Abstract
BackgroundDNA-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.ResultsWe 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.ConclusionThe 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.
Highlights
DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression
To isolate novel proteins that associate with estrogen receptor α (ERα) and might influence estrogen-responsive gene expression, we developed a method that relied on the segregation of proteins on agarose gels and was based on the capacity of these proteins to associate with the estrogen response element (ERE)-bound receptor
We were able to take into consideration DNA-induced modulation of ERα conformation, which we have demonstrated alters recruitment of coregulatory proteins to the DNA-bound receptor [2,3,4,5]
Summary
DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. The p160 proteins steroid receptor coactivator 1 (SRC-1), transcription intermediary factor 2 (TIF-2), and amplified in breast cancer 1 (AIB1) interact with ERα in a hormone-dependent manner and enhance ERα-mediated transcription [10,11,12,13,14,15,16,17] Both SRC-1 and AIB1 as well as CREB binding protein (CBP) and p300/CBP-associated factor (pCAF) possess intrinsic histone acetyltransferase activity that has been implicated in enhancing gene expression by modifying chromatin structure [18,19,20,21,22,23,24]. ERα-associated coregulatory proteins have positive and negative effects on the ability of the receptor to activate transcription
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