Chromosomes of most eukaryotic organisms end in distinctive nucleoprotein structures, called telomeres, which are essential for protection of chromosomes from degradation and fusions by the DNA repair machinery (de Lange, 2018). Short G/C-rich telomeric DNA repeats provide a platform for loading of a specific set of telomeric proteins forming complex dynamic assemblies at the ends of the chromosomes. Dividing cells are constantly losing telomeric repeats due to incomplete replication unless counteracted by either recombination or activity of the ribonucleoprotein complex telomerase. Replenishing of telomeric DNA by telomerase is crucial for long-term proliferation of the majority of eukaryotic cell types. This process is highly coordinated, and plenty of positive and negative regulators of telomerase have been identified to date, many of which are components of the telomeric chromatin (Lee et al., 2021; Lim and Cech, 2021; Shay and Wright, 2019).

In budding yeast Saccharomyces cerevisiae, the double-stranded portion of telomeres is bound by Rap1 through its duplicated MYB domain (Henry et al., 1990; Shore and Nasmyth, 1987). Two additional telomeric proteins – Rif1 and Rif2 – are recruited to telomeres via interaction with Rap1 C-terminus (RCT) (Hardy et al., 1992; Wotton and Shore, 1997). Both of them restrict telomerase action and their double deletion (as well as deletion of the RCT) leads to telomere hyperelongation (Teng et al., 2000; Wotton and Shore, 1997). Rap1 also recruits the histone deacetylase complex Sir2/Sir3/Sir4 (through an interaction between Sir4 and RCT) providing the basis for the silencing of telomere-proximal genes (Moretti et al., 1994). The C-terminal portion of Rif1 contains a Rap1-binding motif (Rif1_RBM) and a tetramerization module (Rif1_CTD), while Rif2 contains two Rap1-binding sites, therefore Rap1/Rif1/Rif2 can form complex DNA-protein structures at the chromosome ends, inhibiting both telomerase recruitment and telomere silencing (Shi et al., 2013). In addition, Rif1 can localize at telomeres in a Rap1-independent fashion through its large HEAT repeats containing N-terminal domain (Rif1_NTD) that exhibits strong affinity to DNA with some preference towards 3’-overhang containing ss-ds junctions (Mattarocci et al., 2017). This binding mode is important for telomerase regulation and attenuation of end resection at telomeres. Recently, the ScRif1_NTD was proposed to contain an interaction site for a yet unidentified protein partner at telomeres (Shubin et al., 2021).

The single-stranded region of telomeres (3’-overhang) is bound and protected by the Cdc13 protein (Garvik et al., 1995; Lin and Zakian, 1996; Nugent et al., 1996) together with its partners Stn1 and Ten1 (the CST complex; Grandin et al., 2001; Grandin et al., 1997; Mersaoui and Wellinger, 2019; Wellinger and Zakian, 2012). Cdc13 is a central hub for telomeric DNA synthesis as it mediates the main pathway of telomerase recruitment (synthesis of the G-strand), as well as assists in loading of the Polα(synthesis of the C-strand) (Qi and Zakian, 2000; Wellinger and Zakian, 2012). Telomerase recruitment in S. cerevisiae also depends on another telomeric complex – the Ku70/Ku80 heterodimer (Ku): it binds a stem-loop structure within telomerase RNA and the Sir4 protein at telomeres (Hass and Zappulla, 2015; Peterson et al., 2001; Stellwagen et al., 2003). In addition, Ku can bind DNA directly and apparently does so at sub-telomeric regions and, perhaps, at the ds-ss junction of the telomere (Larcher et al., 2016; Lopez et al., 2011). Ku loss leads to telomere shortening and Exo1-dependent accumulation of single-stranded telomeric DNA (Bonetti et al., 2010; Boulton and Jackson, 1996; Gravel et al., 1998; Polotnianka et al., 1998; Porter et al., 1996).

Almost every telomeric chromatin component plays important roles outside telomeres. Rap1 protein is a central transcription factor, controlling expression of hundreds of genes in budding yeast (Azad and Tomar, 2016). A regulatory role during RNA PolII transcription was recently described for ScCST complex (Calvo et al., 2019). Ku heterodimer is a key component of the non-homologous end joining (NHEJ) machinery (Frit et al., 2019). Rif1 protein binds Glc7 (PP1) phosphatase and this interaction is important for regulation of replication initiation at several origins (Davé et al., 2014; Hiraga et al., 2014; Mattarocci et al., 2014), but not for telomere length control (Shubin and Greider, 2020). Interestingly, this function in replication, but not the telomeric role, is conserved among eukaryotic Rif1 homologues (see Alavi et al., 2021 for a recent review). The only known species outside budding yeast clade which contains Rif1 as a component of ‘normal’ telomeric chromatin is fission yeast Schizosaccharomyces pombe, although SpRif1 is recruited through an interaction with Taz1 protein rather than Rap1 (Kanoh and Ishikawa, 2001). Mammalian Rif1 associates with dysfunctional telomeres (and other types of DSB) together with 53BP1, limiting accumulation of ssDNA through a mechanism involving Rev7, the Shieldin complex, CST and Polα, and driving the DSB repair toward NHEJ (Barazas et al., 2018; Chapman et al., 2013; Dev et al., 2018; Di Virgilio et al., 2013; Escribano-Díaz et al., 2013; Gupta et al., 2018; Mirman et al., 2018; Noordermeer et al., 2018; Tomida et al., 2018; Zimmermann et al., 2013). Interestingly, S. cerevisiae Rif1 also localizes to non-telomeric DSBs and promotes NHEJ through its DNA binding NTD, suggesting that Rif1 may be a conserved NHEJ factor (Mattarocci et al., 2017).

The thermotolerant methylotrophic budding yeast species Hansenula polymorpha DL-1 is distantly related to S. cerevisiae. Several interesting differences in telomere biology between the two species have already been documented. Rap1 has two paralogues in H. polymorpha (Rap1A and Rap1B) with distinct DNA recognition properties (Malyavko et al., 2019). HpRap1A is located at the subtelomeric regions with no reported telomeric role. HpRap1B is the major telomeric dsDNA binder with the ability to control telomere length, although the primary target of its inhibition appears to be recombination rather than telomerase (Malyavko et al., 2019). The RIF2 gene is absent from H. polymorpha genome (and other yeasts outside Saccharomycetaceae family). A shorter version of Cdc13 protein is present in H. polymorpha, which possesses strong affinity for telomeric G-strand in vitro and binds Stn1 protein (Malyavko and Dontsova, 2020). HpCdc13 also interacts with HpTERT in the yeast-two-hybrid (Y2H) assay.

Here, we explored the conservation of the telomeric roles of Rif1 protein within budding yeast clade by investigating Rif1 function in the telomere maintenance of Hansenula polymorpha. We found that Rif1 restricts telomerase action at H. polymorpha telomeres. We demonstrate that the Rif1 N-terminal extension (Rif1_NTE) – a portion of Rif1 present exclusively in yeast homologues of the protein – is an intrinsically disordered domain with the ability to bind DNA in vitro. We show that this DNA-binding domain helps to recruit Rif1 to telomeres. Moreover, we found that the interaction with the Ku heterodimer is required for Rif1 telomeric localization in H. polymorpha. Finally, we found that the conserved N-terminal domain of HpRif1 binds to HpStn1 protein, and this interaction is crucial for HpStn1 recruitment at telomeres.

We found that Rif1 from the methylotrophic yeast H. polymorpha prevents hyperelongation of telomeres, similar to its other yeast counterparts. In S. cerevisiae, Rif1’s function strongly depends on the recruitment by Rap1 protein (specifically, by the Rap1^RCT domain). Hence, telomerase-dependent telomere overelongation can be observed in the strains with either deletion of the RIF1 gene, or mutations that disrupt Rap1-telomere association (Hardy et al., 1992; Shi et al., 2013; Teng et al., 2000). Long telomeres in the ∆rif1 strain of H. polymorpha do appear to be solely maintained by telomerase (Figure 1B and C). H. polymorpha has two Rap1 paralogues, however, HpRap1A does not recognize telomeric DNA and RCT removal from Rap1A has no effect on telomere length (Malyavko et al., 2019). HpRap1B associates with double-stranded telomeric DNA and the 50 a.a. C-terminal truncation (Rap1B^1–526) leads to more than 10-fold drop in Rap1B expression and strong telomere elongation. In contrast to the ∆rif1 strain, longer telomeres in this B^1–526 strain were found to depend primarily on recombination (Malyavko et al., 2019). This phenotypic discrepancy indicates that functions of Rap1 and Rif1 in H. polymorpha may not be as intimately linked as in S. cerevisiae. In addition, we could not identify the Rap1-binding motif within HpRif1. We failed to detect a direct interaction between HpRif1 and either of the two HpRap1 paralogues in the Y2H experiment (Figure 5A). Finally, Rap1B can efficiently localize to internal as well as terminal telomeric dsDNA (as expected from a sequence specific telomeric factor), whereas Rif1 has a clear preference for telomeric repeats located at the end of the chromosome (Figure 1—figure supplement 1). Thus, although we cannot exclude the possibility of an interaction between HpRap1 and HpRif1, we believe that it is highly unlikely that Rif1 binding to Rap1 is the major mechanism of Rif1 telomeric recruitment in H. polymorpha in a manner it is described for its S. cerevisiae homologues.

