Direct transcriptional induction of Gadd45γ by Ascl1 during neuronal differentiation

The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time- and concentration-dependent manner. Interestingly, the gene encoding the cell cycle regulator Gadd45γ was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45γ as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45γ promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45γ promoter. Furthermore, we found that overexpression of Gadd45γ itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.