RESUMEN
Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.
Asunto(s)
Telomerasa/metabolismo , Homeostasis del Telómero/genética , Telómero/enzimología , Células Madre Embrionarias , Activación Enzimática/genética , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Humanos , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Complejo Shelterina , Telomerasa/genética , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína ETS de Variante de Translocación 6RESUMEN
Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.