Estrogen-responsive Promoter Research Articles

Coactivator-mediated estrogen response in human squamous cell carcinoma lines

Steroid hormones such as 17beta-estradiol (E2) are critical to diverse cellular processes including tumorigenesis. A number of cofactors such as nuclear receptor corepressor (NCoR), CREB-binding protein (CBP), and steroid receptor coactivator 1 (SRC-1) interact with estrogen receptors (ERs) to regulate transcriptional repression or activation of target genes. Estrogen signaling in non-reproductive tract tissues such as skin is less well characterized and the effectiveness of anti-estrogen therapy for cancer arising from these tissues is unknown. We show that tamoxifen (TAM) treatment inhibited cell cycle progression and proliferation of human cancer lines derived from stratified squamous epithelium squamous cell carcinoma (SCC). E2 had no effect on proliferation of these lines despite low levels of ERalpha expression. The E2 treatment promoted displacement of the NCoR from ERalpha and recruitment of CBP to the receptor. SRC-1 expression was not detected in these SCC lines; however, transient transfection of SRC-1, CBP, or both coactivators enhanced transactivation of an estrogen responsive promoter in cancer cells treated with E2 or TAM. In stable clones expressing SRC-1, the coactivator was recruited to ERalpha along with CBP in E2 but not in TAM-treated cells. SRC-1 expression restored the E2-mediated proliferative response to human SCC lines. This increased proliferation correlated with increased extracellular signal regulated kinase 1 (ERK1) expression. SRC-1 and CBP were recruited to the proximal ERK1 promoter region in E2 but not in TAM-treated cells. We concluded that SRC-1 was a key molecular determinant of estrogen-mediated proliferation in human SCC lines.

Interplay of Genes Regulated by Estrogen and Diindolylmethane in Breast Cancer Cell Lines

Diindolylmethane (DIM), a biologically active congener of indole-3-carbinol (I3C) derived from cruciferous vegetables, is a promising agent for the prevention of estrogen-sensitive cancers. Both DIM and estrogen affect transcription of genes by binding receptors, such as aryl hydrocarbon receptor (AhR) or estrogen receptors (ER). Gene regulation by DIM and estradiol (E2) can be very complex. While DIM typically binds the AhR, this complex can directly associate with the ER, recruit co-activators that bind to estrogen-responsive promoters, and activate transcription. Alternately, DIM can bind the ER directly. In this study, we have analyzed gene expression using microarray profiling and quantitative real time-polymerase chain reaction in MCF7 breast cancer cells treated with E2 (1 nM) or DIM (25 microM) alone or in combination for 16 h. The interplay of E2 and DIM was reflected in the expression of a subset of genes (<90) in which the combination of E2 and DIM acted either additively or antagonistically to alter gene expression.

WW Domain Binding Protein-2, an E6-Associated Protein Interacting Protein, Acts as a Coactivator of Estrogen and Progesterone Receptors

WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.

Sulindac Sulfide and Exisulind Inhibit Expression of the Estrogen and Progesterone Receptors in Human Breast Cancer Cells

In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)-positive and progesterone receptor (PR)-positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor alpha (RARalpha), RARbeta, or RARalpha in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

Combinatorial Analysis of Transcription Factor Partners Reveals Recruitment of c-MYC to Estrogen Receptor-α Responsive Promoters

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

ErbB Receptor Signaling and Therapeutic Resistance to Aromatase Inhibitors

We have investigated the effect of HER-2 overexpression on resistance to the aromatase inhibitor letrozole in MCF-7 breast cancer cells stably expressing cellular aromatase (MCF-7/CA). MCF-7/CA cells overexpressing HER-2 showed a >2-fold increase in estrogen receptor (ER)-mediated transcriptional reporter activity upon treatment with androstenedione compared with vector-only control MCF-7/CA cells. Co-treatment with letrozole did not abrogate androstenedione-induced transcription and cell proliferation in HER-2-overexpressing cells. Chromatin immunoprecipitation assays using cross-linked protein-DNA from MCF-7/CA/HER-2 cells indicated ligand-independent association of the ERalpha coactivators AIB-1 and CBP to the promoter region of the estrogen-responsive pS2 gene. Upon treatment with androstenedione, there were increased associations of AIB1 and CBP with the pS2 promoter in the HER-2-overexpressing compared with control MCF-7/CA cells. These results suggest that ligand-independent recruitment of coactivator complexes to estrogen-responsive promoters as a result of HER-2 overexpression may play a role in the development of letrozole resistance.

