FIGURE 4.

p300 binds to the core promoter region and enhances transcription. A, ChIP-qPCR assay of p300, Sp3 and acetylated H3. B, luciferase reporter assay. The reporter construct was described in the legend for Fig. 3B, and co-transfected with empty vector (pcDNA3) or the p300 expression vector (pcDNA3-p300) into ANOS (a), Saos2 (b), or TAKAO (c). The fold-increase was calculated based on empty vector activity. C, down-regulation of ChM-I gene expression by siRNA for Sp3 and/or p300. siRNAs for Sp3 and/or p300 were transfected into ANOS, and the expression of ChM-I, Sp3, and p300 was analyzed by RT-PCR. The expression of ChM-I was semi-quantified taking the value for endogenous expression as 1.0 and is demonstrated at the top. D, ChIP-qPCR assay for the modification of H3K9 (D) and for the binding of transcription regulators (E): cross-linked DNA-protein complexes were prepared from ANOS treated with or without siRNA for p300 and used for ChIP-qPCR assay.