<i>Zfp423</i> overexpression represses intron 5 enhancer activity in P19 cells.

<p>(<i>A</i>) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against <i>Zfp423</i> or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (<i>B</i>) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (<i>C</i>) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and <i>Ebf1</i> knockdown shows comparable reductions in enhancer activity (p<10⁻⁷, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and <i>Ebf1</i> knockdown showed further reduction in reporter activity (p = 0.03–0.0000001), while shRNA against endogenous <i>Zfp423</i> showed no additional effect on activity in <i>Ebf1</i> knockdown cells. Each construct was assayed in duplicate for each of three independent DNA preparations. (<i>D–E</i>) Immunofluorescent detection of Zfp423 by either E20 (<i>D</i>) or our custom affinity purified antibody (<i>E</i>) in P19 cells transfected with plasmid expressing EGFP and either scrambled control or Zfp423-targeted shRNA shows specific reduction or loss of immunoreactivity in nuclei of Zfp423 depleted cells (outline in middle panels). (<i>F</i>) Western blotting with either commercial (left panel) or custom (right panel) antibody showed effective reduction of P19 Zfp423 levels after transfection with plasmid expressing <i>Zfp423</i>-directed shRNA compared with internal loading controls. Repeated experiments showed 60–99% reduction. (<i>G</i>) Western blotting with a pan-EBF antibody showed moderate reduction (44–68% of control levels) after transfection with plasmid expressing <i>Ebf1</i>-directed shRNA. (<i>H</i>) Western blotting showed overexpression of ZNF423 in pcDNA-FLAG-ZNF423 transfected cells. The transfected product showed 1.4× endogenous level, indicating 2.4-fold expression level for endogenous and transfected products combined.