Direct Visualization of Estrogen Receptor-Mediated Transcription in Living Cells
Abstract
Estrogen stimulates proliferation of breast cancer cells whereas antagonists oppose their action. To study the molecular mechanisms of ligand-dependent regulation of transcription, we generated a cell line derived from a parent cell line containing an integrated tandem array of a mouse mammary tumor virus/Harvey viral ras (MMTV/v-Ha-ras) reporter and a vector for cherry red fluorescence-estrogen receptor (ER)pbox mutant which recognizes glucocorticoid response elements (GREs). We observed ERpbox binding to the tandem array in an estradiol (E2)-dependent manner. We also observed concurrent transcription by RNA fluorescent in situ hypbridization (FISH). RNA transcription correlated with ERpbox signal on the tandem array until steady state levels were reached. Chromatin immunoprecipitation (ChIP) assays showed recruitment of ERpbox to MMTV promoter and endogenous serum- and glucocorticoid-regulated protein kinase (Sgk), promoter. These studies in live cells demonstrate ERpbox binding to the MMTV promoter and transcription in an E2-dependent manner. Moreover, they demonstrate that ER pbox fusion protein also can bind to the GRE of an endogenous target gene. Live cell imaging using ERpbox and the MMTV tandem array, in combination with ChIP and RNA FISH, are powerful techniques to visualize the mechanisms of transcriptional regulation by E2 and selective estrogen receptor modulators (SERMs) used for treatment of breast cancer. This cell line also may be a rapid and useful tool for drug screening of novel SERMs.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2007
- Accession Number
- ADA573348
Entities
People
- Paul M. Yen
Organizations
- Johns Hopkins University