Pax6 Levels in Neural Stem Cell Self-Renewal Figure 2. In vivo location analysis identifies the set of genes whose promoters are bound by Pax6 in neocortical stem and progenitor cells. (A–D) Identification of Pax6-bound promoters. ChIP array data for four typical Pax6-bound promoters, all of which were calculated as significantly bound above background levels (see Materials and Methods for details). Each graph plots fold-enrichment (in log base 2) on the y-axis, against relative chromosomal position on the x-axis. (E,F) Validation of the specificity and accuracy of the binding of the Pax6 promoter by Pax6, confirmed by qPCR in (G). Scanning PCR primers were designed to amplify DNA from a series of genomic regions in the Pax6 promoter (red bars represent amplified regions in the plot of fold-enrichment, y-axis, against relative chromosomal position, x-axis). The peak of Pax6 binding from the ChIP array analysis is shown in E. Pax6 binding was confirmed in this assay only in the region around the oligonucleotide, as shown in the gel images in F. Pax6, Pax6 ChIP; WCE, whole cell extract. (G) Validation of Pax6-binding to a set of promoters by quantitative PCR in independent ChIPs (not used for the array hybridisations) using two different Pax6 polyclonal antibodies. Enrichment of a region is calculated relative to that observed in control ChIPs without the primary antibody and normalised to a non-bound control region (NBCR). (H) Three independent ChIPs and promoter array hybridisations were carried out for Pax6, in which Pax6-bound material was compared with total genomic DNA (whole cell extract, WCE) to identify regions enriched in the Pax6-ChIP material by raw ratio measurements. Pax6 is found on the promoters of 1560 genes, 1172 (75%) of which are annotated genes (Table S1). doi:10.1371/journal.pgen.1000511.g002 In contrast, genes demonstrating negative dependency on Pax6 included genes with periodic expression in the cell cycle (FoxM1, Mcm3, Mcm5). These genes were reduced in expression in the Pax6-overexpressing cortex, indicating that increasing Pax6 may alter cell cycle length. Together, the transcriptional analyses demonstrate that Pax6 regulates neural stem cell maintenance, basal progenitor cell genesis and neurogenesis. Pax6 Bound and Regulated Genes Define the Network Directly Controlled by Pax6 in Neocortical Stem and Progenitor Cells To identify those genes that are both bound and regulated by Pax6, we analysed the intersection between the sets of Pax6 bound genes and those genes showing Pax6 dependency from the gain and loss of function transcriptional analyses (Figure 5). Of the set of 1560 Pax6-bound genes identified by the in vivo location analysis, 343 (22%) show significantly changed expression in either the Pax6 gain or loss of function cortex at E12.5 (Figure 5A; Table S4). Of the 343 bound and regulated genes, 180 were positively regulated and 143 negatively regulated by Pax6 (Figure 5B and 5C), with 20 genes showing conflicting regulation (either up- or down-regulated upon both gain and loss of Pax6 function). Using publicly available in situ hybridization data (GenePain- t.org; [38]), we assigned cellular expression to 279 of the 343 Pax6 bound and regulated genes in the E14.5 mouse cortex, the mRNAs of 223 of which were detectable in the cortex at that age PLoS Genetics | www.plosgenetics.org 4 June 2009 | Volume 5 | Issue 6 | e1000511