Telomere-Dependent and Telomere-Independent Roles of RAP1 in Regulating Human Stem Cell Homeostasis

RAP1 is well known as a telomere-binding protein; yet its functions in human stem cells remain unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by CRISPR/Cas9 and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 negatively regulated telomere length. In addition, RAP1 acted as a transcriptional regulator of RLEN by tuning the methylation status of its promoter region. Phenotypically, RAP1 deficiency resulted in enhanced self-renewal and delayed senescence in hMSCs rather than in hNSCs, suggesting a complex role of RAP1 in regulating lineage specific stem cell homeostasis. Altogether, these results indicate that RAP1 plays both telomeric and non-telomeric roles in regulating the homeostasis of human stem cells.

RAP1作为端粒结合蛋白（telomere-binding protein）已为学界所熟知，但其在人类干细胞中的功能仍有待阐明。本研究通过CRISPR/Cas9技术构建了RAP1缺陷型人类胚胎干细胞（human embryonic stem cells，hESCs），并通过定向分化获得了RAP1缺陷型人间充质干细胞（human mesenchymal stem cells，hMSCs）与神经干细胞（human neural stem cells，hNSCs）。在hMSCs与hNSCs中，RAP1均负向调控端粒长度。此外，RAP1可通过调控RLEN启动子区域的甲基化状态，充当RLEN的转录调节因子。表型分析显示，RAP1缺陷可增强hMSCs的自我更新能力并延缓其衰老，而对hNSCs无此影响，这提示RAP1在调控谱系特异性干细胞稳态中发挥着复杂作用。综上，本研究结果表明，RAP1在人类干细胞稳态调控中同时发挥端粒相关与非端粒相关的双重功能。