Control of telomere length in yeast by SUMOylated PCNA and the Elg1 PCNA unloader

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the role of Elg1 in the regulation of telomere length. The main role of the Elg1/RLC complex is to unload the processivity factor PCNA, mainly after completion of synthesis of the Okazaki fragment in the lagging strand. They found that Elg1 physically interacts with the CST (Cdc13-Stn1-Ten1) and propose that Elg1 negatively regulates telomere length by mediating the interaction between Cdc13 and Stn1 in a pathway involving SUMOylation of both PCNA and Cdc13. Accumulation of SUMOylated PCNA upon deletion of ELG1 or overexpression of RAD30 leads to elongated telomeres. On the other hand, the interaction of Elg1 with Sten1 is SIM-dependent and occurs concurrently with telomere replication in late S phase. In contrast Elg1-Cdc13 interaction is mediated by PCNA-SUMO, is independent on the SIM of Elg1 but still dependent on Cdc13 SUMOylation. The authors present a model containing two main messages 1) PCNA-SUMO acts as a positive signal for telomerase activation 2) Elg1 promotes Cdc13/Stn1 interaction at the expense of Cdc13/Est1 interaction thus terminating telomerase action.

The manuscript contains a large amount of data that make a major inroad on a new type of link between telomere replication and regulation of the telomerase. Nevertheless, the detailed choreography of the events as well as the role of PCNA-SUMO remain elusive and the data do not fully explain the role of the Stn1/Elg1 interaction. The data presented do not sufficiently support the claim that SUMO-PCNA is a positive signal for telomerase activation.

Reviewer #2 (Public Review):

This paper purports to unveil a mechanism controlling telomere length through SUMO modifications controlling interactions between PCNA unloader Elg1 and the CST complex that functions at telomeres. This is an extremely interesting mechanism to understand, and this paper indeed reveals some interesting genetic results, leading to a compelling model, with potential impact on the field. The conclusions are largely supported by experiments examining protein-protein interactions at low resolution and ambiguous regarding directness of interactions like co-IP and yeast two-hybrid (Y2H) combined with genetics. However, some results appear contradictory and there's a lack of rigor in the experimental data needed to support claims. There is significant room for improvement and this work could certainly attain the quality needed to support the claims. The current version needs substantial revision and lacks the necessary experimental detail. Stronger support for the claims would add detail to help distinguish competing models.

Reviewer #3 (Public Review):

This paper reveals interesting physical connections between Elg1 and CST proteins that suggest a model where Elg1-mediated PCNA unloading is linked to regulation of telomere length extension via Stn1, Cdc13, and presumably Ten1 proteins. Some of these interactions appear to be modulated by sumolyation and connected with Elg1's PCNA unloading activity. The strength of the paper is in the observations of new interactions between CST, Elg1, and PCNA. These interactions should be of interest to a broad audience interested in telomeres and DNA replication.

What is not well demonstrated from the paper is the functional significance of the interactions described. The model presented by the authors is one interpretation of the data shown, and proposes that the role of sumolyation is temporally regulate the Elg1, PCNA and CST interactions at telomeres. This model makes some assumptions that are not demonstrated by this work (such as Stn1 sumolyation, as noted) and are left for future testing. Alternative models that envision sumolyation as a key in promoting spatial localization could also be proposed based on the data here (as mentioned in the discussion), in addition to or instead of a role for sumolyation in enforcing a series of switches governing a tightly sequenced series of interactions and events at telomeres. Critically, the telomere length data from the paper indicates that the proposed model depicts interactions that are not necessary for telomerase activation or inhibition, as telomeres in pol30-RR strains are normal length and telomeres in elg1∆ strains are not nearly as elongated as in stn1 strains. One possibility mentioned in the paper is the PCNAS and Elg1 interactions are contributing to the negative regulation of telomerase under certain conditions that are not defined in this work. Could it also be possible that the role of these interactions is not primarily directed toward modulating telomerase activity? It will be of interest to learn more about how these interactions and regulation by Sumo function intersect with regulation of telomere extension.

Author Response:

Reviewer #1 (Public Review):

[…] The manuscript contains a large amount of data that make a major inroad on a new type of link between telomere replication and regulation of the telomerase. Nevertheless, the detailed choreography of the events as well as the role of PCNASUMO remain elusive and the data do not fully explain the role of the Stn1/Elg1 interaction. The data presented do not sufficiently support the claim that SUMOPCNA is a positive signal for telomerase activation.

