Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1’s telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S KO), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1^L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1^L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1^L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1^L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.

端粒维持对于人类多能干细胞的长期增殖至关重要，而它们的端粒长度设定点决定了它们分化后代的增殖能力。端粒稳定性和伸长需要shelterin蛋白TPP1，但它在建立端粒长度设定点中的作用仍然难以捉摸。在这里，我们描述了 TPP1 (TPP1S) 的较短同种型和 TEL 补丁外的氨基酸 L104（TPP1 的端粒酶相互作用域）对端粒长度控制的贡献。我们证明缺乏 TPP1S (TPP1S KO) 的细胞以及完整的 TPP1 KO 细胞系会发生端粒缩短。然而，TPP1S KO 细胞能够稳定短端粒，而 TPP1 KO 细胞死亡。我们将这些表型与 TPP1 ^{L104A/L104A的表型进行比较}突变细胞，其端粒短而稳定，类似于 TPP1S KO。与 TPP1S KO 细胞相比，TPP1 ^L104A/L104A细胞响应增加的端粒酶水平并维持受保护的端粒。然而，TPP1 ^L104A/L104A显示出对shelterin 蛋白表达变化的敏感性改变，表明该突变导致端粒长度反馈调节缺陷。这共同突出了 TPP1 ^L104A/L104A作为第一个在人类干细胞的内源性基因座处设计的具有改变的端粒长度设定点的庇护素突变体。