In contrast, CHIR 99021 unlabeled mutant oligonucleotides (Figure S6A) were unable to compete effectively with the labeled WT oligonucleotides. These assays demonstrate that Pax6 protein can bind specifically to sequences representing

the predicted Pax6 binding sites BS1–BS5. We extracted chromatin from E12.5 cortex to test for binding of Pax6 to the predicted Pax6 binding sites BS1–BS5 in vivo by quantitative chromatin immunoprecipitation (qChIP; Figures 5A and 5B). Primer pairs were selected to measure, by qPCR, the relative levels of short fragments spanning each predicted binding site (Figure S6B). Primers for sequences from the genomic regions of Gab1 and Syt8 that were previously shown to be Pax6 bound and Pax6 nonbound, respectively, were used to generate positive and negative control data ( Sansom et al., 2009). Following the qPCR, values for Pax6/immunoglobulin G (IgG) normalized enrichment were expressed relative to the average value for Syt8 ( Figure 5B). DNA sequences that included four of the five Cdk6 Pax6 predicted binding sites (BS1, BS2, BS4, and BS5) were significantly enriched by amounts similar to or greater than that of the Gab1 PI3K Inhibitor Library screening positive control ( Figure 5B). There was no evidence for enrichment of the BS3 sequence. Taken together, the EMSA and ChIP results indicate that Pax6 has the potential to bind all five

Cdk6 sites (BS1–BS5), but binds to only four of them in E12.5 cortex in vivo. We next examined the functionality of each of the Pax6 binding sites (BS1–BS5) using luciferase assays in cells that do not express endogenous PAX6 about (HEK293 cells). We generated a set of eight luciferase reporter constructs to test each site individually (Figure 5C). We first cloned a 2.3 kb upstream fragment encompassing the putative Cdk6 promoter and containing only BS1 into the promoterless luciferase

reporter plasmid pGL4.10 to generate the plasmid pBS1-luc. This produced a substantial increase in relative luciferase activity compared with cells transfected with pGL4 vector alone ( Figure 5Di). Cotransfection of increasing amounts of the Pax6 expression construct pCMV-Pax6 ( Figure 5D) led to a dose-dependent reduction in relative luciferase activity ( Figure 5Di). To test whether this reduction was due to Pax6 binding to BS1, we mutated BS1 exactly as done for the EMSAs ( Figure S6A) to generate pBS1mut-luc ( Figure 5C). The mutation abolished Pax6-dependent suppression of luciferase activity ( Figure 5Di), indicating that binding of Pax6 to site BS1 can repress transcription from the Cdk6 promoter. We then evaluated each of the four remaining Pax6 binding sites (BS2–BS5) individually. Short DNA fragments spanning each of the binding sites were cloned into plasmid pBS1mut-luc (which drives reporter expression and is not itself repressed by Pax6). PCR fragments including BS2 or BS3 were placed immediately upstream of the 2.