Figure 8. An oppositional relationship between Olig2 and p53 in cycling neural progenitors.

(A) Endogenous p21 protein (inset) and mRNA (bar graphs) are suppressed by wild type and phospho mimetic Olig2 in cycling neural progenitors. Olig2-/- mouse neural progenitor cells were stably transduced with vector control (eGFP), WT, TPN or TPM. Endogenous protein levels were compared by western blotting with p21 antibody and β-actin as control. RNA was extracted from cells as described above, and p21 mRNA levels were quantified using premade p21 Taqman gene expression assay. Error bars, SD.

(B) Expression of p21 in cycling neural progenitors correlates with p53 loading on promoter/enhancer elements of the p21 promoter. Lysates of Olig2-/- mouse neural progenitor cells stably transduced with vector control (eGFP), WT, TPN or TPM were processed for chromatin immunoprecipitation with p53 antibody. Primers spanning the distal E-box in the p21 promoter/enhancer region were used for qRT-PCR analysis. Bar graph shows p21 promoter binding normalized for non-target site enrichment. Note that the differences in p53 loading, between the four groups, though nuanced, are statistically significant (p values: ** <0.01, *** <0.001) and in good accord with differences noted in acetylated p53 seen in cycling cells (Figure 7B). Error bars, SEM.

(C) Phosphorylation state of Olig2 is irrelevant in p53 null neurospheres. Olig2cre/cre mice were intercrossed to p53fl/fl mice to obtain neural progenitors that were Olig2:p53 double null. These progenitors were transduced with eGFP, wild type Olig2 or the triple phospho null mutant of Olig2 as indicated. Secondary neurosphere assays were performed on these cells and on matched p53 +/+ controls as per Figure 1.

D) Quantitation of neurosphere counts and sizes from C. Neurosphere size was measured using ImageJ. Error bars, SD.