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In recent years, there has been an emphasis on personalizing breast cancer treatment in order to avoid the debilitating side effects caused by broad-spectrum chemotherapeutic drug treatment. Development of personalized medicine requires the identification of proteins that are expressed by individual tumors. Herein, we reveal the identity of plasma membrane proteins that are overexpressed in estrogen receptor α-positive, HER2-positive, and triple negative breast cancer cells. The proteins we identified are involved in maintaining protein structure, intracellular homeostasis, and cellular architecture; enhancing cell proliferation and invasion; and influencing cell migration. These proteins may be useful for breast cancer detection and/or treatment.

Cancer cells secrete factors that influence adjacent cell behavior and can lead to enhanced proliferation and metastasis. To better understand the role of these factors in oncogenesis and disease progression, estrogen and progesterone receptor positive MCF-7 cells, triple negative breast cancer MDA-MB-231, DT22, and DT28 cells, and MCF-10A non-transformed mammary epithelial cells were grown in 3D cultures. A special emphasis was placed on triple negative breast cancer since these tumors are highly aggressive and no targeted treatments are currently available. The breast cancer cells secreted factors of variable potency that stimulated proliferation of the relatively quiescent MCF-10A cells. The conditioned medium from each cell line was subjected to mass spectrometry analysis and a variety of secreted proteins were identified including glycolytic enzymes, proteases, protease inhibitors, extracellular matrix proteins, and insulin-like growth factor binding proteins. An investigation of the secretome from each cell line yielded clues about strategies used for breast cancer proliferation and metastasis. Some of the proteins we identified may be useful in the development of a serum-based test for breast cancer detection, diagnosis, prognosis, and monitoring.

Although substantial evidence has demonstrated that parity and 17β-estradiol (E2) reduce mammary carcinogenesis, it is not clear how this protection is conferred. Thus, we examined the effects of parity and E2 treatment in the mammary glands of ovariectomized 15 week-old virgin mice, 15 week-old primiparous mice, and 9 month-old retired breeders. E2 treatment significantly increased lipid peroxidation, protein carbonylation, and protein nitrosylation in the virgin mice, but not in the age-matched primiparous mice or retired breeders. Mammary gland expression of the oxidative stress response protein Cu/Zn superoxide dismutase was consistently reduced in all of the E2-treated mice regardless of parity. Expression of the oxidative stress and DNA repair protein apurinic endonuclease (Ape1) was significantly increased only in the mammary glands of the E2-treated retired breeders. These findings suggest that E2 and parity help to reduce mammary oncogenesis by maintaining the structure and function of proteins, lipids, and DNA., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Although estrogen receptor alpha (ERα) and 17β-estradiol play critical roles in protecting the cerebral cortex from ischemia-induced damage, there has been some controversy about the expression of ERα in this region of the brain. We have examined ERα mRNA and protein levels in the cerebral cortices of female mice at postnatal days 5 and 17 and at 4, 13, and 18 months of age. We found that although ERα transcript levels declined from postnatal day 5 through 18 months of age, ERα protein levels remained stable. Importantly, expression of the E2-regulated progesterone receptor gene was sustained in younger and in older females suggesting that age-related changes in estrogen responsiveness in the cerebral cortex are not due to the absence of ERα protein., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.

17β-estradiol (E2) plays critical roles in a number of target tissues including the mammary gland, reproductive tract, bone, and brain. Although it is clear that E2 reduces inflammation and ischemia-induced damage in the cerebral cortex, the molecular mechanisms mediating the effects of E2 in this brain region are lacking. Thus, we examined the cortical transcriptome using a mouse model system. Female adult mice were ovariectomized and implanted with silastic tubing containing oil or E2. After 7 days, the cerebral cortices were dissected and RNA was isolated and analyzed using RNA-sequencing. Analysis of the transcriptomes of control and E2-treated animals revealed that E2 treatment significantly altered the transcript levels of 88 genes. These genes were associated with long term synaptic potentiation, myelination, phosphoprotein phosphatase activity, mitogen activated protein kinase, and phosphatidylinositol 3-kinase signaling. E2 also altered the expression of genes linked to lipid synthesis and metabolism, vasoconstriction and vasodilation, cell-cell communication, and histone modification. These results demonstrate the far-reaching and diverse effects of E2 in the cerebral cortex and provide valuable insight to begin to understand cortical processes that may fluctuate in a dynamic hormonal environment.

