Estrogen receptor α interaction with estrogen response element half-sites from the rat prolactin gene

Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ER alpha) protein to study interactions of ER alpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ER alpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ER alpha similar to that between mER alpha and a consensus ERE in gel shift assays. The ER alpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ER alpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ER alpha/half-site complex had a larger molecular weight and higher negative charge than the ER alpha/consensus ERE complex. Similar results were observed with purified human ER alpha, showing that the ER alpha/half-site complex contained only ER alpha and oligonucleotides. These results are best explained by a model in which a dimer of ERa is bound to two half-site oligonucleotides. We propose that two ER alpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene.