(A) Low-passage adult NPCs were cultured in high growth factor signaling (HGFS) conditions (FOXO3 mostly cytoplasmic) or were transiently cultured in low growth factor signaling (LGFS) conditions to induce FOXO3 nuclear accumulation.
(B) hIP-qPCR analysis of FOXO3 binding to the known target gene Cdkn1b (p27) in NPCs in high growth factor signaling (HGFS) versus low growth factor signaling (LGFS) conditions. Neg C: negative control (genomic primers that amplify a region not bound by FOXO3). Mean +/− SD of triplicates of one representative experiment of two independent experiments. Enrichment is calculated relative to the negative control region of the genome.
(C) Numbers of unique Solexa reads, QuEST peaks, corresponding RefSeq genes, unique gene symbols, and QuEST false discovery rates (FDR) for FOXO3 ChIP from adult NPCs. N/A; not applicable. HGFS: high growth factor signaling. LGFS: low growth factor signaling.
(D) UCSC Genome Browser shot showing FOXO3 binding at the Pot1a locus. Forward and reverse ChIP-seq sequence tags are shown in yellow and green, respectively. Normalized QuEST peaks (blue), binding region (light blue), and binding site (red) are shown below the sequence tags.
(E) List of the 10 FOXO3-bound genes in adult NPCs with the highest QuEST scores. Enrichment values by ChIP-qPCR are indicated on the right.
(F)FOXO3 binding sites identified by ChIP-seq are specific FOXO3 binding sites inNPCs. ChIP-qPCR in NPCs from adult FOXO3+/+ and FOXO3−/− littermates in low growth factor signaling conditions (LGFS). QuEST scores are indicated in purple. Based on QuEST enrichment, three FOXO3 targets from the top ChIP-seq quartile and two targets from each of the bottom quartiles are shown. Mean +/− SD of two independent experiments. Enrichment is calculated relative to a negative control region of the genome. ***p < 0.001, **p < 0.01, Two-way ANOVA, Bonferroni correction. Neg C: negative control.
(G) FOXO3 ChIP-qPCR in adult neurogenic niches (subventricular zones [SVZs]) that were microdissected from 3-6 month old mice. Mean +/− SD of two independent experiments. Enrichment is calculated relative to input. *p < 0.05, Two-way ANOVA, Bonferroni correction. Neg C: negative control. Additional genes are presented in .
(H) Regression analysis comparing FOXO3 ChIP enrichments in vivo in the SVZ vs. ChIP-seq peak height in cultured NPCs. Pearson correlation, R = 0.94, p-value = 3.8 × 10-6.
(I) Expression of top FOXO3-bound genes in vivo in FACS-purified astrocytes, activated NSCs, and NPCs freshly isolated from the adult brain. Mean +/− SD of technical replicates of one representative experiment of two independent experiments. Validation of the FACS strategy and additional genes are shown in .
(J) Overlap between FOXO3-bound genes and genes that are downregulated in FOXO3−/− NPCs by microarray analysis (Fisher's exact test, p = 1.18 × 10-17).
See also .