Rif1 homologues from many species contain a DBD, and DNA binding by the NTD of S. cerevisiae Rif1 promotes its accumulation at telomeres in a Rap1-independent fashion (Kanoh et al., 2015; Mattarocci et al., 2017; Moriyama et al., 2018; Sukackaite et al., 2014; Xu et al., 2010). This prompted us to test whether HpRif1 might be recruited at telomeres by means of direct interaction with telomeric DNA. We found that HpRif1 fragments containing amino acids 1–264 possess DNA-binding activity in vitro. The Rif1_NTE^1-264 fragment can be detected at telomeres by ChIP, while its removal (the Rif1^∆NTE strain) strongly diminishes the telomere binding of Rif1. Moreover, mutations that abolish DNA-binding in vitro lead to the substantial (if not complete) loss of HpRif1 from telomeres and telomere elongation comparable to the Rif1^∆NTE strain (Figure 4). These results are in favor of the ‘recruitment via direct telomeric DNA binding’ hypothesis. The identified DNA-binding mode by the intrinsically disordered portion of HpRif1, however, is clearly different from the one utilized by the conserved structured NTD of ScRif1. We could not detect a DNA binding activity of the HpRif1_NTD (residues ~ 310–1000, Figure 3A–C), although this may be due to improper folding of the large polypeptides in the E. coli expression system or suboptimal conditions of the EMSA reactions. Despite the fact that the NTD is a relatively well conserved region of Rif1, we found that of 19 charged amino acids constituting the DBD of ScRif1 only four can be found in HpRif1 and their mutation has no apparent effect on Rif1-telomere association (Figure 4—figure supplement 2). Given the high evolvability of telomeric proteins, we do not, however, dismiss the possibility that HpRif1 may contact DNA through different amino acids within its NTD domain.

Interestingly, there is a discrepancy between our ChIP experiments, which suggest that Rif1_NTE is necessary and sufficient for the telomeric localization of Rif1, and the telomere length analysis, which points to the relatively minor contribution of the Rif1_NTE to the telomere length control (Figure 4D and F). This points to the existence of another mode(s) of HpRif1 recruitment, which may evade detection by ChIP, perhaps, because it is restricted to the short window of the cell cycle or due to its transient nature. It is important to note, that the ChIP method proved not to be sufficiently sensitive in assessment of Rif1 chromatin binding in S. cerevisiae: Rif1 accumulation at replication origins, for example, was revealed only by the chromatin endogenous cleavage (ChEC) method (Hafner et al., 2019; Hafner et al., 2018). Future experiments utilizing more sensitive techniques may reveal other modes of HpRif1 recruitment, which could be attributed to the DNA binding by the NTD (similar to the ScRif1) and/or to the interaction of HpRif1 with other telomeric proteins.

Our search for Rif1’s protein partners at telomeres revealed two hits – Ku80 and Stn1 – and deletion of the KU80 gene does lead to strong reduction in Rif1 telomere occupancy, suggesting that this interaction plays an important role in localizing Rif1 to telomeres (Figure 5). Although in the Y2H assay the Ku80-Rif1 interaction depends primarily on the short motif within Rif1_NTE, our experiments indicate that in H. polymorpha cells the robust Ku80-Rif1 association requires other Rif1 domains. The Rif1_KBM may thus serve only as an auxiliary module, which is consistent with the fact that it is conserved only in five species closest to H. polymorpha. The interaction of NTE-less Rif1 with Ku could therefore represent the second mode of telomeric recruitment of HpRif1 responsible for the telomerase inhibition in the Rif1^∆NTE strain.

In S. cerevisiae, telomere tract length determines the probability of the elongation of a chromosomal end: shorter telomeres are preferred substrates for telomerase (Marcand et al., 1999; Teixeira et al., 2004). At the basis of this regulation is ‘counting’ of telomeric repeats by Rap1 that defines the number of (telomerase-inhibitory) Rif1 and Rif2 molecules present at any given telomere (Levy and Blackburn, 2004; Marcand et al., 1997). H. polymorpha lacks Rif2 entirely, and at this point it is not obvious how mechanistically Rif1 recruitment could be linked to telomere length. In fact, HpRif1 appears to have only limited ability to bind double-stranded telomeric DNA in vivo (Figure 1—figure supplement 1). It should be noted, that Rif1 is a large protein with many functions and, even in S. cerevisiae, it is not fully involved in the ‘counting’ process. The Rif1^RBM mutant lacking the ability to contact Rap1 is almost completely lost from telomeres as judged by ChIP, yet retains a significant portion of its telomerase inhibitory potential (Mattarocci et al., 2017; Shi et al., 2013). Thus, budding yeast Rif1 homologues may operate (at least partially) in a telomere length-independent way.

Being the only known telomeric dsDNA binding factor in H. polymorpha, Rap1B is the obvious candidate for a telomere length sensor within a potential ‘protein-counting model’. However, as described previously (Malyavko et al., 2019), Rap1B’s role in telomerase inhibition (if exists) appears to be minor, and through which protein partners (and whether at all) Rap1B could be involved in inhibition of telomerase action is yet to be determined. The lack of Rif2, our inability to detect a Rap1-Rif1 interaction and the apparent absence of a prominent inhibitory effect of Rap1B on telomerase suggest that ‘protein counting’ mechanism of telomere length regulation described for S. cerevisiae may not be operating in H. polymorpha. Additional studies are needed to establish whether this is indeed the case and which components of the telomeric chromatin are ‘counted’ to provide a negative feedback loop for telomerase.

Similarly to S. cerevisiae (Boulton and Jackson, 1996; Porter et al., 1996), deletion of the subunits of the HpKu heterodimer leads to pronounced telomere shortening (Figure 5—figure supplement 2B). ScKu recognizes a specific stem-loop structure within telomerase RNA, mediating one of the two pathways of telomerase recruitment (Chen et al., 2018; Peterson et al., 2001; Stellwagen et al., 2003). Our previous analysis of the H. polymorpha telomerase RNA structure did not reveal a similar Ku-binding hairpin (Smekalova et al., 2013) and the lack of a stable interaction between Ku and HpTER was confirmed experimentally in this study (Figure 5—figure supplement 2F). Shorter telomeres in the HpKu knock-out strains could be explained, however, by a transient association with telomerase via another telomerase subunit; for example like in human cells, where an interaction between Ku and TERT has been detected (Chai et al., 2002). Indeed, we found that HpTERT binds HpKu80 in the Y2H experiment (Figure 5—figure supplement 2G). Interestingly, we observed only minor telomere elongation upon HpRif1 loss in the ∆ku80 background, suggesting that Rif1 counteracts the positive effect of the Ku at telomeres (Figure 5J). We propose that Rif1’s inhibitory function in H. polymorpha may involve a direct competition with telomerase for Ku binding at telomeres (Figure 6—figure supplement 2).

The second pathway of telomerase recruitment in S. cerevisiae relies on the binding of the Est1 telomerase subunit to the RD (recruitment domain) of the Cdc13 protein (Chen et al., 2018; Evans and Lundblad, 1999; Pennock et al., 2001). Association with Stn1 blocks this interaction, thereby limiting the amount of telomerase at telomeres (Chandra et al., 2001; Li et al., 2009; Pennock et al., 2001). Although HpCdc13 is shorter than ScCdc13 and lacks the RD domain (and, apparently, the ability to bind Est1), we have demonstrated recently, that it can bind both telomerase (via the TERT subunit) and Stn1 in a Y2H assay, indicating that a similar mechanism of telomerase regulation may be operating in H. polymorpha (Malyavko and Dontsova, 2020). Alternatively, Stn1 may influence telomerase binding indirectly, by controlling the amount of ssDNA (and therefore Cdc13, which is a ssDNA binding protein) at telomeres (Grandin et al., 1997). In any case, we found that Stn1 accumulation at telomeres depends strongly on Rif1, and that mutations disrupting Stn1-Rif1 (and Stn1-Cdc13) interaction lead to highly elongated telomeres (Figure 6). Thus, we speculate that the negative effect of Rif1 on telomere length may also be explained by its ability to promote Stn1 telomere localization, thereby attenuating the telomerase recruitment via Cdc13 (Figure 6—figure supplement 2).

