Isolation of Estrogen-Responsive Genes in Human Breast Cancer Cells
Annual rept. 1 Sep 2000-1 Sep 2001
MANITOBA UNIV WINNIPEG
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The purpose of this proposal is to isolate estrogen-responsive genes in human breast cancer cells. Cisplatin and formaldehyde cross-link protein to DNA, formaldehyde also cross-links protein to protein. Chromatin immunoprecipitation ChIP using anti-estrogen receptor ER antibodies was applied to isolate ER-bound DNA in situ. This protocol should isolate all ER-bound DNA fragments. Our first specific aim was to establish a protocol to isolate DNA bound in situ to ER, and characterize this ER-bound DNA. We have carried out chromatin immunoprecipitation procedure using an anti-ER monoclonal antibody and an ER human breast cancer T5 cell to isolate ER-bound DNA. Southern blot analysis shows that ER-bound DNA contains ER-responsive genes such as PR, ER, pS2 and c-myc, but not control lambda-DNA. The pS2 gene promoter was analyzed using electrophoretic mobility shift assay, and three Sp1Sp3 binding sequences were identified. A high resolution mapping protocol to find location of ER along the pS2 gene promoter was developed. PCR results indicated that estradiol increases ER binding to pS2 promoter. We are testing the optimum conditions to construct an ER-bound genomic DNA library.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research