Ectopic expression of wild-type BRCA1 in Brca1-deficient MEFs restores
ligand-independent repression of ERα transactivation in a histone
deacetylase (HDAC)-dependent manner. (A and
B) Brca1−/− MEFs in estrogen-free media were
transfected with pERE-TK-Luc without (−) or with (+) pRSV-ERα,
pCDNA3.1-BRCA1 expressing wild-type human BRCA1 (WT), or pCDNA3.1-BRCA1
derivatives bearing missense mutants A1708E, Q356R, A1708E/Q356R, or
C64G before assay for luciferase activity. Where indicated,
trichostatin A (TSA; 330 nM) was also included. (C)
Brca1−/− MEFs in estrogen-free media were untransfected (lane 1) or
cotransfected with expression vectors for ERα and either wild-type
BRCA1 (lane 2) or various BRCA1 mutant derivatives (lanes 3–6) as
indicated. Cells were lysed, and immunoprecipitated BRCA1 and ERα
were subjected to immunoblot analysis using antibodies specific for
BRCA1 (Top) or ERα (Middle). Immunoblot
analysis of the nuclear matrix protein p84 (Bottom)
indicates that nearly equivalent amounts of each cell lysate were used
in the immunoprecipitations.