Remarkably, the existence of a physical interaction between Rif1 and the CST complex was proposed (but not demonstrated experimentally) in a study by Anbalagan et al., 2011 to explain the specific requirement of Rif1 for viability of S. cerevisiae cells with hypomorphic mutations in Cdc13 and Stn1. Our Y2H experiments show that Stn1 binds the NTD of Rif1 (Figure 6G). Rif1_NTD contains the most conserved portion of the protein (Supplementary file 1) that is present in all Rif1 orthologues (Mattarocci et al., 2017; Sreesankar et al., 2012). Hence it is plausible that the Stn1-Rif1 direct interaction observed in our study may be present in other species.

In summary, in the present study we have identified several novel aspects of Rif1 function in budding yeasts. We demonstrated that the N-terminal extension (NTE) of Rif1 is an accessory domain partially promoting Rif1 telomeric localization in H. polymorpha. Given that DNA binding by Rif1_NTE is weak and lacks clearly defined specificity toward a particular telomeric substrate, we believe that it may simply stabilize Rif1 at telomeres, while the preference for the telomeric chromatin is provided by the ability to contact Ku heterodimer. Consistently, Rif1_NTE^1-264(F225E) and Rif1_NTE^1-264(R230E) mutants (unable to bind Ku80, but with intact DBD) fail to localize to telomeres (Figure 5G). Our results also indirectly point to the possibility of the DNA recognition by the HpRif1_NTD in a manner akin to ScRif1, which recognizes the 3’-overhang containing ss-ds junctions (Mattarocci et al., 2017). This may also be a factor driving HpRif1 to the chromosomal ends. Finally, we uncovered an HpRif1’s role in loading Stn1 protein onto telomeres. Curiously, the mammalian Rif1 homologue is important for the recruitment of CST complex to promote NHEJ at several types of DSBs (although the Rif1-CST interaction is not direct, but mediated via the Shieldin complex; Barazas et al., 2018; Chapman et al., 2013; Dev et al., 2018; Di Virgilio et al., 2013; Escribano-Díaz et al., 2013; Gupta et al., 2018; Mirman et al., 2018; Noordermeer et al., 2018; Tomida et al., 2018; Zimmermann et al., 2013). It is tempting to speculate that Rif1’s role in recruiting Stn1 may be conserved from yeasts to human.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for figures 1B, 1D, 1F, 1E, 2B, 3B, 3C, 3D, 3E, 4B, 4C, 4D, 4E, 4F, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 6A, 6E, 6F, figure 1 - figure supplement 1B and C, figure 3 - figure supplement 1, figure 4 - figure supplement 1, figure 4 - figure supplement 2B, C and D, and for figure 5 - figure supplement 2A, B, C, D, E and F.

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Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Telomere length regulation by Rif1 protein from Hansenula polymorpha" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Raymund J Wellinger as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: David Shore (Reviewer #2).

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife.

Overall, the reviewers were impressed by the quality of the data and appreciated the attention to an important topic that is relevant for genome stability mechanisms in many eukaryotes, including mammals. As such, your interpretation of the data would impy an important deviation from the concepts established for Rif1 function in in budding yeast. However, several aspects of the hypotheses put forward are not enough supported by actual experiments and in the present form, the ideas therefore remain too speculative for publication in eLife. Specifically, reviewers point out that a very important part of the conclusions hinge on negative results of Y2H approaches and an inability to pinpoint a HpRif1 interaction domain on HpRap1. These results are viewed as unconvincing, and this conclusion will need substantial experimental work to be established. Furthermore, the point mutations in the NTE, when analyzed in the FL length protein, appear to retain a significant level of function (see Figure 4F, compare mutants vs rif1 deletion). It therefore remains unclear as to how and what the implications of those a.a. are on HpRfi1 function on telomeres. Finally, in order to broaden the significance and interest for the findings to other organisms and fields, it will be crucial to establish that some of the proposed new interactions and/or recruitment modes be found to be conserved in divergent species (bakers' yeast; mouse; human). As is, this discussed link remains too speculative. The reviewers nevertheless agreed that this aspect of the work too could become part of a highly significant and important contribution, if solidly supported by experimentation. Yet, given that this again implies huge additional efforts, it was convened that this is beyond a revision that could be completed within a reasonable timeframe.

Reviewer #1:

The Dontsova-Malyavko labs have been investigating telomere and telomerase biology in the thermotolerant budding yeast H. polymorpha since many years. While the findings in general parallel those in the classical budding yeast (S. cerevisiae), there already are a few interesting differences between S.c. and H.p. and for some characteristics, H.p. telomere biology appears more like other systems. In this manuscript, they describe their efforts to elucidate the function of the HpRif1 protein. This protein was originally discovered in budding yeast as a factor associating with the telomeric Rap1 protein and acting as a negative regulator for telomere lengthening by telomerase. More recently, a homologue of the protein was described in mammalian cells, including humans, and this protein was shown to be very important for DSB protection and repair choice. Furthermore, the yeast protein also englobes a regulatory function in DNA replication initiation. Hence, Rif1 is a large protein with a number of seemingly disparate functions in the studied systems.

The results of this paper first describe the identification of the HpRif1 and that it is indeed involved in telomere length restriction, as in S.c. The data show that a DBD comprised of two sequence blocks in the N-terminal part can mediate direct telomeric DNA binding in vitro but the consequences of mutating those blocks in the full-length (FL) protein in vivo remains unclear. Several approaches allowed to predict that this N-terminal extension (NTE) structurally appears unique without predictable motifs (unordered domain). Yeast 2-hybrid analyses, ChIPs and co-IP experiments suggest that the HpRif1 protein interacts with two other factors, namely HpKu and HpStn1. Both of these have important functions at telomeres as well as other sites in the genome. There is some evidence that the HpRif1 – HpKu80 interaction occurs via a specific site in the NTE again, but there appear not to be any in vivo consequences of mutating that site in the FL HpRif1 protein, which renders it difficult to rationalise an interaction via this site. On the other hand, in the absence of HpKu, HpRif1 localisation to telomeres appears significantly reduced (4-fold down) and as opposed to the situation in S.c., an absence of HpRif1 appears not to suppress a lack of Ku in terms of telomeric phenotypes. The interaction of HpRif1 with HpStn1 is documented by 2 hybrid and co-IP experiments. Furthermore, the in vivo recruitment of HpStn1 to telomeres appears dependent on HpRif1 (by ChIP). Finally, deletions of the HpStn1 C-terminal domain cause telomere length dysregulation and a loss of HpCdc13 interaction, as in S.c.

Altogether, these results suggest an intricate network of interactions between the HpRif1, HpStn1 and the HpKu proteins. These plus the NTE DNA binding domain would determine recruitment of the proteins to telomeres. In particular, the HpRif1 would be recruited entirely in a HpRap1 independent fashion, which would significantly deviate from other yeast systems. This latter point is not solidly investigated though and needs a much more thorough study. Furthermore, it is said that the HpKu does not interact with its telomerase RNA, yet I could not find any convincing experimental verification of that (RIP or so). Absence of a substructure in a secondary prediction model for the RNA is not sufficient for this point. Most importantly, the results around the HpRif1, HpKu and HpStn1 interactions are the most significant steps forward for our conceptual understanding, but certain details leave alternative explanations possible and those results do need more support for increased robustness of the conclusions.

This reviewer therefore thinks that while there is potential in these results, the core results and most exciting interpretations need to be clarified or more strongly supported by experiments.

Specifically:

1) Telomeric phenotypes in cells that lack HpKu are incompletely documented and not really clear to me. Are the telomeres shorter in the mutants and, if yes, by how much? For the telomere Southern blots, the ref Sohn et al. 1999 should be used as that paper is the one to give the details of telomeric sequence organization in H.p.

2) There is a bit of a disconnect in Figure 4. While the FL Rif1 protein with the 4A and the 8A mutations appears not to bind to telomeres at all anymore (by ChIP, Figure 4D); the telomeric phenotype conferred by these mutations is mild at best (Figure 4F). At any rate, a complete deletion of SpRif1 causes a much more dramatic telomere elongation phenotype. To this reviewer, this suggests that the identified NTE DNA binding domain is only part of the story and the conclusions must be attenuated to reflect that fact.

