TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis
Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar
Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar
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Research Article Genetics Hematology

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

Abstract

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.

Authors

Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar

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Figure 1

Screen to identify the TPP1 mutations that interfere with POT1 and TIN2 binding.

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Screen to identify the TPP1 mutations that interfere with POT1 and TIN2 ...
(A) Schematic representation of the interactions between shelterin proteins and telomerase at chromosome ends. (B) Sequence alignment of the human TPP1 POT1- and TIN2-binding domains with indicated mammalian orthologs. Residues of human TPP1 that were mutated in this screen are shown above the alignment. TPP1 mutants defective in binding POT1 and TIN2 are highlighted in red. Brackets indicate 2 residues simultaneously mutated (double mutant). Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. Cylinders underneath the sequence alignment indicate α helices. The structure of the POT1 C-terminus bound to the TPP1-PBD (Protein Data Bank [PDB]: 5UN7) is shown above the TPP1 domain diagram with POT1 shown in gray and TPP1 shown in yellow. Structure of the TIN2TRFH-TPP1TBM-TRF2TBM complex (PBD: 5XYF) is shown below the TPP1 domain diagram with TIN2TRFH represented in gray, TRF2TBM represented in purple, and TPP1TBM represented in pink. TPP1 amino acids whose mutation resulted in POT1- and TIN2-binding defects are shown in red in the structures.