Here we demonstrate that SUMOylation limits telomere length by directly modifying telomere proteins in budding yeast. The observation that the abolition of Cdc13 SUMOylation led to longer telomeres, whereas Cdc13-SUMO fusion resulted in shorter telomeres, indicates that SUMO negatively regulates telomere length, in part by targeting Cdc13 ( and ). We show that Cdc13 is a main target of Siz SUMO E3s in telomere length regulation, though SUMOylation of other identified substrates probably contribute to the full inhibitory effect of SUMO, because telomere lengthening in cdc13-snm
Δ cells was not as severe as in ubc9-1
cells ( and Supplementary Fig. 4b
). It is noteworthy that a similar multisubstrate strategy has been suggested for Cdk1 kinase, with Cdc13 being one of several targets3–7
As some of the identified SUMO substrates modulate transcriptional silencing and telomere positioning, their SUMOylation could affect these aspects of telomere metabolism. Indeed, Siz2-mediated Ku SUMOylation was found to influence telomere positioning46
. Our finding of MMS- and high temperature–induced SUMOylation may suggest that SUMO can also modulate telomerase behavior both at native telomeres and/or at DNA breaks under these conditions (). For example, SUMO may prevent telomere addition at the DNA breaks in favor of DNA repair, similar to the effect of Mec1 checkpoint kinase-mediated phosphorylation of telomere proteins29,30
. Our identification of telomere SUMO substrates opens up opportunities for further analysis of SUMO's roles in telomere metabolism under various conditions.
SUMOylation specifically affects the actions of Cdc13 in telomerase regulation but not in end protection, as shown by our finding that cdc13-snm
lengthened telomeres in a telomerase-dependent manner without altering end protection in either wild-type or ku
Δ cells ( and Supplementary Figs. 3, 4a, 5c
). Several lines of evidence support the possibility that Cdc13 SUMOylation affects the Stn1 pathway of telomerase inhibition. Abolition of Cdc13 SUMOylation by cdc13-snm
Δ weakened the Cdc13-Stn1 interaction in Y2H assays ( and Supplementary Fig. 4c
). Genetically, cdc13-snm
was epistatic with stn1
alleles, but not with tel1
Δ and hypomorphic est1
alleles, and its telomere defect was suppressed by Stn1 overexpression (, and Supplementary Fig. 6
). Moreover, Cdc13-SUMO was enriched in the Stn1 immunoprecipitate and led to shorter telomeres (). These results strongly support the idea that SUMO promotes the Cdc13-Stn1 interaction to restrain telomerase function. The specific effect of increased Cdc13 SUMOylation by stn1
alleles also points to a functional interaction between Cdc13 SUMOylation and Stn1 and raises the possibility of an inhibitory feedback mechanism (). As SUMO does not interact with Stn1 or Ten1 by Y2H, it is unlikely that SUMO enhances the Cdc13-Stn1 interaction by simply binding to these proteins (data not shown). Rather, SUMO may affect other properties of Cdc13 such as conformation or DNA binding. Further investigation of these ideas requires the development of an in vitro
assay for the Cdc13-Stn1 interaction.
We showed that SUMOylation and phosphorylation of Cdc13 operate independently and act antagonistically (). This result and those of others6
suggest that each role of Cdc13 can be facilitated by a different modification, which helps explain how Cdc13 acts both positively and negatively to regulate telomerase during S phase. Because Cdc13 SUMOylation peaks in early to mid S phase (), whereas Cdc13 phosphorylation and telomerase recruitment peak in late S to G2 phase (refs. 1
and Supplementary Fig. 2
), these modifications may help to restrict telomere addition to a narrow window within the cell cycle (). As Cdc13 was also proposed to inhibit telomerase in late S to G2 phase, Cdc13 may exert a dual-phase inhibition of telomerase11
. Other models, which are not mutually exclusive, may also help to explain the interplay of the two modifications. For example, because telomerase is preferentially recruited to short telomeres47
, phosphorylation and SUMOylation of Cdc13 may predominantly occur at short and long telomere ends, respectively, to promote or restrain telomerase action (). Future studies will be needed to test these models and to examine the coordination of these modifications with other layers of telomerase regulation.
Because SUMOylation was also found to limit telomere length in fission yeast19
, this modification can have an evolutionarily conserved role in inhibiting telomerases. Considering that Cdk1 and Tel1 kinases and their homologs are typically positive regulators of telomerase, SUMOylation and phosphorylation may provide balancing mechanisms in telomere length homeostasis. Further understanding of their interplay could provide new insights into telomere-related human diseases.