A fusion protein of the estrogen receptor (ER) and nuclear receptor corepressor (NCoR) strongly inhibits estrogen-dependent responses in breast cancer cells.
Nuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of co-transfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.
Chien
P Y
PY
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Ito
M
M
Park
Y
Y
Tagami
T
T
Gehm
B D
BD
Jameson
J L
JL
D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D051548:Histone Acetyltransferases; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007668:Kidney; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D056971:Nuclear Receptor Co-Repressor 1; D056920:Nuclear Receptor Coactivator 1; D011960:Receptors, Estrogen / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C539127:NCOR1 protein, human; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C012589:trichostatin A; D004958:Estradiol; D004247:DNA; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1