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI). We demonstrated that treatment of ovariectomized C57BL/6J female mice with E2 increased the mRNA and protein levels of Trx, but decreased SOD1 and Ape1 mRNA and protein expression. In contrast, E2 treatment increased PDI protein levels but had no effect on PDI transcript levels. Interestingly, E2 treatment also increased two markers of cellular damage, lipid peroxidation and protein carbonylation. Our studies suggest that the decreased expression of SOD1 and Ape1 caused by E2 treatment may in the long term result in disruption of ROS regulation and play a role in endometrial carcinogenesis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

While it is well established that 17β-estradiol (E2) protects the rodent brain from ischemia-induced damage, it has been unclear how this neuroprotective effect is mediated. Interestingly, convincing evidence has also demonstrated that maintaining or increasing the expression of the oxidative stress response and DNA repair protein apurinic endonuclease 1 (Ape1) is instrumental in reducing ischemia-induced damage in the brain. Since E2 increases expression of the oxidative stress response proteins Cu/Zn superoxide dismutase and thioredoxin in the brain, we hypothesized that E2 may also increase Ape1 expression and that this E2-induced expression of Ape1 may help to mediate the neuroprotective effects of E2 in the brain. To test this hypothesis, we utilized three model systems including primary cortical neurons, brain slice cultures, and whole animals. Although estrogen receptor α and Ape1 were expressed in primary cortical neurons, E2 did not alter Ape1 expression in these cells. However, immunofluorescent staining and quantitative Western blot analysis demonstrated that estrogen receptor α and Ape1 were expressed in the nuclei of cortical neurons in brain slice cultures and that E2 increased Ape1 expression in the cerebral cortex of these cultures. Furthermore, Ape1 expression was increased and oxidative DNA damage was decreased in the cerebral cortices of ovariectomized female C57Bl/6J mice that had been treated with E2 and exposed to hypoxia. Taken together, our studies demonstrate that the neuronal microenvironment may be required for increased Ape1 expression and that E2 enhances expression of Ape1 and reduces oxidative DNA damage, which may in turn help to reduce ischemia-induced damage in the cerebral cortex and mediate the neuroprotective effects of E2., (Published by Elsevier Ltd.)

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease.

A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E(2), and that the E(2)-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E(2) reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E(2) and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E(2) treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E(2)-mediated neuroprotection may be conferred., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Estrogen receptor α (ERα) is a ligand-activated transcription factor that, upon binding hormone, interacts with specific recognition sequences in DNA. An extensive body of literature has documented the association of individual regulatory proteins with ERα. It has recently become apparent that, instead of simply recruiting individual proteins, ERα recruits interconnected networks of proteins with discrete activities that play crucial roles in maintaining the structure and function of the receptor, stabilizing the receptor-DNA interaction, influencing estrogen-responsive gene expression, and repairing misfolded proteins and damaged DNA. Together these studies suggest that the DNA-bound ERα serves as a nucleating factor for the recruitment of protein complexes involved in key processes including the oxidative stress response, DNA repair, and transcription regulation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants.

Estrogen receptor alpha (ERalpha) binds to specific target DNA sequences, estrogen response elements (EREs), to regulate estrogen-responsive gene expression. The progesterone receptor (PR) gene has been used extensively as a marker of estrogen responsiveness. Although we previously identified cis elements within 1 kb of the PR-B transcription start site that are associated with ERalpha and help to confer estrogen responsiveness, the identification of ERalpha binding sites far removed from the transcription start site suggested that long-range regulation of this gene may occur. We now show that eight regions of the PR gene from 311 kb upstream to 4 kb downstream of the PR-B transcription start site interact with ERalpha and that coactivator proteins and acetylated histones are selectively associated with these gene regions. Specific PR gene regions confer estrogen responsiveness to a heterologous reporter plasmid, and mutation of EREs within these regions diminishes estrogen-induced transactivation. Importantly, chromosome conformation capture assays reveal ERalpha- and ligand-dependent interactions between proximal and distal PR gene regions. Taken together, our studies suggest that distal regions of the PR gene participate in the dynamic regulation of this gene and that the coordinated action of proximal and distal PR gene regions allows cells to respond to changes in hormone levels with extraordinary versatility and sensitivity.