We thank the reviewer for her/his review efforts and opinion. We will resubmit a new version of the manuscript in which we will clarify some of the criticisms presented.

Reviewer #2 (Public Review):

[…] The conclusions are largely supported by experiments examining protein-protein interactions at low resolution and ambiguous regarding directness of interactions like co-IP and yeast two-hybrid (Y2H) combined with genetics. However, some results appear contradictory and there's a lack of rigor in the experimental data needed to support claims. There is significant room for improvement and this work could certainly attain the quality needed to support the claims. The current version needs substantial revision and lacks the necessary experimental detail. Stronger support for the claims would add detail to help distinguish competing models.

We thank the reviewer for her/his positive opinion. We will resubmit a new version of the manuscript in which we will clarify some of the criticisms presented by the referees, and add all the missing experimental details.

Reviewer #3 (Public Review):

This paper reveals interesting physical connections between Elg1 and CST proteins that suggest a model where Elg1-mediated PCNA unloading is linked to regulation of telomere length extension via Stn1, Cdc13, and presumably Ten1 proteins. Some of these interactions appear to be modulated by sumolyation and connected with Elg1's PCNA unloading activity. The strength of the paper is in the observations of new interactions between CST, Elg1, and PCNA. These interactions should be of interest to a broad audience interested in telomeres and DNA replication.

We thank the reviewer for her/his positive opinion. We will resubmit a new version of the manuscript in which we will clarify some of the criticisms presented.

What is not well demonstrated from the paper is the functional significance of the interactions described. The model presented by the authors is one interpretation of the data shown, and proposes that the role of sumolyation is temporally regulate the Elg1, PCNA and CST interactions at telomeres. This model makes some assumptions that are not demonstrated by this work (such as Stn1 sumolyation, as noted) and are left for future testing. Alternative models that envision sumolyation as a key in promoting spatial localization could also be proposed based on the data here (as mentioned in the discussion), in addition to or instead of a role for sumolyation in enforcing a series of switches governing a tightly sequenced series of interactions and events at telomeres. Critically, the telomere length data from the paper indicates that the proposed model depicts interactions that are not necessary for telomerase activation or inhibition, as telomeres in pol30-RR strains are normal length and telomeres in elg1∆ strains are not nearly as elongated as in stn1 strains. One possibility mentioned in the paper is the PCNAS and Elg1 interactions are contributing to the negative regulation of telomerase under certain conditions that are not defined in this work. Could it also be possible that the role of these interactions is not primarily directed toward modulating telomerase activity? It will be of interest to learn more about how these interactions and regulation by Sumo function intersect with regulation of telomere extension.

We present compelling evidence for a role of SUMOylated PCNA in telomere length regulation. Figure 1 shows that this modification is both necessary and sufficient to elongate the telomeres, indicating that PCNA SUMOylation plays a positive role in telomere elongation. The model we present is consistent with all our results. There are, of course, possible alternative models, but they usually fail to explain some of the results. We agree that the fact that pol30-RR presents normal-sized telomeres implies that SUMO-PCNA is not required for telomerase to solve the "end replication problem", but rather is needed for "sustained" activity of telomerase. Since elongated telomeres (by absence of Elg1 or by over-expression of SUMO-PCNA) was the phenotype monitored, this may require sustained telomerase activity. Similar results were seen in the past for Rnr1 (Maicher et al., 2017), and this mode depends on Mec1, rather than Tel1 (Harari and Kupiec, 2018). Telomere length regulation is complex, and we may not yet understand the whole picture. It appears that for normal “end replication problem” solution, very little telomerase activity may be needed, and spontaneous interactions at a low level may suffice. Future work may find the conditions at which telomerase switches from "end replication problem" to "sustained" activity. We will add further explanations on this subject to the Discussion section.

We suspect, but could not prove, a role for Stn1 SUMOylation in the interactions. SUMOylation is usually transient, and notoriously hard to detect, and despite the fact that many telomeric proteins are SUMOylated, Stn1 SUMOylation could not be shown directly by us and others (Hang et al, 2011).