A non-telomeric role of POT1a in regulating HSC metabolism via TIN2 localization

Abstract Hematopoietic stem cells (HSCs) maintain long-term hematopoiesis through tightly regulated self-renewal and quiescence. Protection of telomeres from DNA damage is essential for preserving HSC function over a lifetime. The shelterin complex—composed of TRF1, TRF2, POT1, TIN2, TPP1, and RAP1—protects telomeres from aberrant DNA damage responses. Among these, POT1 binds to single-stranded telomeric DNA, forming protective looped structures. Moreover, POT1 suppresses the ATR-dependent DNA damage response, preventing inappropriate DNA repair. We previously demonstrated that POT1a, one of the two murine POT1 orthologs and functionally analogous to human POT1, is essential for maintaining HSC function (Hosokawa et al., Nat. Commun. 2016). Silencing of POT1a impairs long-term hematopoietic reconstitution and activates mitochondrial metabolism in HSCs. While shelterin components are primarily known for telomere-associated roles, several have been implicated in extratelomeric roles. Notably, mitochondrial localization of TIN2 has been linked to enhanced oxidative metabolism and reactive oxygen species (ROS) production (Chen et al., Mol. Cell. 2012). Since POT1a and TPP1 form heterodimers and connect to TIN2, we hypothesized that POT1a regulates HSC metabolism by modulating the subcellular localization of TIN2. To investigate the non-telomeric function of POT1a, we generated HSC-specific POT1a conditional knockout (KO) mice (POT1afl/fl;eR1-CreERT2). POT1a deletion disrupted HSC quiescence, leading to a reduced frequency of hematopoietic stem and progenitor cells, especially long-term HSCs. Flow cytometric analyses revealed increased mitochondrial membrane potential, elevated ROS production, and enhanced ATP production. To assess how POT1a deletion alters HSC gene expression, we performed RNA sequencing on HSCs isolated from control and POT1afl/fl;eR1-CreERT2 mice. Gene set enrichment analysis revealed the upregulation of pathways related to cell cycle progression, DNA damage response, oxidative phosphorylation, and the electron transport chain in POT1a KO HSCs. Importantly, immunofluorescence staining demonstrated abnormal mitochondrial localization of TIN2 in POT1a KO HSCs. Interestingly, POT1a and TPP1 expression declined, while TIN2 expression increased in HSCs from aged mice and in cultured HSCs under stress. These findings led us to speculate that an imbalance between the POT1a-TPP1 complex and TIN2 underlies the mitochondrial localization of TIN2 and the metabolic activation of HSCs. To assess the functional impact of TIN2 mitochondrial localization, we engineered a TIN2 mutant lacking the TPP1-binding domain, which overlaps with its mitochondrial targeting sequence. Transfection of this mutant into HSCs increased mitochondrial membrane potential and ROS levels, mimicking the phenotype of POT1a-deficient HSCs. Furthermore, transplantation of HSCs transduced with mutant TIN2 resulted in impaired long-term reconstitution. Conversely, overexpression of TPP1 in HSCs redirected TIN2 to the nucleus and prevented its mitochondrial localization, as revealed by immunofluorescence staining. This intervention restored mitochondrial membrane potential and ROS levels. Notably, TPP1-overexpressing HSCs exhibited restored long-term reconstitution capacity in transplantation assays. To further assess the impact of the POT1–TPP1 complex on TIN2 localization, we generated His-tagged POT1a and TPP1 proteins and cultured them with HSCs. Exogenous delivery of POT1a and TPP1 proteins prevented mitochondrial translocation of TIN2 and reduced ROS levels. Notably, under these culture conditions, the proportion of HSCs was better preserved, and transplantation of these cells resulted in improved long-term hematopoietic reconstitution. In conclusion, our study reveals a non-telomeric role of POT1a in preserving HSC quiescence and function. POT1a, through its interaction with TPP1, restricts mitochondrial localization of TIN2, thereby suppressing ROS production and metabolic activation. Disruption of this regulation causes HSC exhaustion, but restoration of the POT1a–TPP1 complex preserves HSC function and reconstitution capacity. These findings uncover a novel mechanism linking shelterin components to metabolic regulation in HSCs and suggest potential strategies to enhance stem cell fitness.