Valproic acid promotes neuronal differentiation by induction of proneural factors in association with H4 acetylation

Abstract

Valproate (VPA) influences the proliferation and differentiation of neuronal cells. However, little is known about the downstream events, such as alterations in gene transcription, that are associated with cell fate choice. To determine whether VPA plays an instructive role in cell fate choice during hippocampal neurogenesis, the expression of genes involved in the cell cycle and neuronal differentiation was investigated. Treatment with VPA during the progenitor stages resulted in strong inhibition of cell proliferation and induction of neuronal differentiation, accompanied by increases in the expression of proneural transcription factors and in neuronal cell numbers. The increased expression of Ngn1, Math1 and p15 points to a shift towards neuronal fate in response to histone deacetylase inhibitors (HDACi). Chromatin immunoprecipitation (ChIP) analysis showed that acetylated histone H4 (Ac-H4) was associated with the Ngn1, Math1 and p15 promoters in cultured hippocampal neural progenitor cells. VPA-induced hippocampal neurogenesis was also accompanied by association of Ac-H4 with the Ngn1 promoter in hippocampal extracts. The discovery of an association between HDACi and the Ngn1, Math1 and p15 promoters extends the importance of HDAC inhibition as a key regulator of neuronal differentiation at the transcriptional level.

Introduction

Valproic acid (VPA) (2-propylpentanoic acid) is a well known anticonvulsant and mood stabilizer (Henry, 2003). Recent experiments in 293T, Neuro2A, and teratocarcinoma F9 cells have demonstrated that VPA can directly inhibit histone deacetylase inhibitor (HDAC) activity and cause hyperacetylation of histones (Göttlicher et al., 2001, Phiel et al., 2001). HDACs modify the N-terminal tails of histones and alter their interaction with DNA, thus serving as epigenetic regulators of gene expression. HDAC inhibition mediated by VPA has been shown to suppress the growth and increase the differentiation of many different tumor cell lines (Blaheta and Cinatl, 2002). In the nervous system, VPA stimulates the proliferation of rat neural progenitor cells prepared from the lateral ganglionic eminence or neocortex, and increases neuronal differentiation associated with γ-amino butyric acid (GABA) expression at the expense of astrocytic differentiation (Laeng et al., 2004). However, it has been reported that VPA inhibits proliferation and induces neuronal differentiation of adult hippocampal neural progenitor cells in vitro and in vivo (Hsieh et al., 2004) and that this effect is associated with hyperacetylation of histones and upregulation of the neurogenic basic helix-loop-helix (bHLH) transcription factor NeuroD (Hsieh et al., 2004).

Because the acetylation state of histones modulates chromatin structure and regulates gene expression, we hypothesized that VPA might influence the expression of other proneural and negative transcription factors responsible for neuronal differentiation in vitro and in vivo. In this study we examined whether the acetylation induced by VPA regulates proneural transcriptional factors and cell cycle-related genes, and whether it affects the timing of neuronal differentiation. HDAC inhibition appears to be an important mechanism controlling the onset of gene expression leading to neuronal differentiation in vitro because VPA was found to be required to upregulate several proneural genes and downregulate differentiation inhibitors.