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. 2014 Nov 19;34(47):15816–15831. doi: 10.1523/JNEUROSCI.1865-14.2014

Figure 5.

Figure 5.

Prox1 regulates Olig2 expression via direct transcriptional suppression of Olig2 gene. A, Schematic representation of the organization of Olig2 gene locus. B, ChIP analysis for the binding of Prox1 to the proximal 1.1 kb Olig2 promoter and K23 enhancer in chromatin prepared from the spinal cords of E12.5 mouse embryos. The immunoprecipitated DNAs from these experiments were analyzed and quantified with real-time qPCR assays. The primer pairs used are indicated with arrows in A; **p < 0.01, ***p < 0.001, n.s: p > 0.05 (t test), n = 4. C, Schematic representation of the five luciferase reporter constructs used for the transcriptional assays, including the murine 1.1 kb Olig2 promoter fused with luciferase (1.1 kb), SV40 promoter fused with luciferase (SV40), K23 enhancer upstream of the 1.1 kb (K23enh), and K23 enhancer upstream of the SV40 (SV40-K23enh), as well as empty vector (PRless). D, Transcriptional assays in NPCs, cotransfected with Prox1 (+) or GFP (−) expression vectors and luciferase reporter constructs 1.1 kb or K23enh, represented in C, as indicated. For both 1.1 kb and K23enh, **p < 0.01 (t test), n = 4. E, Transcriptional assays in NPCs, cotransfected with Prox1 (+) or GFP expression (−) vectors and luciferase reporter constructs (SV40 or SV40-K23enh) represented in C, as indicated. For SV40 reporter: n.s., p > 0.05 (t test), n = 4; for SV40-K23enh: ***p < 0.001 (t test), n = 4. F, Transcriptional assays in mouse NPCs cotransfected with K23enh and GFP, WT-Prox1, ΔDBD-Prox1, or ΔRD-Prox1, as indicated. For both ΔDBD-Prox1 and ΔRD-Prox1 versus Prox1: **p < 0.01 (t test) n = 4; for both ΔDBD-Prox1 and ΔRD-Prox1 versus GFP: p > 0.1 (t test), n = 4. In all luciferase experiments, data are represented as the mean ± SD of quadruplicate assays. G, Transcriptional assays in N2A cells transfected with various promoter constructs as indicated in C. For 1.1 kb versus SV40, **p < 0.01 (t test), n = 4. H, Transcriptional assays in N2A cells cotransfected with PRless or 1.1 kb constructs and Prox1 (+) or GFP (−) expression vectors, respectively, as indicated. For 1.1 kb, ***p < 0.001 (t test), n = 4.