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. 1998 Nov 10;95(23):13501–13506. doi: 10.1073/pnas.95.23.13501

Figure 5.

Figure 5

The SV40 enhancer or the recruitment of PCAF counteracts HDAC-mediated repression. (A) The reporter constructs Gal-TATA-MG (Left, −SVE) and Gal-TATA-MG-SVE (Right, +SVE), which differ through SV40 enhancer sequences (SVE) downstream of the transcribed MG gene, were transfected into mouse B78 cells along with the CMV-hGH gene as an internal control and with Gal4-VP16, Gal4-VP16 mad, or Gal4-VP16 madpro as transactivators. Gal4-VP16 mad contains the Sin3 interaction domain (SID) of the Mad protein, whereas Gal4-VP16 madpro is mutated within SID, and therefore does not interact with Sin3 (30). RNA from transfected cells was analyzed with S1-nuclease assays. Transactivation by Gal4-VP16 mad is significantly less (12-fold) than transactivation by Gal4-VP16 and Gal4-VP16 madpro. (B) Gal4-PCAF counteracts Mad-mediated repression of VP16 transactivation. The LEX-TATA-MG-Gal reporter construct was cotransfected into B78 cells with expression vectors encoding Lex-VP16 madpro (left lane), LexVP16 mad (middle and right lanes), and Gal4-PCAF (right lane). The CMV-hGH plasmid was included as an internal control for transfection efficiency. RNA was analyzed in S1-nuclease assays.