SummaryStil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil -/-embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. Journal of Cell Scienceleads to degradation of mitotic kinases including Plk1, delaying the activation of Cdc25c phosphatase, and consequently delayed activation of Cdk1 and entry into mitosis (Kang et al., 2002;Shtivelman, 2003;Yu et al., 2005). Ubiquitination of target proteins is required for Chfr activity also in a proteosome-independent manner (Matsusaka and Pines, 2004). Its subcellular localization might be cell-cycle-dependent. It has been shown to localize in nuclear promyelocytic leukemia (PML) bodies and to regulate nuclear dynamics and genomic stability (Daniels et al., 2004;Kwon et al., 2009;Oh et al., 2009). Other studies have demonstrated localization at the spindle poles during metaphase and interaction with microtubule-associated proteins (Burgess et al., 2008;Maddika and Chen, 2009). Chfr might auto-regulate itself by autoubiquitination (Chaturvedi et al., 2002). However, the upstream negative regulators of Chfr are unknown. Thus, the mechanisms of Chfr activity and regulation are incompletely understood. Because Stil is required for entry into mitosis and Chfr is known to regulate the G 2 -M transition, we hypothesized that Stil might be involved in the regulation of Chfr. The present study demonstrates that Stil has a role in centrosome organization, entry into mitosis and cell proliferation, and that these functions are at least partially mediated by Chfr. In this regard, we show that overexpression of Stil results in increased auto-ubiquitination and reduced protein stability of Chfr. Results Mitotic defects in mouse embryonic fibroblasts lacking StilMouse embryonic fibroblasts (MEFs) derived from Stil knockout embryos (1022M) are characterized by a low prolifer...