Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

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 set point establishment remains elusive. Here, we characterize the contribution of TPP1 isoforms and residues outside the TEL patch, TPP1’s telomerase interaction domain, to telomere length control. We demonstrate that TPP1L, the longer minor isoform of TPP1, can partially compensate for loss of the more abundant shorter isoform, TPP1S. Both TPP1S knockout and complete TPP1 knockout cell lines (TPP1 KO) show telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres while TPP1 KO cells do not and die. We compare these phenotypes with that of TPP1 L104A/L104A mutant cells that like the TPP1S KO have short stable telomeres. In contrast to TPP1S KO, TPP1 L104A/L104A cells respond to increased telomerase. However, TPP1 L104A/L104A ’s sensitivity to shelterin-mediated feedback is altered, revealing TPP1 L104A/L104A as a new type of shelterin mutant with aberrant set point regulation.