Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment.

EWS/FLI1 is a fusion gene product generated by a chromosomal translocation t(11;22)(q24;q12) found in Ewing sarcoma. EWS/FLI1 encodes an aberrant transcription factor with oncogenic properties in vitro. Paradoxically, expression of EWS/FLI1 in nontransformed primary cells results in apoptosis, but the exact mechanism remains unclear. In primary mouse embryonic fibroblasts derived from conditional EWS/FLI1 knock-in embryos, expression of EWS/FLI1 resulted in apoptosis with concomitant increase in the endogenous Caspase 3 (Casp3) mRNA. EWS/FLI1 directly bound and activated the CASP3 promoter, whereas small interfering RNA-mediated knockdown of EWS/FLI1 led to a marked decrease in CASP3 transcripts in Ewing sarcoma cell lines. Ectopic expression of EWS/FLI1 resulted in an increased expression of CASP3 protein in heterologous cell lines. Importantly, expression of EWS/FLI1 in the mouse triggered an early onset of apoptosis in kidneys and acute lethality. These findings suggest that EWS/FLI1 induces apoptosis, at least partially, through the activation of CASP3 and show the cell context-dependent roles of EWS/FLI1 in apoptosis and tumorigenesis.

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