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. 2012 Feb 22;7(2):e32449. doi: 10.1371/journal.pone.0032449

Figure 4. P300/PCAF complexes with ZEB1 on the miR-200c/141 promoter and requires the CH3 domain of P300 for transcription.

Figure 4

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) (A). Immunoprecipitation experiments show PCAF acetylates ZEB1 following transfection with ZEB1 ± PCAF expression vectors (24 hrs) (B). DNA pull-down assay using a wild type (wt) or mutational E-box/Z-box sequence for the miR-200c/141 promoter after co-transfection of HuCCT-1 shows binding of ZEB1 and PCAF to the wt promoter sequence (C), and p300/PCAF/ZEB1 binding to the wt promoter, which is unaffected by TGF-β1 treatments (5 ng/mL; 24 hrs) (D). Luciferase assay for miR-200c/141 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; ***, P<0.001; Student's t -test) (E).