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. 2012 Sep 20;8(9):e1002924. doi: 10.1371/journal.pgen.1002924

Figure 9. Molecular interactions between the VSX2 variants and components of the pigmentation circuitry.

Figure 9

(A) p27 mRNA expression in E12.5 retinas of the indicated genotypes as determined by qRT-PCR. Samples were normalized to orJ. Only the R227W retina was significantly different. (B) Luciferase activities from HEK293 cells transfected with the indicated expression vectors (x-axes) and ∼1.1 kb of the p27 promoter region (pGL3B-p27). Graph I: H-MITF repressed reporter activity in a DNA binding-dependent manner. Graph II: VSX2 and VSX2[R227W] enhanced reporter activity. Graph III: H-MITF combined with VSX2[R227W] elicited a specific and synergistic increase in reporter activity that depended on DNA binding as revealed by the abrogated activity of the VSX2[R200Q, R227W] double mutant (RQRW). Graph IV: Expression of the mi version of H-MITF had no effect on reporter activity resulting from VSX2 or its variants. (C) Schematic of p27 5′-intergenic region (∼1.1 kb). Positions of putative Mitf binding sites (M) and homeodomain core sequences (H) are shown. Positions are relative to p27 transcriptional start site. Position of primers that constitute p27 primer set 2 (Table S1) is also shown. Graphs show quantification of ChIP-qPCR assays using MITF or VSX2 antibodies reacted with E12.5 lysates from wild-type, R200Q and R227W retinas. MITF binding was detected in R200Q and R227W lysates. VSX2 binding was detected in R227W lysate. (D) Co-IPs of E12.5 R227W and R200Q retinal protein lysates with a negative control sheep IgG or VSX2 antibodies followed by western blot probed with MITF antibody (top panel). Co-IPs of HEK293 cells transfected with VSX2 or its variants (listed below images) plus H-MITF (middle panel) or its mi variant (bottom panel). IPs were performed with sheep IgG or VSX2 antibodies followed by western blot probed with MITF antibody. input refers to the 20% of whole protein lysate used for co-IP. (E) Luciferase assays in HEK293 cells transfected with the indicated expression vectors (x-axes) and the pGL3B-HMitf. Left graph: effects of VSX2 and its variants on reporter activity were not statistically significant. Right graph: H-MITF repressed reporter activity, whereas OTX1 enhanced reporter activity. Reporter activity in cells co-expressing of OTX1 and H-MITF was significantly higher than the sum of the factors expressed individually (** associated with lines over bars). H-MITF[mi] enhanced reporter activity, but reporter activity in cells co-expressing OTX1 and H- MITF[mi] was not significantly different than the sum of the two factors expressed individually. * P≤0.05; ** P≤0.01; *** P≤0.001.