Background: During the course of normal cellular metabolism, oxygen is consumed and reactive oxygen species (ROS) are produced. If not effectively dissipated, ROS can accumulate and damage resident proteins, lipids, and DNA. Enzymes involved in redox regulation and DNA repair dissipate ROS and repair the resulting damage in order to preserve a functional cellular environment. Because increased ROS accumulation and/or unrepaired DNA damage can lead to initiation and progression of cancer and we had identified a number of oxidative stress and DNA repair proteins that influence estrogen responsiveness of MCF-7 breast cancer cells, it seemed possible that these proteins might be differentially expressed in normal mammary tissue, benign hyperplasia (BH), ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC)., Methods: Immunohistochemistry was used to examine the expression of a number of oxidative stress proteins, DNA repair proteins, and damage markers in 60 human mammary tissues which were classified as BH, DCIS or IBC. The relative mean intensity was determined for each tissue section and ANOVA was used to detect statistical differences in the relative expression of BH, DCIS and IBC compared to normal mammary tissue., Results: We found that a number of these proteins were overexpressed and that the cellular localization was altered in human breast cancer tissue., Conclusions: Our studies suggest that oxidative stress and DNA repair proteins not only protect normal cells from the damaging effects of ROS, but may also promote survival of mammary tumor cells.

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERalpha, Trx, and TrxR interact and ERalpha and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17beta-estradiol, Trx, and TrxR alter hydrogen peroxide (H(2)O(2)) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H(2)O(2) levels and transcription factor activity, aid ERalpha in regulating the expression of estrogen-responsive genes in target cells.

Apurinic/apyrimidinic endonuclease 1 or redox factor-1 (Ape1/Ref-1) is a pleiotropic cellular protein involved in DNA repair and, through its redox activity, enhances the binding of a select group of transcription factors to their cognate recognition sequences in DNA. Thus, we were intrigued when we identified Ape1/Ref-1 and a number of DNA repair and oxidative stress proteins in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Because Ape1/Ref-1 interacts with a number of transcription factors and influences their activity, we determined whether it might also influence ERalpha activity. We found that endogenously expressed Ape1/Ref-1 and ERalpha from MCF-7 human breast cancer cells interact and that Ape1/Ref-1 enhances the interaction of ERalpha with estrogen-response elements (EREs) in DNA. More importantly, Ape1/Ref-1 alters expression of the endogenous, estrogen-responsive progesterone receptor and pS2 genes in MCF-7 cells and associates with ERE-containing regions of these genes in native chromatin. Interestingly, knocking down Ape1/Ref-1 expression or inhibiting its redox activity with the small molecule inhibitor E3330 enhances estrogen responsiveness of the progesterone receptor and pS2 genes but does not alter the expression of the constitutively active 36B4 gene. Additionally, the reduced form of Ape1/Ref-1 increases and E3330 limits ERalpha-ERE complex formation in vitro and in native chromatin. Our studies demonstrate that Ape1/Ref-1 mediates its gene-specific effects, in part, by associating with endogenous, estrogen-responsive genes and that the redox activity of Ape1/Ref-1 is instrumental in altering estrogen-responsive gene expression.

Regulating gene expression is a complex process requiring the interaction of multiple transcription factors with their cognate recognition sequences. While these DNA-bound transcription factors are the primary drivers of gene expression, the capacity of a transcription factor to alter gene expression is tempered by its association with a host of coregulatory proteins that are recruited to the DNA-bound transcription factor. We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound estrogen receptor alpha (ERalpha) using an agarose-based electrophoretic mobility shift assay (EMSA). This method should be readily adapted to a variety of cultured cell lines, DNA sequences, and transcription factors and has the potential to provide valuable information about a wide variety of regulatory proteins involved in influencing gene expression.

RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the effect of a putative regulatory protein on gene expression can be delineated. Methods of assessing the effectiveness of the siRNA procedure using real time quantitative PCR and Western analysis are also included.

Background: DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor alpha (ERalpha) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERalpha using full-length receptor and endogenously-expressed nuclear proteins., Results: We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERalpha. Purified ERalpha and HeLa nuclear extracts were combined with oligos containing ERalpha binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERalpha, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERalpha, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERalpha activity and expression of estrogen-responsive genes., Conclusion: The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.

The differential recruitment of coregulatory proteins to the DNA-bound estrogen receptor alpha (ERalpha) plays a critical role in mediating estrogen-responsive gene expression. We previously isolated and identified retinoblastoma-associated proteins 46 (RbAp46) and 48 (RbAp48), which are associated with chromatin remodeling, histone deacetylation, and transcription repression, as proteins associated with the DNA-bound ERalpha. We now demonstrate that RbAp46 and RbAp48 interact with ERalphain vitro and in vivo, associate with ERalpha at endogenous, estrogen-responsive genes, and alter expression of endogenous, ERalpha-activated and -repressed genes in MCF-7 breast cancer cells. Our findings reveal that RbAp48 limits expression of estrogen-responsive genes and that RbAp46 modulates estrogen responsiveness in a gene-specific manner. The ability of RbAp46 and RbAp48 to interact with ERalpha and influence its activity reveals yet another role for these multifunctional proteins in regulating gene expression.

Estrogen receptor alpha (ERalpha) plays an important role in several human cancers. Most current ERalpha antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERalpha binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERalpha binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency, and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI) (TPBM), inhibited ERalpha binding to the flcERE (IC(50) approximately 3 microm) and inhibited ERalpha-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER-specific, because progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ERalpha, TPBM inhibited 17beta-estradiol (E(2))-ERalpha (IC(50) 9 microm) and 4-hydroxytamoxifen-ERalpha-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E(2).ERalpha recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E(2)-dependent growth of ERalpha-positive cancer cells (IC(50) of 5 microm). TPBM is not toxic to cells and does not affect estrogen-independent cell growth. TPBM acts outside of the ER ligand binding pocket, does not act by chelating the zinc in ER zinc fingers, and differs from known ERalpha inhibitors. Using a simple high throughput screen for inhibitors of ERalpha binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERalpha-mediated gene expression and estrogen-dependent growth of cancer cells.

The effects of estrogen on gene expression in mammary cells are mediated by interaction of the estrogen receptor (ER) with estrogen response elements in target DNA. Whereas the ER is the primary initiator of transcription, the recruitment of coregulatory proteins to the DNA-bound receptor influences estrogen responsiveness. To better understand how estrogen alters gene expression, we identified proteins associated with the DNA-bound ERalpha. Surprisingly, the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1), which is known primarily as a scavenger of superoxide, was associated with the DNA-bound receptor. We have now demonstrated that SOD1 interacts with ERalpha from MCF-7 cell nuclear extracts and with purified ERalpha and that SOD1 enhances binding of ERalpha to estrogen response element-containing DNA. Although SOD1 decreases transcription of an estrogen-responsive reporter plasmid in transiently transfected U2 osteosarcoma cells, RNA interference assays demonstrate that SOD1 is required for effective estrogen responsiveness of the endogenous pS2, progesterone receptor, cyclin D1, and Cathepsin D genes in MCF-7 breast cancer cells. Furthermore, ERalpha and SOD1 are associated with regions of the pS2 and progesterone receptor genes involved in conferring estrogen-responsive gene expression. Interestingly, when MCF-7 cells are exposed to 17beta-estradiol and superoxide generated by addition of potassium superoxide (KO2) to the cell medium, SOD1 levels are increased and tyrosine nitration, which is an indicator of oxidative stress-induced protein damage, is significantly diminished. Our studies have identified a new role for SOD1 in regulating estrogen-responsive gene expression and suggest that the 17beta-estradiol- and KO2-induced increase in SOD1 may play a role in the survival of breast cancer cells and the progression of mammary tumors.