3) Similar to pt 2, the identified Ku-interaction domain on the NTE appears not important when put in the context of the FL Rif1 (Figure 5E. 5F). Furthermore, the co-IP experiment in Figure 5I shows a Ku80 band in lane 5; but according to my reading, there should not be one. Finally, the Southern blot (Figure 5J) may show an absence of telomere lengthening in the rfi1 ku80 double mutant, but the differences appear small and in the absence of a quantification of telomere lengths, the conclusions drawn are tantalizing, but not convincing.

4) All two hybrid analyses with the FL Rif1 or subfragment constructs were made with the Rif1 protein (or variants) fused to the AD. Does the reverse order 2 hybrid (Rif1 fused to GBD) yield the same results?

5) In strains lacking Rif1, does Ku still bind to telomeres (by ChIP)?

6) In the absence of Ku, Rif1 binding to telomeres appears down, but is this just an effect of the shorter telomeres? Thus, in another short telomere mutant (not Ku), is Rif1 bound at the wt level or reduced too? In S.c., telomere length does correlate with Rif1 binding, which would easily explain the result in Figure 5D.

7) For Figure 6D, it must be mentioned that such C-terminal truncations with a loss of Cdc13 interaction have been shown to cause exactly the same phenotype in S.c. (Petreaca et al.; NCB 2007). Whether or not this has anything to do with a potential Rif1 interaction remains to be determined. From the Southern blot in Figure 6D, it would also appear that the FLAG epitope on Stn1 causes some impairment, given the longer telomeres in that strain.

Reviewer #2:

Malyavko et al. present a biochemical and functional analysis of the telomere regulator Rif1 in a budding yeast species (Hansenula polymorpha; Hp) distantly related to the well-studied Saccharomyces cerevisiae (Sc), where Rif1 was first identified and is best characterized. They present convincing evidence for a DNA-binding function of HpRif1 encoded in a conserved N-terminal domain predicted to be intrinsically disordered, as well as a region in the N-terminus that promotes interaction with the Hp Ku heterodimer. Based upon these findings and a series of negative results related to known functional domains and interacting partners of ScRif1, they propose that Hp and its close relatives have evolved a strikingly different mode of recruitment of Rif1 at telomeres compared to Sc. The novel interactions reported are of interest, but the data so far presented indicate that they make only a minor contribution to Rif1's overall role in telomere length regulation. In this regard, there is concern that the authors place too much weight on a single negative result related to the possible role of Rap1 in the recruitment of Rif1 in Hp, an issue that needs to be more carefully addressed.

As a whole, the experiments presented here are well designed and executed and the data are clearly presented and appear to be of high quality. This is a substantial piece of experimental work that will be of high interest to those in the telomere field. My concerns (detailed below) are primarily related to interpretation. Put briefly, I am worried that the authors have missed a potentially important role for HpRap1 in HpRif1 binding and telomere length regulation simply because they failed to detect a Y2H interaction between the two. This is a negative result that carries little weight. The novel findings reported here, as the authors acknowledge, still provide an incomplete picture of HpRif1 telomere recruitment and function. As such, the present manuscript, though interesting, is still rather preliminary. Specific points and questions are as follows:

1. The conclusion that the HpRif1 NTE is required for telomere recruitment is questionable. The data clearly indicate an important role for the two basic motifs in DNA binding both in vitro and in vivo, but the functional assay of the mutants (the telomere blot in Figure 4F) clearly shows that the mutants retain a significant telomere length regulation function compared to a rif1Δ strain. (Indeed, the mutants display more heterogeneity in the lower telomere-specific (?) bands compared to wild-type but nothing like the essentially uniform and pronounced extension of the deletion strain). In any event, these data suggest that Rif1 is still functioning at telomeres in the K/R cluster mutant strains. The simplest explanation of these observations is that the mutant proteins are still recruited to telomeres, even though the ChIP assay no longer reports this binding. This may be due either to a more transient binding, not capture by ChIP, or to a decrease in crosslinking efficiency provoked by the mutations themselves. In short, the effect of this putative DNA-binding domain is rather minor.

2. The authors have somewhat hastily dismissed the notion that the ScRif1 NTD homology region in HpRif1 (which is relatively conserved) might be involved in DNA binding. As best I can tell, they may have simply failed to express this part of HpRif1 in a soluble or properly folded form that would allow for biochemical analysis. This point needs to be clarified and if possible, resolved experimentally. The authors should also comment on the conservation of amino acid residues shown to be involved in ScRif1 DNA binding by Mattarocci et al. (2017). If present in HpRif, these residues should be mutated and tested in functional assays.

3. Identification and analysis of the novel Ku-binding domain in HpRif1 suffers from several of the problems cited above concerning the DNA-binding domain. In this case, though, only the Y2H analysis indicates a loss of binding for the mutants that were generated, at least in the context of full-length HpRif1. The failure of Rif1NTE1-264 fragments carrying these mutations to bind (as measured by ChIP) does support an in vivo role for the Ku interaction but again highlights a relatively minor functional role, at least concerning telomere length regulation.

4. The authors should test the functional redundancy of DNA- and Ku-binding by generating and characterizing double mutants of the relevant motifs.

5. The authors' conclusion that HpRap1 plays no role in HpRif1 telomere recruitment, if true, is a very significant finding given our present understanding of telomere length regulation. The so-called "protein counting" model posits that telomere length is "read out" by a system that is sensitive to the number of bound Rap1 molecules. If HpRap1 plays no role in recruiting HpRif1, it is unclear how Rif1 would operate to control telomere length. The authors need to at least discuss this issue.

6. Regarding the above (HpRap1's role in telomere length regulation), the authors should discuss HpRap1's homology with ScRap1 and if not already done, make mutations in the C-terminal domain of HpRap1 that might be likely to play a role in telomere length regulation. Surprisingly, I find no discussion of this issue, apart from their claim that a Rap1-binding motif (RBM) cannot be identified in HpRif1. In Sc, the RBM interacts with a groove in the C-terminal domain of ScRap, and the same groove is used by the silencing protein Sir3 for ScRap1 binding. Furthermore, even human TRF2 interacts with human Rap1 through a similar mechanism (see Chen et al. 2011 NSMB). I would thus be surprised if something similar does not occur in Hp, though it might be difficult to uncover by sequence homology searches. This is something that needs to be addressed experimentally.

Reviewer #3:

Rif1 has been implicated in DNA replication and DNA damage repair in mammals and yeast. In Saccharomyces cerevisiae (and other yeast species) it was also shown to negatively regulate telomere length by inhibiting telomerase action. The authors aimed at understanding the interactions and function of Rif1 in a distant budding yeast species, Hansenula polymorpha. The authors show that deleting Rif1 in H. polymorpha indeed causes telomere elongation, confirming the conserved function of Rif1 as a negative regulator of telomerase. However, while S. cerevisiae Rif1 is recruited to telomeres by Rap1, here the authors describe two other types of interactions that contribute to the recruitment of H. polymorpha Rif1 to telomeres: a direct interaction of its unstructured N-terminal domain with the telomeric DNA and another interaction with the Ku heterodimer, also mediated (at least in part) by the N-terminal domain of Rif1. The authors also show that H. polymorpha Rif1 recruits Stn1 to telomeres, and Stn1 negatively regulates telomerase, as shown in other yeasts. Altogether, the authors portray a network of interactions at the telomere, which are different from what has been reported in other yeasts, but they are also important for the regulation of telomerase action at the telomere. Possibly, some of these interactions also take place at the telomeres of S. cerevisiae and other yeasts, and they have not been detected because of redundancy. Interestingly, disrupting these interactions results in effects on telomere length that are not readily explained by a simple recruitment function, suggesting that the roles of these interactions are more complex and/or other unknown interactions are yet to be discovered. The experiments are well designed and conducted with the proper controls, the results are convincing and the manuscript at large is clearly written. The manuscript would be interesting to readers in the telomere field as they portray a more complex network of interactions taking place at the telomere, which is likely to occur also in other species. In my opinion, however, this work still leaves open questions. It could have a stronger impact, especially outside the yeast telomere field, if the mechanism of telomerase regulation was explored in more detail or if the interactions found in H. polymorpha were shown to be conserved also in other species.

1. I believe that the introduction would benefit from mentioning what is known about other telomeric proteins in H. polymorpha, such as the CST complex and its interactions with telomerase, Rif2 homolog (if exists). As well as mentioning the more recent papers about Rif1 from the Greider group.

