Research Paper Volume 3, Issue 2 pp 108—124

Figure 7. FoxO3 binds to a site in the first intron of miR-106b~25/Mcm7. (A) Location of the FoxO binding site (FHRE) within the first intron of the miR-106b~25/Mcm7 gene, and the sequence locations used for EMSA, ChIP, and luciferase experiments. (B) EMSA with recombinant FoxO3-GST and a radioactively-labeled (hot) probe corresponding to the FoxO binding site in miR-106b~25/Mcm7 (FHRE WT). + Ctrl: FoxO3-GST incubated with a probe for a known FoxO binding site. The specificity of the interaction was tested by increasing amounts of unlabeled (cold) probe or cold probe with mutations in the FoxO consensus binding sequence (FHRE Mut). (C) Wild-type and FoxO3-null NSPCs were dissociated and the next day treated with 4 hours growth factor removal followed by addition of LY294002 for 1 hour. Antibodies to FoxO3 or control IgG antibodies were used for ChIP. qPCR was used to assess the enrichment of FHRE and of a negative control site (- Ctrl). Shown is the relative enrichment for 1 experiment (age 12 weeks, passage 10). These results were confirmed in ChIP-Seq studies (Webb et al. submitted). (D) HEK 293T cells were co-transfected with a plasmid to express FoxO3 (empty control, wild-type FoxO3, FoxO3 lacking the DNA binding domain, or constitutively nuclear FoxO3), a firefly luciferase reporter containing FHRE with or without the FoxO consensus sequence mutated, and a Renilla luciferase reporter to normalize for transfection efficiency. As a positive control, a luciferase reporter containing a known FoxO3-activated site was used (+ Ctrl); as a negative control, a luciferase reporter without an enhancer site was used (- Ctrl). Luciferase activity was assessed two days after transfection. Mean and SEM for 4 independent experiments (- Ctrl, + Ctrl, and FHRE WT) or 2 independent experiments (FHRE Mut) are shown. Unpaired two-tailed t-test, **: p<0.01. (E) NSPCs from wild-type and FoxO3-null mice were isolated and cultured. Total RNA was collected, and the levels of mature miR-106b~25 members (relative to 5S RNA) and Mcm7 mRNA (relative to β-actin mRNA) were assessed by RT-qPCR. Mean and SEM of the FoxO3-null/wild-type fold change for 5-6 independent cultures (age 10-13 weeks, passage 2-5) are shown. One-sample two-tailed t-test, *: p<0.05.