Metastasis of cancer cells from the primary tumor is associated with poor prognosis and decreased overall survival. One protein implicated in inhibiting metastasis is the tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1). NM23-H1 is a multifunctional protein, which, in addition to limiting metastasis, has DNase and histidine protein kinase activities. We have identified new functions for NM23-H1 in influencing estrogen receptor alpha (ER alpha)-mediated gene expression. Using a battery of molecular and biochemical techniques, we show that NM23-H1 interacts with ER alpha and increases the ER alpha-estrogen response element (ERE) interaction. When NM23-H1 expression is increased in U2 osteosarcoma and MDA-MB-231 breast cancer cells, transcription of a transiently transfected, estrogen-responsive reporter plasmid is decreased. More importantly, when endogenous NM23-H1 expression is knocked down in MCF-7 human breast cancer cells using small interfering RNA, estrogen responsiveness of the progesterone receptor (PR), Bcl-2, cathepsin D, and cyclin D1 genes, but not the pS2 gene, is enhanced. Furthermore, NM23-H1 associates with the region of the PR gene containing the +90 activator protein 1 site, but not with the ERE-containing region of the pS2 gene, indicating that NM23-H1 mediates gene-specific effects by association with endogenous chromatin. Our studies suggest that the capacity of NM23-H1 to limit the expression of estrogen-responsive genes such as cathepsin D and Bcl-2, which are involved in cell migration, apoptosis, and angiogenesis, may help to explain the metastasis-suppressive effects of this protein. The complementary abilities of ER alpha and NM23-H1 together to influence gene expression, cell migration, and apoptosis could be key factors in helping to determine tumor cell fate.

Estrogen receptor alpha (ER alpha) is a ligand-activated transcription factor that regulates expression of estrogen-responsive genes. Upon binding of the ligand-occupied ER alpha to estrogen response elements (EREs) in DNA, the receptor interacts with a variety of coregulatory proteins to modulate transcription of target genes. We have isolated and identified a number of proteins associated with the DNA-bound ER alpha. One of these proteins, Rho guanosine diphosphate (GDP) dissociation inhibitor alpha (RhoGDI alpha), is a negative regulator of the Rho family of GTP-binding proteins. In this study, we demonstrate that endogenously expressed RhoGDI alpha is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, and that RhoGDI alpha binds directly to ER alpha, alters the ER alpha-ERE interaction, and influences the ability of ER alpha to regulate transcription of a heterologous estrogen-responsive reporter plasmid in transient transfection assays as well as endogenous, estrogen-responsive genes in MCF-7 cells. Our studies suggest that, in addition to the activity of RhoGDI alpha in the cytoplasm, it also influences ER alpha signaling in the nucleus.

The ligand-occupied estrogen receptor alpha (ERalpha) initiates changes in gene expression through its interaction with target DNA. The capacity of ERalpha to modulate gene expression is influenced by the association of the receptor with a variety of coregulatory proteins. To further understand the role of these coregulatory proteins in ERalpha-mediated transcription, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus estrogen response element. One of the proteins identified in this complex, flap endonuclease-1 (FEN-1), is required for DNA replication and repair. We show that FEN-1 interacts directly with ERalpha and enhances the interaction of ERalpha with estrogen response element-containing DNA. More importantly, chromatin immunoprecipitation and RNA interference assays demonstrate that endogenously expressed FEN-1 associates with the native pS2 gene in MCF-7 cells and influences estrogen-responsive gene expression. Interestingly, estrogen differentially regulates expression of FEN-1 in mouse uterine epithelial, stromal, and myometrial cells. Together, our studies help to elucidate the functional consequence of the ERalpha-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.

