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. 2014 Jul 31;10(7):e1004520. doi: 10.1371/journal.pgen.1004520

Figure 6. Clims and LMO4 directly target and the Fgfr2 promoter to induce gene expression.

Figure 6

(A–B) ChIP-qPCR assays of Myc-tagged DN-Clim, Clim2, LMO4, and H3K4me3 enrichment at the indicated locations of the ±2 kb surrounding the Fgfr2 promoter (top panel) in (A) WT and (B) DN-Clim MECs. MECs were collected from three littermate mice then pooled for ChIP-qPCR experiments. The data suggest Clim2 binds approximately 0.5–1.0 kb upstream of the Fgfr2 TSS. When DN-Clim is bound to this region, LMO4 binding is lost and H3K4me3 levels are decreased. (C) Luciferase assays from MCF10A cells transiently expressing the Clim-binding promoter region of Fgfr2 in the pGL3-promoter luciferase reporter vector indicate Clim acts as an inducer of Fgfr2 expression. The DN-Clim expression vector was transiently titrated to demonstrate reduction of luciferase activity with increasing DN-Clim expression. (D) Expression of Fgfr2 in the MCF10A and MCF7 cells is reduced upon transient siRNA knockdown of Clim1, Clim2, and LMO4. Expression is normalized to GAPDH. Data represent mean ± SEM from three replicate experiments (C–D).