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Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

ABSTRACT: 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 Gadd45gamma was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45gamma as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45gamma promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45gamma promoter. Furthermore, we found that overexpression of Gadd45gamma itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.

SUBMITTER: Huang HS

PROVIDER: S-EPMC2905796 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

Huang Holly S HS Kubish Ginger M GM Redmond Tanya M TM Turner David L DL Thompson Robert C RC Murphy Geoffrey G GG Uhler Michael D MD

Molecular and cellular neurosciences 20100409 3

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 regulat ...[more]

PMID: 20382226

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Project description:Neuroblastoma (NB), although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs) and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1 in its multiply phosphorylated state might prevent terminal differentiation in NB, which could offer new approaches for differentiation therapy in NB.

Dataset Information

Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

Publications

Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

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