Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer.

Estrogens, acting through their nuclear receptors have a broad impact on target cells, eliciting a transcriptional response program that involves gene repression as well as gene stimulation. While much is known about the mechanisms by which the estrogen-occupied estrogen receptor (ER) stimulates gene expression, the molecular events that lead to gene repression by the hormone-ER complex are largely unknown. Because estradiol represses expression of the cyclin G2 gene, which encodes a negative regulator of the cell cycle, our aim was to understand the mechanism by which cyclin G2 is repressed by estrogen. We show that cyclin G2 is a primary ER target gene in MCF-7 breast cancer cells that is rapidly and robustly down-regulated by estrogen. Promoter analysis reveals a responsive region containing a halfestrogen response element and GC-rich region that interact with ER and Sp1 proteins. Mutation of the half-ERE abrogates hormone-mediated repression. Mutational mapping of receptor reveals a requirement for its N-terminal region and DNA binding domain to support cyclin G2 repression. Following estradiol treatment of cells, chromatin immunoprecipitation analyses reveal recruitment of ER to the cyclin G2 regulatory region, dismissal of RNA polymerase II, and recruitment of a complex containing N-CoR and histone deacetylases, leading to a hypoacetylated chromatin state. Our study provides evidence for a mechanism by which the estrogen-occupied ER is able to actively repress gene expression in vivo and indicates a role for nuclear receptor corepressors and associated histone deacetylase activity in mediating negative gene regulation by this hormone-occupied nuclear receptor.In target cells, estrogen hormones act via their nuclear receptors to elicit a diverse transcriptional response program that involves gene repression as well as gene stimulation (1-3). Estrogen receptors (ERs) 3 share their modular domain structure with the other members of the steroid and nuclear receptor superfamily and are comprised of an N-terminal domain characterized by a ligand-independent activation function (AF-1), a central DNA binding domain (DBD) followed by a hinge region, and a C-terminal ligand binding domain (LBD), which contains a ligand-dependent activation function (AF-2) important for coregulator recruitment (4, 5).In the mammary gland, the actions of estrogens are essential for normal growth and development. In breast cancer, the presence of ERs is associated with likely response to endocrine therapy, most usually treatment with antiestrogens such as tamoxifen, or estrogen depletion by the use of aromatase inhibitors (6). Estrogens enhance the proliferation of ER-positive breast cancer cells, and recent studies have shown that this enhancement of proliferation involves both stimulation of the expression of many genes associated with cell cycle progression, as well as the suppression of genes that block the cell cycle (7). Most previous studies, focusing on the mechanisms that lead to stimulation of gene expression, have demonstrat...

Estrogen-regulated gene expression is dependent on interaction of the estrogen receptor (ER) with the estrogen response element (ERE). We assessed the ability of the ER to activate transcription of reporter plasmids containing either the consensus vitellogenin A2 ERE or the imperfect pS2, vitellogenin B1, or oxytocin (OT) ERE. The A2 ERE was the most potent activator of transcription. The OT ERE was significantly more effective in activating transcription than either the pS2 or B1 ERE. In deoxyribonuclease I (DNase I) footprinting experiments, MCF-7 proteins protected A2 and OT EREs more effectively than the pS2 and B1 EREs. Limited protease digestion of the A2, pS2, B1, or OT ERE-bound receptor with V8 protease or proteinase K produced distinct cleavage products demonstrating that individual ERE sequences induce specific changes in ER conformation. Receptor interaction domains of glucocorticoid receptor interacting protein 1 and steroid receptor coactivator 1 bound effectively to the A2, pS2, B1, and OT ERE-bound receptor and significantly stabilized the receptor-DNA interaction. Similar levels of the full-length p160 protein amplified in breast cancer 1 were recruited from HeLa nuclear extracts by the A2, pS2, B1, and OT ERE-bound receptors. In contrast, significantly less transcriptional intermediary factor 2 was recruited by the B1 ERE-bound receptor than by the A2 ERE-bound receptor. These studies suggest that allosteric modulation of ER conformation by individual ERE sequences influences the recruitment of specific coactivator proteins and leads to differential expression of genes containing divergent ERE sequences.

