Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.     

The plasticity of estrogen receptor-DNA complexes: binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3bp. This study investigates the extent to which estrogen receptors, ERalpha and ERbeta can bind target sequences not considered as conventional EREs. We determined the effect of spacer size (n=0-4) on the binding affinity and conformation of ERalpha and ERbeta in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cEREn, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cEREn complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.     

Selective recognition of distinct classes of coactivators by a ligand-inducible activation domain

How nuclear receptors (NRs) coordinate the sequential, ligand-dependent recruitment of multiple coactivator complexes (e.g., SRC complexes and Mediator) that share similar receptor binding determinants is unclear. We show that although the receptor binding subunits of these complexes (i.e., SRCs and Med220, respectively) share overlapping binding sites on estrogen receptor alpha (ERalpha), information contained in the receptor-coactivator interface allows the receptor to distinguish between them. In support of this conclusion, we have identified an ERalpha AF-2 point mutant (L540Q) that selectively binds and recruits Med220, but not SRCs, both in vitro and in vivo. In cells expressing this mutant, the recruitment of Med220 to the pS2 promoter is delayed, and the expression of the vast majority of estrogen target genes is impaired, suggesting a nearly global functional interdependence of these coactivators. Collectively, our results suggest that "facilitated recruitment," rather than competition, drives the sequential recruitment of SRC complexes and Mediator by NRs.