P107 negatively regulates the neural precursor pool by repressing Hes1 transcription

Abstract

Stem cells are defined by their multipotentiality and their long-term ability to self-renew. P107, a member of the pocket protein family of cell cycle regulators has previously been shown in our laboratory to negatively regulate neural precursor cell number and self-renewal (Vanderluit et al., 2004). In this study, we investigated the mechanism by which p107 regulates the neural precursor pool by examining interactions between p107 and the Notch pathway, which has also been shown to regulate the neural stem cell population (Nakamura et al., 2000; Ohtsuka et al., 2001; Hitoshi et al., 2002b). We found an increase in both the transcript and protein levels of Hes1 in p107-/- brains using in situ hybridization and western blot analysis. Examination of the Hes1 promoter revealed three putative E2F binding sites, which were subsequently found to bind E2F3 and E2F4 using chromatin immunoprecipitation. P107 was found to significantly repress Hes1 promoter activity in the luciferase reporter assay, and finally, using the primary neurosphere assay we showed that removal of Hes1 from p107-/- neurospheres restores the number of neurosphere forming cells to wildtype levels. Our results suggest that p107 represses Hes1 transcription through E2F, and demonstrate that an upregulation of Hes1 is responsible for the increased neural precursor pool in p107-/- mice.