RESUMEN
The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.
Asunto(s)
Autorrenovación de las Células , Células Madre Hematopoyéticas , Proteínas Nucleares , Animales , Femenino , Humanos , Masculino , Ratones , Células Cultivadas , Endocitosis , Endosomas/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Sangre Fetal/citología , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/embriología , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Oncogene-induced replication stress is a crucial driver of genomic instability and one of the key events contributing to the onset and evolution of cancer. Despite its critical role in cancer, the mechanisms that generate oncogene-induced replication stress remain not fully understood. Here, we report that an oncogenic c-Myc-dependent increase in cohesins on DNA contributes to the induction of replication stress. Accumulation of cohesins on chromatin is not sufficient to cause replication stress, but also requires cohesins to accumulate at specific sites in a CTCF-dependent manner. We propose that the increased accumulation of cohesins at CTCF site interferes with the progression of replication forks, contributing to oncogene-induced replication stress. This is different from, and independent of, previously suggested mechanisms of oncogene-induced replication stress. This, together with the reported protective role of cohesins in preventing replication stress-induced DNA damage, supports a double-edge involvement of cohesins in causing and tolerating oncogene-induced replication stress.