1. A B C D Supplementary Figure 1 CMV p300
Luc activity (x104)
+ p300
- p300 p YY
(1g) (2g) (3g) 8 4
Luc activity (x104)
+ p300
- p300 p
HDAC
(1g) (2g) (3g) 8 4 p
HDAC
HDAC
(3g) (3g)
CMV
p300 D
HDAC1
HDAC2
HDAC3
-tubulin g
-FLAG
p300 Cooperation with HDAC3 and YY1 in Myc Repression. To demonstrate that p300 cooperates with YY1 and HDAC3 in Myc repression, rat fibroblasts were transfected with del-6 promoter plasmid along with a constant amount of p300 expression plasmid, and increasing concentrations of either HDAC3 or YY1 expression plasmids. Forty-eight hours after starvation, cells were serum stimulated for 2h then the luciferase activity in the cell extracts was determined. As shown in Fig. 6A, with a gradual increase of HDAC3, there was a progressive decrease in promoter activity of control and p300-containing samples. However, p300 and HDAC3 together inhibited the promoter more severely than either of the proteins alone. For example, in the absence of HDAC3, 1.0ug of p300 plasmid reduced the promoter activity to about 50% of the control (compare bar 2 with 1). With p300 and HDAC3 (3ug) together in the assay, the promoter activity dropped to about 6% (bar 8) of the initial activity (bar 1), or to about 12% of the activity observed with p300 in the absence of HDAC3 (bar 2). In the absence of p300, 3ug of HDAC3 only reduced the promoter activity by about 50% (compare bar 7 with bar 1). Thus, expressing p300 and HDAC3 together was more effective in inhibiting the Myc promoter than either plasmid alone indicating that p300 and HDAC3 cooperate with YY1 in repressing the Myc promoter. A similar dose dependent effect of YY1 was also observed when YY1 was assayed in the presence of p300 (Fig. 6B; compare bars 7 and 8 with bars 1 and 2, respectively). The above transfection assay was repeated with plasmids expressing either HDAC1 or HDAC2. In contrast to HDAC3, HDAC1 had only a modest effect in this assay while HDAC2 had no effect al all (Fig. 6C). Western immunoblotting showed that the three proteins were expressed at similar levels (Fig. 6D). Thus, consistent with ChIP assay results shown in Fig. 2D, only HDAC3 cooperated with p300 at significant levels in repressing Myc promoter