We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

The ability of estrogen receptor alpha (ERalpha) to modulate gene expression is influenced by the recruitment of a host of co-regulatory proteins to target genes. To further understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to DNA containing the consensus estrogen response element (ERE). One of the proteins identified in this complex, proliferating cell nuclear antigen (PCNA), is required for DNA replication and repair. We show that PCNA interacts with ERalpha in the absence and in the presence of DNA, enhances the interaction of ERalpha with ERE-containing DNA, and associates with endogenous estrogen-responsive genes. Interestingly, rather than altering hormone responsiveness of endogenous, estrogen-responsive genes, PCNA increases the basal expression of these genes. Our studies suggest that in addition to serving as a platform for the recruitment of DNA replication and repair proteins, PCNA may serve as a platform for transcription factors involved in regulating gene expression.

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.

Estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function. Although it is widely accepted that the estrogen-occupied receptor mediates its effects by interacting with estrogen response elements (EREs) residing in target genes, a number of estrogen-responsive genes contain no identifiable ERE. To understand how estrogen-responsive genes lacking EREs but containing activator protein 1 (AP-1) and Sp1 sites respond to hormone treatment, we have identified four discrete regions of the human progesterone receptor gene that contain AP-1 or Sp1 sites and examined their abilities to modulate transcription in the presence of 17 beta-estradiol, ICI 182,780, tamoxifen, raloxifene, genistein, or daidzein. Transient cotransfection assays demonstrated that ER alpha was a more potent activator of transcription than ER beta in bone, uterine, and mammary cells. The Sp1-containing promoters were substantially more potent transcriptional enhancers than the AP-1-containing promoters, but a 1.5-kb region of the human progesterone receptor gene containing both AP-1 and Sp1 sites was the most hormone-responsive promoter tested. The ability of ligands to modulate transcription of AP-1- or Sp1-containing promoters was dependent on cell context, but the expression of AP-1 or Sp1 proteins was not necessarily related to transcriptional response. Taken together, these studies have helped to delineate the roles of ER alpha and ER beta in modulating transcription of genes containing AP-1 and Sp1 sites and define the effects of widely used, pharmacologic agents in target cells with distinct cellular environments.

Estrogen receptor alpha (ER alpha) serves as a ligand-activated transcription factor, turning on transcription of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with the receptor to influence ER alpha-mediated transactivation. In this study, we have identified pp32, which interacts with the DNA binding domain of ER alpha when the receptor is free, but not when it is bound to an estrogen response element. Coimmunoprecipitation experiments demonstrate that endogenously expressed pp32 and ER alpha from MCF-7 breast cancer cells interact. Although pp32 substantially enhances the association of the receptor with estrogen response element-containing DNA, overexpression of pp32 in MCF-7 cells decreases transcription of an estrogen-responsive reporter plasmid. pp32 Represses p300-mediated acetylation of ER alpha and histones in vitro and inhibits acetylation of ER alpha in vivo. pp32 Also binds to other nuclear receptors and inhibits thyroid hormone receptor beta-mediated transcription. Taken together, our studies provide evidence that pp32 plays a role in regulating transcription of estrogen-responsive genes by modulating acetylation of histones and ER alpha and also influences transcription of other hormone-responsive genes as well.

Estrogen receptor alpha (ERalpha) interacts with basal transcription factors, coregulatory proteins, and chromatin modifiers to initiate transcription of the target genes. We have identified a novel interaction between ERalpha and the DNA repair protein 3-methyladenine DNA glycosylase (MPG) thereby providing a functional link between gene expression and DNA repair. Interestingly, the ERalpha-MPG interaction was enhanced by the presence of estrogen response element (ERE)-containing DNA. In vitro pull-down assays indicated that the interaction of ERalpha with MPG was direct and occurred through the DNA- and ligand-binding domains and the hinge region of the receptor. More importantly, endogenously expressed ERalpha and MPG from MCF-7 cells coimmunoprecipitated with ERalpha- and MPG-specific antibodies. The ERalpha-MPG interaction had functional consequences on the activities of both proteins. ERalpha 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 ERalpha 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.