2. The authors did not find the Rap1 interacting domain within Rif1, but have not excluded that Rif1 interacts with Rap1, perhaps through a different domain. Experiments testing this potential interactions would strengthen the conclusions.

3. Figure 1C: It appears that TER deletion is lethal. Is it indeed the case? Are there no survivors by the alternative recombination-based mechanism?

4. Figure 1B,E: some of the lanes display altered pattern of telomeric restriction fragments. Do the authors consider how are they formed? Is it sub-telomeric recombination, and is it related to Rif1?

5. Figure 4: The Rif1 8A mutant abolished the interaction with the telomeric DNA, and yet it did not cause a significant telomere elongation. This is puzzling. Is this interaction dispensable for telomere length regulation? Can Rif1 inhibit telomerase if only transiently localized to telomeres? Or even without binding to telomeres?

6. Lines 258-260: Could the reduced Rif1 association with telomeres of the Ku deletion strains be the result of the shorter telomeres, rather than the loss of interaction with Ku? The explanation given in lines 265-269 is not clear.

7. Figure 6F: The ChIP is not clearly described. Is Stn1-HA immunoprecipitated here? Please indicate the target of the pull-down.

8. Figure 6D: Could be interesting to test double mutants such as Stn1delta C with ku or rif1.

9. Can the authors draw a schematic model to illustrate the identified interactions and how they might regulate telomerase?

10. In several places 'the' or 'a' are missing (e.g., line 48 – THE C-terminal portion, line 212, line 300…).

11. Line 51: Does Rif1 inhibit or mediate telomere silencing?

12. Figure 1A, Supplementary file 1, give reference to T-Coffee.

13. Line 155: 'is' is missing.

14. The figure legends could benefit from a better description of the figures, for example, In Figure 2A, what is the difference between the lines MetaDisorder, MetaDisorderMD and MetaDisorderMD2?

15. Line 179: May be remove 'and' and start a new sentence?

16. Line 190 and Figure 3E: When titrating a C4 competitor in a reaction with a G4 probe, first it eliminates the single stranded G4 by generating a double stranded GC4 probe. Then the excess C4 competes with the GC4 probe on the binding of the protein. Thus the results are consistent with a preference for the single stranded G4 as seen in 3D.

17. Line 371: Not in all yeasts the role of Ku is conserved (e.g. K. lactis). I'm not convinced that the binding of Ku to telomerase is conserved and we can assume that it occurs also in H. polymorpha.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

The Dontsova-Malyavko labs have been investigating telomere and telomerase biology in the thermotolerant budding yeast H. polymorpha since many years. While the findings in general parallel those in the classical budding yeast (S. cerevisiae), there already are a few interesting differences between S.c. and H.p. and for some characteristics, H.p. telomere biology appears more like other systems. In this manuscript, they describe their efforts to elucidate the function of the HpRif1 protein. This protein was originally discovered in budding yeast as a factor associating with the telomeric Rap1 protein and acting as a negative regulator for telomere lengthening by telomerase. More recently, a homologue of the protein was described in mammalian cells, including humans, and this protein was shown to be very important for DSB protection and repair choice. Furthermore, the yeast protein also englobes a regulatory function in DNA replication initiation. Hence, Rif1 is a large protein with a number of seemingly disparate functions in the studied systems.

The results of this paper first describe the identification of the HpRif1 and that it is indeed involved in telomere length restriction, as in S.c. The data show that a DBD comprised of two sequence blocks in the N-terminal part can mediate direct telomeric DNA binding in vitro but the consequences of mutating those blocks in the full-length (FL) protein in vivo remains unclear. Several approaches allowed to predict that this N-terminal extension (NTE) structurally appears unique without predictable motifs (unordered domain). Yeast 2-hybrid analyses, ChIPs and co-IP experiments suggest that the HpRif1 protein interacts with two other factors, namely HpKu and HpStn1. Both of these have important functions at telomeres as well as other sites in the genome. There is some evidence that the HpRif1 – HpKu80 interaction occurs via a specific site in the NTE again, but there appear not to be any in vivo consequences of mutating that site in the FL HpRif1 protein, which renders it difficult to rationalise an interaction via this site. On the other hand, in the absence of HpKu, HpRif1 localisation to telomeres appears significantly reduced (4-fold down) and as opposed to the situation in S.c., an absence of HpRif1 appears not to suppress a lack of Ku in terms of telomeric phenotypes. The interaction of HpRif1 with HpStn1 is documented by 2 hybrid and co-IP experiments. Furthermore, the in vivo recruitment of HpStn1 to telomeres appears dependent on HpRif1 (by ChIP). Finally, deletions of the HpStn1 C-terminal domain cause telomere length dysregulation and a loss of HpCdc13 interaction, as in S.c.

Altogether, these results suggest an intricate network of interactions between the HpRif1, HpStn1 and the HpKu proteins. These plus the NTE DNA binding domain would determine recruitment of the proteins to telomeres. In particular, the HpRif1 would be recruited entirely in a HpRap1 independent fashion, which would significantly deviate from other yeast systems. This latter point is not solidly investigated though and needs a much more thorough study. Furthermore, it is said that the HpKu does not interact with its telomerase RNA, yet I could not find any convincing experimental verification of that (RIP or so). Absence of a substructure in a secondary prediction model for the RNA is not sufficient for this point. Most importantly, the results around the HpRif1, HpKu and HpStn1 interactions are the most significant steps forward for our conceptual understanding, but certain details leave alternative explanations possible and those results do need more support for increased robustness of the conclusions.

This reviewer therefore thinks that while there is potential in these results, the core results and most exciting interpretations need to be clarified or more strongly supported by experiments.

We thank the reviewer for the appreciation of our work and for the careful reading of the manuscript. To obtain new data on the potential HpRif1-HpRap1 relationship, we constructed the intTEL18 strain of H. polymorpha by inserting 18 telomeric repeats upstream of the WSC3 gene on the chromosome I (Figure 1 —figure supplement 1A). As expected, Rap1B binds telomeric repeats with similar efficiency at the internal locus or at the chromosome end (only ~2-fold difference between WSC3 and TEL signals for the intTEL18 strain, Figure 1 —figure supplement 1B). On the contrary, Rif1 association with the internal telomeric repeats is weak and close to the background levels (ChIP WSC3 signal is ~8-fold lower than TEL, Figure 1 —figure supplement 1C). We believe that this experiment together with our previous results (the phenotypic discrepancy between Rap1 and Rif1 mutants, the absence of the Y2H interaction) allows us to conclude that “it is highly unlikely that Rif1 binding to Rap1 is the major mechanism of Rif1 telomeric recruitment in H. polymorpha in a manner it is described for its S. cerevisiae homologues.”

Specifically:

1) Telomeric phenotypes in cells that lack HpKu are incompletely documented and not really clear to me. Are the telomeres shorter in the mutants and, if yes, by how much? For the telomere Southern blots, the ref Sohn et al. 1999 should be used as that paper is the one to give the details of telomeric sequence organization in H.p.

To provide a quantitative estimate of the telomere length changes observed in Ku mutants, we calculated the lengths of the brightest TRFs on the Southern blots and report them in Figures and in the text. Since it is not clear what is the exact length of the subtelomere region, we used the value ~160 bp (~20 telomeric repeats) as an estimate of the mean WT telomere length (18-23 telomeric repeats reported in Sohn et al. 1999). We found that Ku loss leads to telomere shortening (~25-50% reduction in telomere length, Figure 5 —figure supplement 2B, Figure 5J).

2) There is a bit of a disconnect in Figure 4. While the FL Rif1 protein with the 4A and the 8A mutations appears not to bind to telomeres at all anymore (by ChIP, Figure 4D); the telomeric phenotype conferred by these mutations is mild at best (Figure 4F). At any rate, a complete deletion of SpRif1 causes a much more dramatic telomere elongation phenotype. To this reviewer, this suggests that the identified NTE DNA binding domain is only part of the story and the conclusions must be attenuated to reflect that fact.

We have changed our conclusions accordingly (see for example, page 22 lines 421-423; page 25 lines 485-486).

3) Similar to pt 2, the identified Ku-interaction domain on the NTE appears not important when put in the context of the FL Rif1 (Figure 5E. 5F).

We agree and we state that “The Rif1_KBM may thus serve only as an auxiliary module” (please see pages 22-23 lines 430-434 of the Discussion).

Furthermore, the co-IP experiment in Figure 5I shows a Ku80 band in lane 5; but according to my reading, there should not be one.

