Estrogen receptor alpha requires no accessory factors for high-affinity binding to a consensus response element

Area of Science:

• Molecular Biology
• Endocrinology
• Genetics

Background:

• Estrogen receptor (ER) alpha is a key regulator of gene expression.
• ERalpha is believed to bind DNA as a homodimer to estrogen response elements (EREs).
• Previous studies could not definitively exclude the presence of other proteins in the ERalpha/ERE complex.

Purpose of the Study:

• To rigorously characterize the protein composition of the ERalpha/ERE complex.
• To determine if additional proteins are required for high-affinity ERalpha binding to DNA.
• To investigate if ERalpha binding in mammalian tissue extracts mirrors in vitro findings.

Main Methods:

• Overexpression of mouse ERalpha in a baculovirus system.
• Gel shift assays to analyze ERalpha-ERE complex formation.
• Molecular weight determination of the complex.
• Purification of recombinant ERalpha and analysis by silver stain.
• Ferguson analysis of purified ERalpha and nuclear extracts.
• High-affinity binding studies (Kd determination).
• Analysis of rat uterine cytosol by Ferguson analysis.

Main Results:

• Recombinant ERalpha bound 17beta-estradiol and a consensus vitellogenin ERE with high affinity.
• Gel shift assays consistently showed a single complex with a 2:1 ERalpha:ERE ratio.
• Molecular weight and Ferguson analysis indicated the complex consisted solely of two ERalpha proteins and one ERE.
• Purified ERalpha demonstrated high-affinity binding (Kd = 0.92 ± 0.20 nM) independently.
• ERalpha from rat uterine cytosol exhibited identical behavior to purified ERalpha, suggesting homodimerization.

Conclusions:

• Estrogen receptor alpha binds to consensus estrogen response elements predominantly as a homodimer.
• No other proteins are necessary for high-affinity ERalpha binding to DNA.
• ERalpha functions as a homodimer in estrogen-responsive mammalian tissues, consistent with in vitro findings.