Estrogen receptor ␤ (ER␤) activates transcription by binding to estrogen response elements (EREs) and coactivator proteins that act as bridging proteins between the receptor and the basal transcription machinery. Although the imperfect vitellogenin B1, pS2, and oxytocin (OT) EREs each differ from the consensus vitellogenin A2 ERE sequence by a single base pair, ER␤ activates transcription of reporter plasmids containing A2, pS2, B1, and OT EREs to different extents. To explain how these differences in transactivation might occur, we have examined the interaction of ER␤ with these EREs and monitored recruitment of the coactivators amplified in breast cancer (AIB1) and transcription intermediary factor 2 (TIF2). Protease sensitivity, antibody interaction, and DNA pull-down assays demonstrated that ER␤ undergoes ERE-dependent changes in conformation resulting in differential recruitment of AIB1 and TIF2 to the DNA-bound receptor. Overexpression of TIF2 or AIB1 in transient transfection assays differentially enhanced ER␤-mediated transcription of reporter plasmids containing the A2, pS2, B1, and OT EREs. Our studies demonstrate that individual ERE sequences induce changes in conformation of the DNA-bound receptor and influence coactivator recruitment. DNA-induced modulation of receptor conformation may contribute to the ability of ER␤ to differentially activate transcription of genes containing divergent ERE sequences.Transcription activation requires the coordinated interaction of multiple transacting factors with DNA recognition sites and other regulatory proteins. In response to cellular signals, transcription factors bind to specific DNA sequences residing in target genes and interact with numerous regulatory proteins to form an active transcription complex and initiate changes in gene expression. This multistep process provides a mechanism by which cells expressing different populations of proteins can differentially regulate expression of target genes.The nuclear receptor superfamily is composed of a large number of transcription factors that bind to hormone response elements and modulate transcription. Estrogen receptors (ERs) 1 ␣ and ␤ are members of this nuclear receptor superfamily (1-5) and function as ligand-induced transcription factors that modulate expression of estrogen-responsive genes. Upon binding hormone, the ER undergoes a conformational change and binds to estrogen response elements (EREs) residing in target genes to initiate changes in transcription (6). The hormone-induced modulation of receptor conformation has been documented in the ligand-binding domains (LBDs) of 17␤-estradiol-and raloxifene-bound ER␣ (7) and in genistein-and raloxifene-bound ER␤ (8), with the most striking changes in conformation occurring in the positioning of helix 12 of the LBD. In addition to modulating receptor conformation, ligand binding influences the interaction of the ER with coactivator proteins such as steroid receptor coactivator 1 (SRC1) (9), transcription intermediary factor 2 (TIF2/GRIP1) (10 -12), amplif...

Estrogen receptor ␣ (ER␣) interacts with basal transcription factors, coregulatory proteins, and chromatin modifiers to initiate transcription of the target genes. We have identified a novel interaction between ER␣ and the DNA repair protein 3-methyladenine DNA glycosylase (MPG) thereby providing a functional link between gene expression and DNA repair. Interestingly, the ER␣-MPG interaction was enhanced by the presence of estrogen response element (ERE)-containing DNA. In vitro pull-down assays indicated that the interaction of ER␣ with MPG was direct and occurred through the DNAand ligand-binding domains and the hinge region of the receptor. More importantly, endogenously expressed ER␣ and MPG from MCF-7 cells coimmunoprecipitated with ER␣-and MPG-specific antibodies. The ER␣-MPG interaction had functional consequences on the activities of both proteins. ER␣ increased MPG acetylation, stabilized the binding of MPG with hypoxanthine-containing oligos, and enhanced MPG-catalyzed removal of hypoxanthine from DNA. In turn, MPG dramatically stabilized the interaction of ER␣ with ERE-containing oligos, decreased p300-mediated acetylation of the receptor, and reduced transcription of simple and complex ERE-containing reporter plasmids in a dose-dependent manner. Our studies suggest that recruitment of MPG to ERE-containing genes influences transcription and plays a role in maintaining integrity of the genome by recruiting DNA repair proteins to actively transcribing DNA.Estrogen is critical for the growth, development, and homeostasis of neural, skeletal, cardiovascular, and reproductive tissues (1, 2). The actions of estrogen are mediated by two members of the nuclear receptor family, estrogen receptors (ERs) 1 ␣ and ␤. ER␣ is, however, generally a more potent transcriptional activator than ER␤ (3-5). Like other nuclear receptor family members, ER␣ has a modular structure. At the amino terminus is the A/B region with its autonomous activation function 1 (6). Region C encompasses the DNA binding domain and is linked by the hinge domain to region E, which contains the ligand-binding domain (LBD, Ref. 7). The DNAbinding domain has two zinc finger motifs that are involved in DNA binding. The LBD contains a hydrophobic pocket that interacts with estrogens and antiestrogens. The LBD also contains a ligand-dependent activation function 2, which is responsible for interaction of the receptor with coregulatory proteins (8 -10).ER␣ binds to estrogen response elements (EREs) in target genes to initiate changes in transcription. The consensus ERE is comprised of the palindromic sequence GGTCAnnnTGACC and is found in the Xenopus laevis vitellogenin A2 gene (11). In addition to interacting with EREs, ER␣ modulates transcription through its interaction with components of the basal transcription machinery, regulatory proteins, and chromatin modifiers (12). ER␣ interacts with proteins in the basal transcription complex including TATA-binding protein (13). The coregulatory proteins steroid receptor coactivator 1, transcription interm...