A Novel Repressor of Estrogen-Regualted Genes for Breast Cancer Growth Suppression
Abstract
The objective of this project was to develop a system for generating estrogen receptor mutants targeting DNA sequences in estrogen receptor regulated genes in breast cancer cells so that they could be experimentally suppressed ("turned off") or activated ("turned on") in response to small molecules. These estrogen receptor mutants would allow testing of the effect of activating or suppressing specific estrogen regulated genes on the growth of breast cancers. To achieve this objective we set out to develop a novel type of genetically selected mutant estrogen receptor able to bind to the genes of specific estrogen receptor-regulated genes. These mutant receptors could then be converted into chimeric receptors to efficiently and quantitatively suppress both estrogen-dependent and estrogen-independent expression of estrogen-regulated growth stimulatory genes. We used information from a genetic selection done using our modified P222 challenge phage system to identify mutations of interest which were then incorporated into full-length estrogen receptor (ER) for further testing. To repress transcription of estrogen receptor regulated genes we created chimeras in which full length ER, or the ER DNA binding domain, is fused to a powerful KRAB repressor domain. We showed that these chimeras form powerful, easily- regulated, ligand-dependent repressors. Wild-type ER, fused to KRAB domains, was unable to repress transcription of the native p52 gene, while a KRAB-ER chimera containing a set of up-binding mutations we identified in our challenge phage selections was a powerful ligand-dependent repressor of both basal and estrogen-induced transcription of the pS2 gene. Further analysis of wild and mutant ERs revealed the surprising finding that the affinity of wild type ER for the imperfect ERE half site in the pS2 gene was 100- 200 fold lower than its affinity for the consensus ERE half site.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2000
- Accession Number
- ADA387868
Entities
People
- David J. Shapiro
Organizations
- University of Illinois Urbana–Champaign