We have demonstrated that multiple biological processes influence the ability of telomeres to form a higher-order fold-back structure. High-throughput screening coupled to stringent bioinformatic analysis, has revealed that class I (Rpd3) and class II (Hda1) KDAC activities were among the most significantly enriched biological processes required to promote the formation/maintenance of a telomere loop. In addition, the Rap1 binding proteins, Rif1 and Rif2, which localize directly to telomeres, were implicated in telomere fold-back establishment. Through genetic epistasis analysis, we found that rif2Δ
and mutants of the Rpd3L complex did not have additive structural defects at telomeres, suggesting that they may function together in a single pathway. This genetic interaction was confirmed by demonstrating that Rpd3L was no longer able to localize to telomeres in rif2Δ
cells. Furthermore, we have shown that the telomeres in mutants with looping defects are more susceptible to uncapping, nucleolytic degradation, telomere loss and promote accelerated rates of cellular senescence in the absence of telomere maintenance. A recent study has reported that the Rpd3 complex is required to prevent chromosome end fusions in Drosophila melanogaster
. Together, these data suggest that the regulation of such telomere fold-back structures may be conserved, and furthermore indicate that multiple cellular processes, apart from those that directly impinge on DNA metabolism, may have effects on telomere structure/function.
Although we have focused on the telomere dysfunction phenotypes associated rpd3
mutants, it is of interest that many of the other mutants recovered in our telomere structure screen have been previously identified to have negative synthetic interactions with cdc13-1
(rrd1Δ, swr1Δ, arp6Δ, spe1Δ, spe3Δ, oca1Δ, oca2Δ, oca5Δ, elp4Δ, pep8Δ, rif1Δ, yme1Δ, htd2Δ, ski3Δ, rim101Δ, ede1Δ
as well as an increased rate of replicative senescence (hda1Δ, sin3Δ, sec28Δ, med1Δ, asf1Δ, rif1Δ, rif2Δ, arp6Δ, elp4Δ
(see for a complete overview of overlaps between our screen and other selected telomere function screens). Moreover, the Sir2/3/4 complex, which is required to form a telomere fold-back structure 
also prevents premature senescence 
. This overlap between our screen and previously published data suggests that telomere structure may make significant contributions towards preserving telomere integrity when telomere function is compromised (). Consistently, sin3Δ
single mutants do not exhibit increased ssDNA accumulation at telomeres nor do they have any changes in telomere length in comparison to isogenic wild type strains. This would suggest that in the absence of a telomere fold-back, the CST complex and other capping factors are sufficient to maintain a protected state (). However, when CST function is compromised (e.g. cdc13-1
) in combination with an inability to fold-back, resection becomes accelerated (, ). In terms of telomere-induced cellular senescence in the absence of telomere maintenance, the increased resection in non-looped mutants would also lead to increased telomere shortening (). It will be of interest to perform an extensive genetic epistasis of all mutants isolated in the loop screen in order to determine if the negative interactions with cdc13-1
are epistatic (i.e. due to a fold-back defect).
The fold-back may contribute to telomere protection.
There was also a large overlap between mutants found in our screen and mutants that have been implicated in both the positive and negative regulation of telomere length (vps28Δ, trk1Δ, ctk1Δ, mrt4Δ, rif1Δ, sur4Δ, nut1Δ, siw14Δ, leo1Δ, hit1Δ, pcp1Δ, pdx3Δ
. This would suggest that either telomere looping has a direct effect on telomere length regulation, or conversely, may indicate that telomere length changes impinge on the ability to form a fold-back. Our results suggest that telomere looping does not affect telomere length homeostasis directly, as many of the mutants recovered in our screen, even those with “strong” looping defects, have wild type telomere length. On the other hand, telomere length changes could indeed have a drastic effect on telomere structure in terms of the chromatin alterations that occur with respect to length changes. Telomere shortening, for example, results in de-silencing in the subtelomeric region 
due to the decreased capacity of shortened telomeres to recruit the Sir2/3/4 histone deacetylase complex, which is required for loop formation in yeast 
. Long telomeres, in contrast, promote a hyper-silenced state in the subtelomere 
, much like what occurs in rif1Δ
mutants, where, in the case of the latter mutant, Rpd3L fails to localize to telomeres. Indeed, Rpd3 mutants (L and S) are hypersilenced in the subtelomeric zone. One possibility would be that long telomeres (as seen in rif2Δ
) mutants fail to properly localize Rpd3L, which leads to a subsequent fold-back defect. Although our screen implicates proper length regulation as a key regulator of telomere looping, they remain correlative and require further investigation in order to draw concrete conclusions.
Whereas both the Rpd3S and Hda1 complexes were additive with rif2Δ
mutants in terms of a looping defect, the Rpd3L complex was epistatic. This relationship was confirmed mechanistically as we noticed that Rpd3L is not able to properly localize to telomeres in rif2Δ
cells. These epistasis analyses revealed that multiple KDACs contribute to telomere looping (). Rpd3L, Rpd3S and Hda1 all promote telomere looping in parallel pathways whereas the relationship between the Sir2/3/4 complex and the other KDACs in terms of telomere structure remains enigmatic. The KDACs are best known for their deacetylation of histones and they are known to contribute significantly to silencing at subtelomeric loci 
. Consistent with a connection between chromatin modification and the telomere fold-back structure, we also found that many members of the Swr1 chromatin remodeling complex as well as the histone chaperone Asf1 () were important for telomere folding. A future challenge will be to determine the targets of the KDACs. Although subtelomeric histones are prime candidates, telomeric proteins themselves may be targets. Furthermore, it will be important to characterize how the other mutants revealed in the screen contribute to telomere looping and to understand if these mutants are epistatic with the KDACs. Indeed, it has been difficult to understand why mutations that affect such diverse biological pathways may have synthetic growth defects with cdc13-1
or in some cases, senesce rapidly 
. Since many signaling pathways activate effectors via activation/repression of target genes through chromatin remodeling and/or histone acetylation/deacetylation, we propose that activation or repression of these pathways may influence the ability of the KDACS (Rpd3/Hda1 and Sir2) to act at telomeres and in turn directly or indirectly influence telomere structure.
This work has uncovered multiple regulators of the telomere fold-back structure, including lysine deactylation and chromatin remodeling. The results of our screen correlate well with screens that have been performed to elucidate genes implicated in telomere function and cellular senescence suggesting that the fold-back structure may be important for chromosome end protection. Previous models have speculated that the fold-back in yeast may be important to establish silent chromatin within the telomeric/subtelomeric loci 
. As an alternative, it was also suggested that silent telomeric chromatin may be required to establish a particular architecture that contributes to chromosome end protection 
. Our results indicate that silent chromatin can be established in the absence of a telomere fold-back since many of the mutants recovered in our screen are not compromised for silencing () or even have slightly enhanced silencing (e.g. rpd3
mutants). Sir2/3/4-mediated silencing as well as other chromatin modifications are however important to establish a telomere loop, which likely promotes end protection. Interestingly, Rpd3 dependent histone deacetylation has been shown to prevent Sir2/3/4 protein spreading towards the centromere 
, which may potentially deplete SIR protein levels immediately adjacent to the telomere, raising the possibility that SIR2/3/4
disruption and RPD3L/S
disruption may be one in the same in terms of a fold-back defect. Further characterization of the yeast fold-back structure and the relationship to silent chromatin will be essential in order to clarify these issues. In summary, by understanding how different biological processes impinge on chromosome end structure, we increase the possibilities to manipulate telomere function, both positively and negatively, which may have important implications for diseases that stem from telomere dysfunction.