Interplay of SOX and POU Factors in Regulation of the Nestin Gene in Neural Primordial Cells

Abstract

Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed as markers for neural primordium, suggesting their regulatory link. We have identified adjacent and essential SOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer including these sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimeric forms in transfection assays and was utilized in assessing the specificity of the synergism; combinations of either group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mouse spinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3 expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subventricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) was expressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderate expression was observed in the intermediate zone, distributed in the cytoplasm. These data support the model that synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestin expression. Evidence also suggests that such interactions are involved in the regulation of neural primordial cells.Specific gene regulation for the determination of neural primordial cells is of high interest for its possible contribution to stem cell biology, where neural tissues are of primary importance. The majority of the neural primordial cells present in the central nervous system (CNS) from embryonic to adult stages (2, 47) have been documented to express the intermediate filament protein Nestin (11,28,41). Given that transcriptional regulation determining a cell type is largely reflected by the regulation of genes specific to the cell type, we focused on the regulation of the Nestin gene to better understand gene regulation that is determinative for the neural primordial state.Previous studies of rat and human Nestin genes indicated that Nestin expression in the CNS, at least during embryonic stages, is largely regulated by a nervous system-specific enhancer located in the 3Ј half of the second intron (21,29,58,61). After an analysis of a 257-bp-long Nestin enhancer in the rat, Josephson et al. (21) identified putative binding sites for POU, AP2, and a hormone-responsive element; further assessment of their involvement in Nestin enhancer activity was performed using transgenic mouse embryos. Mutational alterations of the downstream POU factor sites, bound by class III POU factors in vitro, largely eliminated the activity of the enhancer; mutations of other sites altered the domain of the CNS in which the enhancer showed activity (21). These previous results indicated that the activation of the Nestin neural enhancer is fundamentally dependent on the binding of a POU factor to the downstream site and that other nuclear factors may cooperate in est...

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