Tpz1-Ccq1 and Tpz1-Poz1 Interactions within Fission Yeast Shelterin Modulate Ccq1 Thr93 Phosphorylation and Telomerase Recruitment

In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3ATR and Tel1ATM kinases are redundantly required for telomerase recruitment, since Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1) contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these findings, we suggest that the negative regulatory function of Tpz1-Poz1 interaction works upstream of Rad3ATR kinase, while Tpz1-Ccq1 interaction works downstream of Rad3ATR kinase to facilitate Ccq1 Thr93 phosphorylation and telomerase recruitment.在裂殖酵母与人类中，端粒保护蛋白复合体（shelterin complex）在调控端粒酶招募、保护端粒免受DNA损伤应答因子侵扰以及端粒区异染色质形成等过程中发挥核心作用。尽管端粒保护蛋白复合体可抑制端粒处DNA损伤检验点激酶ATR与ATM的激活，这两种激酶却是稳定维持端粒所必需的。在裂殖酵母中，Rad3ATR与Tel1ATM激酶对端粒酶的招募存在冗余需求，因为Rad3ATR/Tel1ATM依赖的端粒保护蛋白复合体亚基Ccq1的苏氨酸93位点磷酸化，可促进Ccq1与端粒酶亚基Est1的相互作用。然而，此前尚不明确由Taz1、Rap1、Poz1、Tpz1、Pot1与Ccq1组成的端粒保护蛋白复合体内部的蛋白质-蛋白质相互作用，如何调控Ccq1苏氨酸93位点的磷酸化以及端粒酶的招募过程。本研究中，我们鉴定出裂殖酵母端粒保护蛋白复合体中介导Tpz1-Ccq1与Tpz1-Poz1相互作用的关键结构域与氨基酸残基。我们利用维持Tpz1-Pot1相互作用，但特异性破坏Tpz1-Ccq1或Tpz1-Poz1相互作用的功能分离型Tpz1突变体，证实Tpz1-Ccq1相互作用可促进Ccq1苏氨酸93位点磷酸化、端粒酶招募、检验点抑制以及端粒区异染色质形成。此外，我们证实Tpz1-Poz1相互作用可促进Poz1的端粒定位；而端粒处Poz1的缺失会导致Ccq1苏氨酸93位点磷酸化水平与端粒酶招募活性升高，并引发端粒区异染色质形成缺陷。此外，我们的研究证实Tpz1-Poz1与Tpz1-Ccq1相互作用可冗余行使端粒保护蛋白复合体的核心端粒保护功能：当两种相互作用同时丧失时，绝大多数细胞会立即丧失细胞活力，并产生携带环状染色体的存活细胞。基于上述发现，我们提出：Tpz1-Poz1相互作用的负调控功能作用于Rad3ATR激酶的上游，而Tpz1-Ccq1相互作用则作用于Rad3ATR激酶的下游，以促进Ccq1苏氨酸93位点磷酸化与端粒酶招募。