Isolation of novel coregulatory protein networks associated with DNA-bound estrogen receptor alpha

Jennifer R. Schultz-Norton, Yvonne S. Ziegler, Varsha S. Likhite, John R. Yates, Ann M. Nardulli

Research output: Contribution to journalArticlepeer-review

Abstract

Background: DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor α (ERα) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERα using full-length receptor and endogenously-expressed nuclear proteins. Results: We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERα. Purified ERα and HeLa nuclear extracts were combined with oligos containing ERα binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERα, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERα, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERα activity and expression of estrogen-responsive genes. Conclusion: The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.

Original languageEnglish (US)
Article number97
JournalBMC Molecular Biology
Volume9
DOIs
StatePublished - Oct 30 2008

ASJC Scopus subject areas

  • Molecular Biology

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