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

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 alpha (ERalpha) 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 ERalpha using full-length receptor and endogenously-expressed nuclear proteins.

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.

We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERalpha. Purified ERalpha and HeLa nuclear extracts were combined with oligos containing ERalpha 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 ERalpha, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERalpha, 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 ERalpha activity and expression of estrogen-responsive genes.