The progesterone receptor (PR) gene is activated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is typically thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the human PR gene lacks a palindromic ERE sequence. We have identified an activating protein-1 (AP-1) site at +745 in the human PR gene that bound purified Fos and Jun and formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts. Surprisingly, mutating the +745 AP-1 site in the context of a 1.5-kb region of the PR gene significantly enhanced estrogen receptor (ER) alpha-mediated transactivation, suggesting that the wild-type +745 AP-1 site plays a role in inhibiting PR gene expression in the presence of hormone. In support of this idea, transient transfection assays demonstrated that increasing levels of Fos and Jun repressed transcription of a reporter plasmid containing the +745 AP-1 site. Fos levels were transiently increased, ERalpha levels were decreased, and Jun was dephosphorylated after MCF-7 cells were treated with estrogen. Chromatin immunoprecipitation assays demonstrated that Jun was associated with the +745 AP-1 site in the endogenous PR gene in the presence and in the absence of estrogen, but that ERalpha and Fos were only associated with the +745 AP-1 site after estrogen treatment of MCF-7 cells. Our studies suggest that the human PR gene is regulated by multiple transcription factors and that the differential binding of these dynamically regulated trans-acting factors influences gene expression.

The progesterone receptor (PR) gene is regulated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated by interaction of the ligand-occupied estrogen receptor (ER) with estrogen response elements (EREs) in target genes, the human progesterone receptor (PR) gene lacks a palindromic ERE. Promoter A of the PR gene does, however, contain an ERE half site upstream of two adjacent Sp1 sites from +571 to +595, the +571 ERE/Sp1 site. We have examined the individual contributions of the ERE half site and the two Sp1 sites in regulating estrogen responsiveness. Transient transfection assays demonstrated that both Sp1 sites were critical for estrogen-mediated activation of the PR gene. Interestingly, rather than decreasing transcription, mutations in the ERE half site increased transcription substantially suggesting that this site plays a role in limiting transcription. Chromatin immunoprecipitation assays demonstrated that Sp1 was associated with the +571 ERE/Sp1 site in the endogenous PR gene in the absence and in the presence of estrogen, but that ERalpha was only associated with this region of the PR gene after MCF-7 cells had been treated with estrogen. Our studies provide evidence that effective regulation of transcription through the +571 ERE/Sp1 site requires the binding of ERalpha and Sp1 to their respective cis elements and the appropriate interaction of ERalpha and Sp1 with other coregulatory proteins and transcription factors.

The progesterone receptor (PR) gene is induced by estrogen in reproductive and mammary tissues and in MCF-7 human breast cancer cells even though the human PR gene lacks an estrogen response element. We have identified a region from -80 to -34 in the PR gene that contains two Sp1 sites and confers estrogen responsiveness to a heterologous promoter in an estrogen and estrogen receptoralpha (ERalpha)-dependent manner. Sp1 present in MCF-7 nuclear extracts and purified Sp1 bind to and protect both Sp1 sites from DNase I cleavage, but the proximal Sp1 site is preferentially protected. Mutation of either Sp1 site decreases Sp1-DNA complex formation and ERalpha-mediated transactivation. ERalpha enhances Sp1 binding, but does not interact directly with the -80/-34 region. Our studies suggest that ERalpha confers estrogen responsiveness to the PR gene by enhancing Sp1 interaction with the Sp1 site in the -80/-34 region of the human PR gene.

Estrogen receptor-alpha (ERalpha) functions as a ligand-activated transcription factor that alters expression of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with ERalpha to influence estrogen-mediated transactivation. We have identified a novel coregulatory protein, template-activating factor-Ibeta (TAF-Ibeta), which binds to ERalpha in vitro when the receptor is not complexed with an estrogen response element. The central region of TAF-Ibeta interacts with both the DNA-binding domain and the carboxy-terminal region of ERalpha. Coimmunoprecipitation experiments demonstrate that TAF-Ibeta is associated with the unoccupied, but not the estrogen-occupied, ERalpha in MCF-7 breast cancer cells. Overexpression of TAF-Ibeta inhibits ERalpha-mediated transcription in a dose- dependent manner. TAF-Ibeta represses p300-mediated acetylation of histones and ERalpha in vitro and decreases ERalpha acetylation in vivo. TAF-Ibeta also binds to other nuclear receptor superfamily members and represses thyroid hormone receptor beta- induced transcription in transient transfection assays. Taken together, these data provide evidence that TAF-Ibeta regulates transcription of estrogen- responsive genes by modulating acetylation of histones and ERalpha and that the effects of TAF-Ibeta extend to other nuclear receptor superfamily members as well.

