We previously identified a coregulator, repressor of estrogen receptor activity (REA), that directly interacts with estrogen receptor (ER) and represses ER transcriptional activity. Decreasing the intracellular level of REA by using small interfering RNA knockdown or antisense RNA approaches in cells in culture resulted in a significant increase in the level of up-regulation of estrogen-stimulated genes. To elucidate the functional activities of REA in vivo, we have used targeted disruption to delete the REA gene in mice. The targeting vector eliminated, by homologous recombination, the REA exon sequences encoding amino acids 12 to 201, which are required for REA repressive activity and for interaction with ER. The viability of heterozygous animals was similar to that of the wild type, whereas homozygous animals did not develop, suggesting a crucial role for REA in early development. Female, but not male, heterozygous animals had an increased body weight relative to age-matched wild-type animals beginning after puberty. REA mRNA and protein levels in uteri of heterozygous animals were half that of the wild type, and studies with heterozygous animals revealed a greater uterine weight gain and epithelial hyperproliferation in response to estradiol (E2) and a substantially greater stimulation by E2 of a number of estrogen up-regulated genes in the uterus. Even more dramatic in REA heterozygous animals was the loss of down regulation by E2 of genes in the uterus that are normally repressed by estrogen in wild-type animals. Mouse embryo fibroblasts derived from heterozygous embryos also displayed a greater transcriptional response to E2. These studies demonstrate that REA is a significant modulator of estrogen responsiveness in vivo: it normally restrains estrogen actions, moderating ER stimulation and enhancing ER repression of E2-regulated genes.The biological activity of estrogens, acting through estrogen receptors (ERs), is critically dependent on coregulator proteins (coactivators or corepressors) that are recruited to the ligand-receptor complex at various gene regulatory sites. Recent work indicates that this is a highly dynamic situation, with the equilibrium between corepressors and coactivators in a cell and the nature of the hormonal ligand determining the state of nuclear receptor activation or inhibition (4,8,11,17,21,30,32,34,35,40,43,44,47).Coactivator and corepressor proteins assemble into distinct, dynamic multiprotein complexes. In the case of coactivators, these complexes are constituted of the SRC/p160 family of proteins, CREB binding protein (CBP) and/or p300, and other factors that are recruited in a temporally ordered fashion (4, 44) and up-regulate nuclear receptor activity, at least in part, through enhanced histone acetyltransferase activity (8,30,32). ATP-dependent chromatin remodeling complexes, such as SWI/SNF, and the TRAP-DRIP-ARC (mediator) complex, which act sequentially or combinatorially, also enhance gene transcription by facilitating RNA polymerase II recruitment to promoters and ...

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ER␣ are coexpressed in mammary epithelial cells. REA heterozygous (REA ؉͞؊ ) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA ؉͞؊ mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT͞ ERE-Luc and REA ؉͞؊ ͞ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in humans.prohibitin 2 ͉ nuclear receptor ͉ coregulator ͉ breast ͉ gene expression T he mammary gland is a dynamic tissue in which morphogenesis, epithelial differentiation, and physiological function are tightly regulated by estrogen and progesterone in accordance with pubertal development and reproductive cycles (1). Estrogen regulates mammary gland development and function through binding to the estrogen receptor ␣ (ER␣) and inducing the expression of ER␣ target genes such as progesterone receptor (PR) and cyclin D1. The phenotype of ER knockout mice has clearly shown that ER␣ plays a mandatory role in promoting mammary epithelial proliferation and mammary ductal growth after birth (2). Recent studies have demonstrated that the transcriptional activity of nuclear receptors, including ER␣ and PR, is determined not only by hormone binding but also by relative activities of nuclear receptor-associated coactivators and corepressors (3-5). Repressor of ER activity (REA) is a recently identified corepressor that interacts with select nuclear receptors such as ER (6, 7) and the orphan nuclear receptors chicken ovalbumin upstream promoter transcription factors I and II (COUP-TFI and II) (8). Unlike most other known corepressors that are recruited to nuclear receptors mainly in the absence of ligand or in the presence of antagonists, REA belongs to a subclass of corepressors that interacts dynamically with both agonist-and antagonist-occupied ER (6). REA and ot...

The Na+/H+ exchanger regulatory factor (NHE-RF; also known as ezrin-radixin-moesin binding protein 50) is a primary response gene under estrogen receptor (ER) control that may provide a link between estrogen action and the regulation of cytoskeletal and cell-signaling pathways. These studies were undertaken to define the human NHE-RF genomic regions and regulatory sequences mediating its robust estrogen responsiveness. Screening of a human genomic library yielded NHE-RF clones comprising the full gene, including the 5'-regulatory region and first exon, which were found to contain a large number (13) of consensus half-estrogen response elements (EREs), but to lack palindromic full EREs. Transfection-transactivation assays with wild-type and mutant ERs and reporter gene constructs linked to progressive deletions, or containing mutations, of the 5'-flanking region including a portion of exon I, and electrophoretic mobility and competitive gel shift assays were performed. These demonstrated direct ER interaction with the multiple half-ERE sites and the importance of the one proximal half-ERE and the multiple upstream half-EREs for eliciting the robust transcription activation of the NHE-RF gene by the estrogen-ER complex. Our findings highlight a paradigm for gene regulation via numerous half-ERE sites that expands the range of modes by which DNA recognition sites mediate the actions of this nuclear receptor.