Indeed, in the old version of the Figure 5I (Author response image 1) the background level of Ku80-HA associated with the anti-Flag resin is higher for the ∆ku70 strain (lanes 5 and 6) compared to the KU70 strain (lane 7). Our intention was to demonstrate that Ku80-HA is no longer associated with Rif1-Flag (relatively to the “no Flag tag” control, since lanes 5 and 6 are the same). We do not know the reason for this higher non-specific binding; however, we understand that it may be confusing and removed the experiment with the ∆ku70 strain. We note that the pull down from the KU70 strain does support our conclusion that Rif1 interacts with Ku80 in H. polymorpha cells (the Ku80 signal in lane 8 is much higher than in lane 7, and lanes 5,6) and we show it in the new version of the manuscript (new Figure 5D).

Author response image 1

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Finally, the Southern blot (Figure 5J) may show an absence of telomere lengthening in the rfi1 ku80 double mutant, but the differences appear small and in the absence of a quantification of telomere lengths, the conclusions drawn are tantalizing, but not convincing.

We quantified the lengths of the brightest TRFs on the Southern blot in Figure 5J and found that the length increase upon the Rif1 loss in ∆ku80 is ~10 bp (399±1 bp for RIF1∆ku80 vs 410±11 bp for ∆rif1∆ku80). This constitutes ~13% (10/80) of the ∆ku80 telomere length (KU80 TRF is ~480 bp, ∆ku80 TRF is ~400 bp; WT telomere tract is ~160 bp, therefore in ∆ku80 telomere tract is ~80 bp). We did not quantify the TRFs in the rif1 mutants, because of the high heterogeneity and it is not clear which TRF corresponds to the brightest in the WT strain. However, from the Southern blot in Figure 5J it can be seen that the mean length of the brightest TRFs is close to 750 bp. Therefore, the length increase upon the Rif1 loss in the KU80 strain should be ~270 bp, which is more than 100% of the WT telomere length. Thus, we believe that the following statement is justified: “RIF1 deletion in a ∆ku80 background does not lead to strong telomere elongation compared to the parental ∆ku80 strain, suggesting that inhibition of telomerase by Rif1 is attenuated in the absence of Ku (Figure 5J).” (lines 323-325).

4) All two hybrid analyses with the FL Rif1 or subfragment constructs were made with the Rif1 protein (or variants) fused to the AD. Does the reverse order 2 hybrid (Rif1 fused to GBD) yield the same results?

We chose not to perform the reverse order 2 hybrid experiments because we believe that our main conclusions based on the Y2H assay are corroborated by more informative complementary approaches (Co-IPs, ChIPs and Southern blots).

5) In strains lacking Rif1, does Ku still bind to telomeres (by ChIP)?

Yes, Ku80-HA binds telomeres in the ∆rif1 strain, with only ~2-fold reduced efficiency compared to the RIF1 strain (Figure 5 —figure supplement 2C).

6) In the absence of Ku, Rif1 binding to telomeres appears down, but is this just an effect of the shorter telomeres? Thus, in another short telomere mutant (not Ku), is Rif1 bound at the wt level or reduced too? In S.c., telomere length does correlate with Rif1 binding, which would easily explain the result in Figure 5D.

To verify that Rif1 telomere localization defect upon Ku loss is not simply a consequence of the telomere shortening, we measured Rif1 telomere binding in a strain lacking telomerase RNA (new Figure 5 —figure supplement 2D, E). TER knock-out resulted in ~40% reduction in telomere length, whereas Rif1-HA telomere occupancy diminished only ~2-fold (new Figure 5 —figure supplement 2D, E); contrasting to ~4-fold Rif1 ChIP signal drop in case of Ku mutants (new Figure 5E).

7) For Figure 6D, it must be mentioned that such C-terminal truncations with a loss of Cdc13 interaction have been shown to cause exactly the same phenotype in S.c. (Petreaca et al.; NCB 2007). Whether or not this has anything to do with a potential Rif1 interaction remains to be determined. From the Southern blot in Figure 6D, it would also appear that the FLAG epitope on Stn1 causes some impairment, given the longer telomeres in that strain.

We included this and two other references with descriptions that Stn1 C-terminal truncations have been shown to cause exactly the same phenotype in S. cerevisiae. (lines 362-363). We agree that FLAG epitope on Stn1 causes some functional impairment, however we could not see how this fact changes any of our conclusions.

Reviewer #2:

Malyavko et al. present a biochemical and functional analysis of the telomere regulator Rif1 in a budding yeast species (Hansenula polymorpha; Hp) distantly related to the well-studied Saccharomyces cerevisiae (Sc), where Rif1 was first identified and is best characterized. They present convincing evidence for a DNA-binding function of HpRif1 encoded in a conserved N-terminal domain predicted to be intrinsically disordered, as well as a region in the N-terminus that promotes interaction with the Hp Ku heterodimer. Based upon these findings and a series of negative results related to known functional domains and interacting partners of ScRif1, they propose that Hp and its close relatives have evolved a strikingly different mode of recruitment of Rif1 at telomeres compared to Sc. The novel interactions reported are of interest, but the data so far presented indicate that they make only a minor contribution to Rif1's overall role in telomere length regulation. In this regard, there is concern that the authors place too much weight on a single negative result related to the possible role of Rap1 in the recruitment of Rif1 in Hp, an issue that needs to be more carefully addressed.

As a whole, the experiments presented here are well designed and executed and the data are clearly presented and appear to be of high quality. This is a substantial piece of experimental work that will be of high interest to those in the telomere field. My concerns (detailed below) are primarily related to interpretation. Put briefly, I am worried that the authors have missed a potentially important role for HpRap1 in HpRif1 binding and telomere length regulation simply because they failed to detect a Y2H interaction between the two. This is a negative result that carries little weight. The novel findings reported here, as the authors acknowledge, still provide an incomplete picture of HpRif1 telomere recruitment and function. As such, the present manuscript, though interesting, is still rather preliminary. Specific points and questions are as follows:

We thank the reviewer for the time and effort spent on reading of our manuscript and for the valuable comments and critique. We wanted to stress that our conclusion on the absence of the major role for HpRap1 in HpRif1 recruitment stems mainly not from the negative results of the Y2H experiments but from the different phenotypes of the strains with HpRap1 C-truncations and RIF1 knock-out (please see the detailed response to the point #6). We understand that in our previous version of the study this was not discussed properly and tried to extend the description of our previous results with HpRap1 C-truncations (page 21 lines 381-393). In addition, we included the results with the newly created intTEL18 strain containing an array of telomeric repeats at the internal WSC3 locus (Figure 1 —figure supplement 1A). As expected, HpRap1B binds telomeric repeats with similar efficiency at the internal locus or at the chromosome end (only ~2-fold difference between WSC3 and TEL signals for the intTEL18 strain, Figure 1 —figure supplement 1B). On the contrary, HpRif1 association with the internal telomeric repeats is weak and close to the background levels (ChIP WSC3 signal is ~8-fold lower than TEL, Figure 1 —figure supplement 1C). We strongly believe that these results (together with the Y2H assay) make enough evidence to conclude that “it is highly unlikely that Rif1 binding to Rap1 is the major mechanism of Rif1 telomeric recruitment in H. polymorpha in a manner it is described for its S. cerevisiae homologues.”

1. The conclusion that the HpRif1 NTE is required for telomere recruitment is questionable. The data clearly indicate an important role for the two basic motifs in DNA binding both in vitro and in vivo, but the functional assay of the mutants (the telomere blot in Figure 4F) clearly shows that the mutants retain a significant telomere length regulation function compared to a rif1Δ strain. (Indeed, the mutants display more heterogeneity in the lower telomere-specific (?) bands compared to wild-type but nothing like the essentially uniform and pronounced extension of the deletion strain). In any event, these data suggest that Rif1 is still functioning at telomeres in the K/R cluster mutant strains. The simplest explanation of these observations is that the mutant proteins are still recruited to telomeres, even though the ChIP assay no longer reports this binding. This may be due either to a more transient binding, not capture by ChIP, or to a decrease in crosslinking efficiency provoked by the mutations themselves. In short, the effect of this putative DNA-binding domain is rather minor.

We agree with the reviewer. HpRif1 mutants with K/R cluster mutations (or even lacking the entire NTE) retain large portion of Rif1’s functional potential. We discuss this in the manuscript (see, for example, page 22 lines 421-427). Indeed, the contribution of the NTE region to telomere length regulation is relatively small, although it does appear to be important for “full” inhibition of telomerase by HpRif1. We attenuated our conclusions throughout the text to reflect that fact. We now state: “We demonstrated that the N-terminal extension (NTE) of Rif1 is an accessory domain partially promoting Rif1 telomeric localization in H. polymorpha.” (page 25, lines 485-486).