The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.

The human estrogen receptor (ER) induces transcription of estrogen-responsive genes upon binding to estrogen and the estrogen response element (ERE). To determine whether receptor-induced changes in DNA structure are related to transactivation, we compared the abilities of ER alpha and ER beta to activate transcription and induce distortion and bending in DNA. ER alpha induced higher levels of transcription than ER beta in the presence of 17 beta-estradiol. In circular permutation experiments ER alpha induced greater distortion in DNA fragments containing the consensus ERE sequence than ER beta. Phasing analysis indicated that ER alpha induced a bend directed toward the major groove of the DNA helix but that ER beta failed to induce a directed DNA bend. Likewise, the ER alpha DNA binding domain (DBD) and hinge region induced a bend directed toward the major groove of the DNA helix, but the ER beta DBD and hinge region failed to bend ERE-containing DNA fragments. Using receptor chimeras we demonstrated that the ER alpha DBD C-terminal extension is required for directed DNA bending. Transient transfection assays revealed that appropriately oriented DNA bending enhances receptor-mediated transactivation. The different abilities of ER alpha and ER beta to induce change in DNA structure could foster or inhibit the interaction of regulatory proteins with the receptor and other transcription factors and help to explain how estrogen-responsive genes are differentially regulated by these two receptors.

Estrogen receptor beta (ERbeta) 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, ERbeta 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 ERbeta 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 ERbeta 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 ERbeta-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 ERbeta to differentially activate transcription of genes containing divergent ERE sequences.

To understand how estrogen-responsive genes are regulated, we compared the abilities of estrogen receptors (ERs) alpha and beta to bind to and activate transcription through the consensus vitellogenin A2 ERE and the imperfect pS2, vitellogenin B1, and oxytocin (OT) EREs. Transient transfection experiments demonstrated that ERalpha and ERbeta induced the highest levels of transcription with the A2 ERE, intermediate levels of transcription with the OT ERE, and low levels of transcription with the pS2 and B1 EREs. ERalpha and ERbeta had higher affinities for the A2 ERE than for any of the three imperfect EREs but similar affinities for the pS2, B1, and OT EREs in gel mobility shift assays. ERalpha had a higher affinity and was a more potent activator of transcription than ERbeta. Interestingly, protease sensitivity assays demonstrated that A2, pS2, B1, and OT EREs induced distinct changes in ERalpha and ERbeta conformation thereby providing different functional surfaces for interaction with regulatory proteins involved in control of estrogen-responsive genes.

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.

To understand how hormones and antihormones regulate transcription of estrogen-responsive genes, in vivo footprinting was used to examine the endogenous pS2 gene in MCF-7 cells. While the consensus pS2 estrogen response element (ERE) half site was protected in the absence of hormone, both the consensus and imperfect ERE half sites were protected in the presence of estrogen. 4-Hydroxytamoxifen and ICI 182,780 elicited distinct footprinting patterns, which differed from those observed with vehicle- or with estrogen-treated cells suggesting that the partial agonist/antagonist and antagonist properties of 4-hydroxytamoxifen or ICI 182,780, respectively, may be partially explained by modulation of protein-DNA interactions. Footprinting patterns in and around the TATA and CAAT sequences were identical in the presence and in the absence of estrogen suggesting that the basal promoter is accessible and poised for transcription even in the absence of hormone. In vitro DNase I footprinting experiments demonstrated that the estrogen receptor bound to the pS2 ERE and that adjacent nucleotides were protected by MCF-7 nuclear proteins. These findings indicate that transcription of the pS2 gene is modulated by alterations in protein binding to multiple sites upstream of the basal promoter, but not by changes in protein-DNA interactions in the basal promoter.

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE). In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X. laevis vitellogenin A2 ERE and the human pS2 ERE. Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE. However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations. Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed. Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns. Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation. The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells.

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis-acting EREs.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.