Prox1 regulates the notch1-mediated inhibition of neurogenesis.

Activation of Notch1 signaling in neural progenitor cells (NPCs) induces self-renewal and inhibits neurogenesis. Upon neuronal differentiation, NPCs overcome this inhibition, express proneural genes to induce Notch ligands, and activate Notch1 in neighboring NPCs. The molecular mechanism that coordinates Notch1 inactivation with initiation of neurogenesis remains elusive. Here, we provide evidence that Prox1, a transcription repressor and downstream target of proneural genes, counteracts Notch1 signaling via direct suppression of Notch1 gene expression. By expression studies in the developing spinal cord of chick and mouse embryo, we showed that Prox1 is limited to neuronal precursors residing between the Notch1+ NPCs and post-mitotic neurons. Physiological levels of Prox1 in this tissue are sufficient to allow binding at Notch1 promoter and they are critical for proper Notch1 transcriptional regulation in vivo. Gain-of-function studies in the chick neural tube and mouse NPCs suggest that Prox1-mediated suppression of Notch1 relieves its inhibition on neurogenesis and allows NPCs to exit the cell cycle and differentiate. Moreover, loss-of-function in the chick neural tube shows that Prox1 is necessary for suppression of Notch1 outside the ventricular zone, inhibition of active Notch signaling, down-regulation of NPC markers, and completion of neuronal differentiation program. Together these data suggest that Prox1 inhibits Notch1 gene expression to control the balance between NPC self-renewal and neuronal differentiation.

Ubiquitin Specific Peptidase 15 (USP15) suppresses glioblastoma cell growth via stabilization of HECTD1 E3 ligase attenuating WNT pathway activity.

Expression based prediction of new genomic alterations in glioblastoma identified the de-ubiquitinase Ubiquitin Specific Peptidase 15 (USP15) as potential tumor suppressor gene associated with genomic deletions (11%). Ectopic expression of USP15 in glioblastoma cell-lines reduced colony formation and growth in soft agar, while overexpression of its functional mutant had the opposite effect. Evaluation of the protein binding network of USP15 by Mass Spectrometry in glioblastoma cells uncovered eight novel interacting proteins, including HECT Domain Containing E3 Ubiquitin Protein Ligase 1 (HECTD1), whose mouse homologue has been associated with an inhibitory effect on the WNT-pathway. USP15 de-ubiquitinated and thereby stabilized HECTD1 in glioblastoma cells, while depletion of USP15 led to decreased HECTD1 protein levels. Expression of USP15 in glioblastoma cells attenuated WNT-pathway activity, while expression of the functional mutant enhanced the activity. Modulation of HECTD1 expression pheno-copied the effects observed for USP15. In accordance, human glioblastoma display a weak but significant negative correlation between USP15 and AXIN2 expression. Taken together, the data provide evidence that USP15 attenuates the canonical WNT pathway mediated by stabilization of HECTD1, supporting a tumor suppressing role of USP15 in a subset of glioblastoma.