2. The authors have somewhat hastily dismissed the notion that the ScRif1 NTD homology region in HpRif1 (which is relatively conserved) might be involved in DNA binding. As best I can tell, they may have simply failed to express this part of HpRif1 in a soluble or properly folded form that would allow for biochemical analysis. This point needs to be clarified and if possible, resolved experimentally. The authors should also comment on the conservation of amino acid residues shown to be involved in ScRif1 DNA binding by Mattarocci et al. (2017). If present in HpRif, these residues should be mutated and tested in functional assays.

We agree with the reviewer and discuss the limitations of our approach and the potential conservation of the DNA binding by HpRif1 NTD (page 13 lines 262-265, page 22 lines 413415; page 22 lines 426-427; page 25 lines 490-493). We tried to find whether DNA-contacting residues from ScRif1 NTD are conserved in HpRif1 and present the results in this version of the manuscript. According to the published ScRif1_NTD/DNA structure, 19 charged residues have the potential to contact the DNA backbone (Mattarocci et al., 2017). Of these 19, only four are conserved in H. polymorpha Rif1 (new Figure 4 —figure supplement 2A, Supplementary file 1).

Double mutant HpRif1_K658E/K666E is expressed at considerably lower levels than WT HpRif1, indicating that residues K658 and K666 are important for protein stability (new Figure 4 —figure supplement 2B). This would complicate interpretation of the functional assays and these residues were excluded from further analysis. Substitution of the other two conserved amino acids (K504 and R539) for glutamines does not lead to detectable changes in Rif1 telomere occupancy or telomere length (Figure 4 —figure supplement 2C, D). These results suggest that the HpRif1 might lack the DNA-binding activity described for ScRif1. However, since the telomere-related proteins (especially in yeast) are highly evolvable we still do not dismiss the possibility of DNA binding by HpRif1 NTD (page 22, lines 418-420).

3. Identification and analysis of the novel Ku-binding domain in HpRif1 suffers from several of the problems cited above concerning the DNA-binding domain. In this case, though, only the Y2H analysis indicates a loss of binding for the mutants that were generated, at least in the context of full-length HpRif1. The failure of Rif1NTE1-264 fragments carrying these mutations to bind (as measured by ChIP) does support an in vivo role for the Ku interaction but again highlights a relatively minor functional role, at least concerning telomere length regulation.

Indeed, our experiments indicate that other residues within HpRif1 (its NTE-less/265-1521 portion) make crucial contacts with Ku in vivo. The inability to detect the interaction between Ku80 and Rif1^264-1521 in the Y2H experiment might even suggest that this interaction requires Ku as a whole, or protein modifications on Ku/Rif1 or other Ku/Rif1-bound proteins. Unfortunately, we could not provide more information on this issue. We would like to note, however, that we discuss that in the manuscript (page 16 lines 307-308; page 23, lines 430-436).

4. The authors should test the functional redundancy of DNA- and Ku-binding by generating and characterizing double mutants of the relevant motifs.

We thank the reviewer for an interesting suggestion, however we believe that the outcome of the experiments would be similar to the Rif1^∆NTE strain (since both DNA- and Ku-binding motifs are located within the NTE region). Rif1^∆NTE mutant is “not present” at telomeres according to ChIP and has moderately elongated telomeres (Figure 1D, E). The DNA-binding mutant Rif1^8A has the identical phenotype (Figure 4D, F).

5. The authors' conclusion that HpRap1 plays no role in HpRif1 telomere recruitment, if true, is a very significant finding given our present understanding of telomere length regulation. The so-called "protein counting" model posits that telomere length is "read out" by a system that is sensitive to the number of bound Rap1 molecules. If HpRap1 plays no role in recruiting HpRif1, it is unclear how Rif1 would operate to control telomere length. The authors need to at least discuss this issue.

Following the suggestion by the reviewer, we introduced the paragraph on this matter in the Discussion section (page 23 lines 437-448). At this point it is not obvious how mechanistically Rif1 recruitment could be linked to telomere length. HpRif1 appears to have only limited ability to bind double-stranded telomeric DNA in vivo (Figure 1 —figure supplement 1). We also would like to note that ScRif1^NTD interacts with DNA in a Rap1-independent (and telomere length independent) manner. This suggests that Rif1 homologues operate (at least partially) in a telomere length-independent way.

6. Regarding the above (HpRap1's role in telomere length regulation), the authors should discuss HpRap1's homology with ScRap1 and if not already done, make mutations in the C-terminal domain of HpRap1 that might be likely to play a role in telomere length regulation. Surprisingly, I find no discussion of this issue, apart from their claim that a Rap1-binding motif (RBM) cannot be identified in HpRif1. In Sc, the RBM interacts with a groove in the C-terminal domain of ScRap, and the same groove is used by the silencing protein Sir3 for ScRap1 binding. Furthermore, even human TRF2 interacts with human Rap1 through a similar mechanism (see Chen et al. 2011 NSMB). I would thus be surprised if something similar does not occur in Hp, though it might be difficult to uncover by sequence homology searches. This is something that needs to be addressed experimentally.

The functional characterization of HpRap1 and the homology with ScRap1 was the focus of our earlier study (Malyavko et. al 2019, Sci Rep), and the experiments therein are indeed useful for rationalizing potential Rif1-Rap1 interaction. We discovered that H. polymorpha has two Rap1 homologues and deletion of the entire RCT from one of them (Rap1A) has no effect on the telomere length, consistent with its lack of preference for the telomeric DNA sequence. RCT deletion from Rap1B (the major dsTBP in H. polymorpha) was found to be impossible (presumably lethal). Removal of the 50 a.a. from its C-terminus was found tolerated by cells but strongly (more than 10-fold) reduced the expression level of Rap1B, creating thus effectively the strain with a Rap1B knock-down. In this (B^1-526) strain telomeres were indeed long and extremely heterogeneous, but the telomere overelongation was found to be telomerase-independent. In contrast, telomeres in the ∆rif1 strain cannot elongate upon telomerase loss (Figure 1B, C). These phenotypic differences actually formed our initial premise that Rif1 and Rap1 functions may be (largely) independent in H. polymorpha.

Reviewer #3:

Rif1 has been implicated in DNA replication and DNA damage repair in mammals and yeast. In Saccharomyces cerevisiae (and other yeast species) it was also shown to negatively regulate telomere length by inhibiting telomerase action. The authors aimed at understanding the interactions and function of Rif1 in a distant budding yeast species, Hansenula polymorpha. The authors show that deleting Rif1 in H. polymorpha indeed causes telomere elongation, confirming the conserved function of Rif1 as a negative regulator of telomerase. However, while S. cerevisiae Rif1 is recruited to telomeres by Rap1, here the authors describe two other types of interactions that contribute to the recruitment of H. polymorpha Rif1 to telomeres: a direct interaction of its unstructured N-terminal domain with the telomeric DNA and another interaction with the Ku heterodimer, also mediated (at least in part) by the N-terminal domain of Rif1. The authors also show that H. polymorpha Rif1 recruits Stn1 to telomeres, and Stn1 negatively regulates telomerase, as shown in other yeasts. Altogether, the authors portray a network of interactions at the telomere, which are different from what has been reported in other yeasts, but they are also important for the regulation of telomerase action at the telomere. Possibly, some of these interactions also take place at the telomeres of S. cerevisiae and other yeasts, and they have not been detected because of redundancy. Interestingly, disrupting these interactions results in effects on telomere length that are not readily explained by a simple recruitment function, suggesting that the roles of these interactions are more complex and/or other unknown interactions are yet to be discovered. The experiments are well designed and conducted with the proper controls, the results are convincing and the manuscript at large is clearly written. The manuscript would be interesting to readers in the telomere field as they portray a more complex network of interactions taking place at the telomere, which is likely to occur also in other species. In my opinion, however, this work still leaves open questions. It could have a stronger impact, especially outside the yeast telomere field, if the mechanism of telomerase regulation was explored in more detail or if the interactions found in H. polymorpha were shown to be conserved also in other species.

We are grateful to the reviewer for the time and effort in evaluating our manuscript and for the valuable comments. Following your and other reviewers’ suggestions we explored the interactions between H. polymorpha telomeric proteins in more detail.

