P300/PCAF complexes with ZEB1 on the <i>miR-200c/141</i> promoter and requires the CH3 domain of P300 for transcription.

<p>Immunoprecipitation of P300 following transfection with the indicated protein expression vectors verifies P300/ZEB1 and P300/PCAF interactions, which were unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>A</b>). Immunoprecipitation experiments show PCAF acetylates ZEB1 following transfection with ZEB1 ± PCAF expression vectors (24 hrs) (<b>B</b>). DNA pull-down assay using a wild type (wt) or mutational E-box/Z-box sequence for the <i>miR-200c/141</i> promoter after co-transfection of HuCCT-1 shows binding of ZEB1 and PCAF to the wt promoter sequence (<b>C</b>), and p300/PCAF/ZEB1 binding to the wt promoter, which is unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (<b>D</b>). Luciferase assay for <i>miR-200c/141</i> promoter activity following transfection with wild type or CH3 deleted P300 expression vector in HuCCT-1 parent cells shows the CH3 domain is required for miR transcription. (n = 3 independent experiments; ***, <i>P</i><0.001; Student's <i>t</i> -test) (<b>E</b>).