Figure 9.

CITED1 is recruited to the TGF-α promoter in an estrogen-dependent manner but not to the pS2 promoter. In vivo binding of ERα or CITED1 to either the pS2 promoter or the TGF-α promoter was examined by chromatin immunoprecipitation (ChIP) assay. (A) MCF-7 cells infected with vector or CITED1-expressing retroviruses were treated with (+) or without (−) 100 nM 17β-estradiol (E2) for 45 min and fixed immediately by formaldehyde. Soluble chromatin was prepared from the fixed cells by sonication followed by density-gradient centrifugation and subjected to immunoprecipitation using anti-ERα or anti-CITED1 antibodies. Co-precipitated genomic DNA was released from the cross-linked protein by heat and then amplified by polymerase chain reaction (PCR) using primers that annealed to the proximal region of the pS2 or TGF-α promoters. Positions of the PCR primers are indicated by pairs of arrows in the diagrams of each promoter; the transcriptional initiation site is defined as nucleotide number 1. The presence of the pS2 and TGF-α promoter DNA in the chromatin preparations before immunoprecipitation was confirmed by PCR (Input). (B) Quantitative aspects of recruitment of ERα and CITED1 to the pS2 and TGF-α promoters. Signal intensities of the PCR products shown in panel A were quantitated by densitometry and expressed as proportion of input.