1. I believe that the introduction would benefit from mentioning what is known about other telomeric proteins in H. polymorpha, such as the CST complex and its interactions with telomerase, Rif2 homolog (if exists). As well as mentioning the more recent papers about Rif1 from the Greider group.

Thank you for the suggestion. We added a paragraph describing the telomeric proteins in H. polymorpha in the Introduction section (page 4 lines 88-97) and included the description of the results from the works Shubin and Greider, 2020; Shubin et al., 2021 (page 3 lines 55-56 and line 76)

2. The authors did not find the Rap1 interacting domain within Rif1, but have not excluded that Rif1 interacts with Rap1, perhaps through a different domain. Experiments testing this potential interactions would strengthen the conclusions.

We added new data with the strain intTEL18 containing the array of 18 telomeric repeats at the internal WSC3 locus. We found that HpRap1B (the major dsTBP in H. polymorpha) efficiently binds the internal telomeric DNA. At the same time HpRif1 binding at the internal telomeric array is strongly reduced compared to the native chromosomal end (Figure 1 —figure supplement 1B). We also note that removal of the 50 a.a. from its C-terminus was found tolerated by cells but strongly (more than 10-fold) reduced the expression level of Rap1B, creating thus effectively the strain with Rap1B knock-down (Malyavko et. al 2019, Sci Rep). In this (B^1-526) strain telomeres were indeed long and extremely heterogeneous, but the telomere overelongation was found to be telomerase-independent. In contrast, telomeres in the ∆rif1 strain cannot elongate upon telomerase loss (Figure 1B, C). Both of these experiments suggest that Rif1 and Rap1 functions may be (largely) independent in H. polymorpha, and even if they interact through any of their domains such an interaction would play only a limited role in Rif1 recruitment.

3. Figure 1C: It appears that TER deletion is lethal. Is it indeed the case? Are there no survivors by the alternative recombination-based mechanism?

No, TER deletion is not lethal, but leads to ever shorter phenotype as in other yeast species, although ∆ter in H. polymorpha does senesce faster due to shorter telomeres (~160 bp WT length). When the ∆ter culture is propagated in liquid medium the survivors will eventually emerge (Smekalova et al., 2013). In the Figure 1C it appears lethal because we re-streaked ∆ter colonies on YPD plates (as described in Materials and methods), and it turned out that in the colonies we re-streaked no survivor cells appeared.

4. Figure 1B,E: some of the lanes display altered pattern of telomeric restriction fragments. Do the authors consider how are they formed? Is it sub-telomeric recombination, and is it related to Rif1?

This is an interesting question the answer to which we do not have yet. The altered pattern of TRFs can result both from the sub-telomeric recombination and telomere length change. It is important to note that telomeres can no longer elongate after TER deletion in the ∆rif1 strain, therefore the TRF length changes must be primarily due to telomerase activity.

5. Figure 4: The Rif1 8A mutant abolished the interaction with the telomeric DNA, and yet it did not cause a significant telomere elongation. This is puzzling. Is this interaction dispensable for telomere length regulation? Can Rif1 inhibit telomerase if only transiently localized to telomeres? Or even without binding to telomeres?

This is indeed puzzling. We do not think (neither do we state that in the text) that 8A mutation actually completely abolishes interaction with telomeres, we think it is just reduced to the levels we can’t detect by our ChIP assay. Perhaps, Rif1 8A mutant binds only at a specific cell cycle stage or its binding is transient (as we discuss that on page 22 lines 421-425). It also should be mentioned that in S. cerevisiae the Rif1^RBM mutant lacking the ability to contact Rap1 is almost completely lost from telomeres as judged by ChIP, yet retains a significant portion of its telomerase inhibitory potential (Mattarocci et al., 2017; Shi et al., 2013).

6. Lines 258-260: Could the reduced Rif1 association with telomeres of the Ku deletion strains be the result of the shorter telomeres, rather than the loss of interaction with Ku? The explanation given in lines 265-269 is not clear.

To clarify this issue, we performed the ChIP assay with the Rif1-HA∆ter strain (new Figure 5 —figure supplement 2D, E). TER knock-out resulted in ~40% reduction in telomere length, whereas Rif1-HA telomere occupancy diminished only ~2-fold (new Figure 5 —figure supplement 2D, E); contrasting to ~4-fold Rif1 ChIP signal drop in case of Ku mutants (new Figure 5E) and comparable telomere length reduction (~25-50% reduction in telomere length, Figure 5 —figure supplement 2B, Figure 5J). Thus, shorter telomeres cannot fully explain the reduction of Rif1 in the Ku deletion strains

7. Figure 6F: The ChIP is not clearly described. Is Stn1-HA immunoprecipitated here? Please indicate the target of the pull-down.

Yes, Stn1-HA is the target of IP in this experiment. We added this to the figure legend.

8. Figure 6D: Could be interesting to test double mutants such as Stn1delta C with ku or rif1.

We agree with the reviewer that these experiments could be interesting. We however chose not to perform them at this point, because we think that the results will be difficult to interpret and it is not clear how these could change the conclusions of this study. The functions of Ku, Rif1 and Stn1 only partially overlap. Stn1 has Rif1-independent functions (Stn1deltaC telomere phenotype is “stronger” than ∆rif1, Figures 1E and 6D) and can associate with telomeres with Cdc13 protein (which has strong affinity towards the telomeric G strand). We observe only limited role of Ku in Stn1 telomere recruitment (Figure 6F). Rif1 can associate with telomeres independently of Stn1 via Ku.

9. Can the authors draw a schematic model to illustrate the identified interactions and how they might regulate telomerase?

We introduced a schematic representation of the putative protein-protein interactions at H. polymorpha telomeres (Figure 6 —figure supplement 2).

10. In several places 'the' or 'a' are missing (e.g., line 48 – THE C-terminal portion, line 212, line 300…).

Thank you for pointing this out, we corrected these mistakes.

11. Line 51: Does Rif1 inhibit or mediate telomere silencing?

ScRif1 inhibit telomere silencing by competition with Sir3 for Rap1 RCT binding (as demonstrated in Shi et al., 2013).

12. Figure 1A, Supplementary file 1, give reference to T-Coffee.

We added reference to T-Coffee.

13. Line 155: 'is' is missing.

Thank you for pointing this out, we corrected the mistake.

14. The figure legends could benefit from a better description of the figures, for example, In Figure 2A, what is the difference between the lines MetaDisorder, MetaDisorderMD and MetaDisorderMD2?

The MetaDisorder, MetaDisorderMD and MetaDisorderMD2 are three different algorithms (each is a combination of several algorithms) for disorder prediction. We added this explanatory note to the Figure 2A along with the reference with the detailed description.

15. Line 179: May be remove 'and' and start a new sentence?

We removed “and” and started a new sentence (new line 208).

16. Line 190 and Figure 3E: When titrating a C4 competitor in a reaction with a G4 probe, first it eliminates the single stranded G4 by generating a double stranded GC4 probe. Then the excess C4 competes with the GC4 probe on the binding of the protein. Thus the results are consistent with a preference for the single stranded G4 as seen in 3D.

Thank you for pointing this out, we added this interpretation in the Figure 3E legend. This however does not change the conclusion that “HpRif1^NTE-DNA interaction is weak and lacks clearly defined specificity”, since it poorly differentiates between Tel4(G) and Tel4(GC) (and the non-telomeric dsrnd1 oligo; Figure 3D, E).

17. Line 371: Not in all yeasts the role of Ku is conserved (e.g. K. lactis). I'm not convinced that the binding of Ku to telomerase is conserved and we can assume that it occurs also in H. polymorpha.

We apologize for this mistake. The paragraph is re-written now (lines 449-462). We also added the new result showing that HpKu80 interact with HpTERT in the Y2H assay (Figure 5 —figure supplement 2G). This result may help to explain the telomere shortening in the HpKu mutants and is in agreement with the proposition that transient telomerase binding to telomeres could be mediated via Ku heterodimer.

Author details

1. Alexander N Malyavko

   Faculty of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing - original draft

   For correspondence

   malyavkoan@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5064-2704

2. Olga A Petrova

   Faculty of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation

   Contribution

   Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

3. Maria I Zvereva

   Faculty of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation

   Contribution

   Funding acquisition, Investigation, Project administration, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

4. Vladimir I Polshakov

   Center for Magnetic Tomography and Spectroscopy, Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russian Federation

   Contribution

   Funding acquisition, Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3216-5737

5. Olga A Dontsova

   1. Faculty of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation
   2. Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russian Federation
   3. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russian Federation

   Contribution

   Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

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