF1 and RIF2 are deleted display extremely elongated telomeres (Wotton and Shore, 1997), with no apparent equi- librium length, similar to that observed in RAP1 C-terminus deletion mutants. These ﬁndings suggested that Rif1 and Rif2 might operate through different mechanisms, which together are essential for feedback regulation on telomerase. Indeed, tethering experiments carried out with Rif1 and Rif2, similar to those described above for the Rap1 C-terminus, have & 2009 European Molecular Biology Organization