Fig. 5.

MACROD2 overexpression can induce estrogen regulated genes and increases p300 binding to estrogen response elements in response to tamoxifen. (A) The parental MCF-7 and TamR clones were seeded in assay media without estrogen and tamoxifen and then RNA was harvested from these cells and RT-PCR was performed on a subset of estrogen responsive genes (shown). GAPDH was used as a loading control. (B) Parental MCF-7 cells and TamR cells were seeded in assay conditions and then vehicle (ethanol) or tamoxifen was added to the cells. The cells were then harvested and subjected to chromatin immunoprecipitation (ChIP) as described in the text using antibodies against MACROD2, ER, p300, and a control [IgG(−)]. Quantitative PCR was then performed using primers that encompass estrogen response elements in promoter or enhancer regions of known ER target genes (primers in SI Appendix, Table S5). Results are representative of duplicate samples comparing quantitative real time PCR results after controlling for input DNA and then analyzing relative fold differences between tamoxifen and vehicle control. Parental MCF-7 and two representative TamR clones are shown.