, 2005 and Meletis et al., 2006). As shown in Figure 8, wild-type and phosphomimetic Olig2 suppress basal expression of p21 protein and mRNA in cycling cells. These effects are reflected, at least in part, BAY 73-4506 research buy by diminished levels of p53 protein associated with promoter/enhancer

elements of the p21 gene. However, the pronounced impact of Olig2 phosphorylation on expression of p21 protein and mRNA ( Figure 8A) stands somewhat in contrast to the more nuanced differences in total p53 bound to p21 promoter/enhancer elements ( Figure 8B). Expression of p21 protein and mRNA is likely to reflect the cumulative impact of reduced p53 recruitment to the p21 promoter, reduced transcriptional function of hypoacetylated p53 at the promoter ( Barlev et al., 2001), and possible posttranscriptional effects on stability of the p21 protein ( Coleman et al., 2003 and Gong et al., 2003). In addition we have shown in previous studies that Olig2 can interact directly with the promoter/enhancer elements of p21 ( Ligon et al., 2007). Because Olig2 has been characterized as a transcription repressor

( Novitch et al., 2001 and Zhou et al., 2001), it is possible that the attenuation of p53 transcriptional functions is further augmented by direct Bortezomib clinical trial Olig2-mediated suppression of p21 expression (however, see below). Notwithstanding the complexity of p21 regulatory mechanisms, the experiment summarized in Figures 8C and 8D shows clearly that p53 is a prime target of the Olig2 proliferative phenotype. How does phosphorylation of the triple serine motif affect transcriptional functions of Olig2? Members of the bHLH transcription factor family function as homodimers or as heterodimers with E12/E47 proteins to bind to canonical E box elements in the promoter/enhancer

regions of their target genes (Ross et al., 2003). The bHLH motif is almost exclusively responsible for both heterodimerization and DNA targeting to the E box. It seems unlikely that phosphorylation much events in the amino terminus would have any direct effect on these functions of Olig2, and preliminary chromatin immunoprecipitation (ChIP) studies failed to show changes in Olig2:p21 targeting that could account for the effects on basal p21 expression of p21 seen in Figure 8A. The fact that all three serine residues at positions 10, 13, and 14 must be mutated to achieve a strong loss-of-function or gain-of-function phenotype suggests that the proliferative function associated with the Olig2 phosphorylation state involves a significant conformational change in the amino terminus of Olig2. This conformational change could affect the interaction of Olig2 with DNA or important coregulator proteins and, thus, affect the transcriptional activity of Olig2 upon target genes that affect p53 function. One final possibility that cannot be ruled out by the data is that the p53 antagonist function of Olig2 is independent of its function as a bHLH transcription factor.