MUC1 Oncoprotein Stabilizes and Activates Estrogen Receptor α

The MUC1 protein is aberrantly overexpressed by most human breast carcinomas. We report that the MUC1 C-terminal subunit associates with estrogen receptor alpha (ERalpha) and that this interaction is stimulated by 17beta-estradiol (E2). MUC1 binds directly to the ERalpha DNA binding domain and stabilizes ERalpha by blocking its ubiquitination and degradation. Chromatin immunoprecipitation assays further demonstrate that MUC1 (1) associates with ERalpha complexes on estrogen-responsive promoters, (2) enhances ERalpha promoter occupancy, and (3) increases recruitment of the p160 coactivators SRC-1 and GRIP1. In concert with these results, we show that MUC1 stimulates ERalpha-mediated transcription and contributes to E2-mediated growth and survival of breast cancer cells. These findings provide evidence that MUC1 stabilizes ERalpha and that this oncoprotein is of importance to the activation of ERalpha function.

Stimulatory Cross-talk between NFAT3 and Estrogen Receptor in Breast Cancer Cells

Estrogen receptors (ERalpha and ERbeta) are ligand-regulated transcription factors that play critical roles in the development and progression of breast cancer by regulating target genes involved in cellular proliferation. The transcriptional activity of ERalpha and ERbeta is known to be modulated by cofactor proteins. We used a yeast two-hybrid system and identified NFAT3 as a novel ERbeta-binding protein. NFAT3 interacted with ERalpha and ERbeta both in vitro and in mammalian cells in a ligand-independent fashion. NFAT3 bound specifically to the ERbeta region containing the activation function-1 domain, a ligand-independent transactivation domain. Overexpression of NFAT3 enhanced both ERalpha and ERbeta transcriptional activities in a ligand-independent manner and up-regulated downstream estrogen-responsive genes including pS2 and cathepsin D. Reduction of endogenous NFAT3 with NFAT3 small interfering RNA or overexpression of NFAT3 deletion mutants that lack the ER-binding sites reduced the NFAT3 coactivation of ERalpha and ERbeta. NFAT3 increased binding of ERalpha to the estrogen-responsive element and was recruited to endogenous estrogen-responsive promoters. NFAT3 was expressed differentially in many breast cancer cell lines and overexpressed in a subset of breast cancer patients. Knockdown of endogenous NFAT3 reduced the growth of human breast cancer ZR75-1 cells in a ligand-independent manner. Taken together, these results suggest that NFAT3 may play important roles in ER signaling and represent a novel target for breast cancer therapy.

Estrogen Receptor (ER) β Modulates ERα-Mediated Transcriptional Activation by Altering the Recruitment of c-Fos and c-Jun to Estrogen-Responsive Promoters

In this study, an estrogen receptor (ER) alpha-expressing T47D cell line containing an inducible tet-off FLAG-ERbeta was used to examine the influence of ERbeta on ERalpha activity. Real-time PCR analysis of mRNA levels of two well-studied estrogen-responsive genes, pS2 and progesterone receptor (PR), showed that the expression levels of both genes were reduced in the presence of ERbeta. Chromatin immunoprecipitation assays showed that the 17beta-estradiol (E2)-induced recruitment patterns to the pS2 and PR promoters were similar for both ERalpha and ERbeta. ERbeta expression did not significantly influence the kinetic recruitment profile of ERalpha to the pS2 promoter, but it was evident that ERalpha occupancy at the PR promoter was reduced. The E2-induced recruitment of c-Fos to a 12-O-tetradecanoylphorbol-13-acetate response element site in the PR promoter was significantly reduced in the presence of ERbeta, whereas only a slight reduction in the recruitment of c-Fos to the pS2 promoter was observed. ERbeta expression resulted in a significant reduction in the E2-induced expression of c-Fos mRNA. The recruitment pattern of c-Jun was also altered by ERbeta, although the expression levels of c-Jun were not. Expression of ERbeta caused a further 30-50% decrease of the E2-induced reduction in ERalpha protein after 3 h of E2 treatment, showing that ERbeta influences ERalpha protein levels. The altered recruitment of the activating protein-1 complex, combined with the reduction in ERalpha protein levels, may partly explain the antagonistic effect of ERbeta on ERalpha-mediated transcription.

Formation of an IKKα-Dependent Transcription Complex Is Required for Estrogen Receptor-Mediated Gene Activation

The IkappaB kinases IKKalpha and IKKbeta regulate distinct cytoplasmic and nuclear events that are critical for cytokine-mediated activation of the NF-kappaB pathway. Because the IKKs have previously been demonstrated to associate with the nuclear hormone receptor coactivator AIB1/SRC-3, the question of whether either IKKalpha or IKKbeta may be involved in increasing the expression of hormone-responsive genes was addressed. We demonstrated that IKKalpha, in conjunction with ERalpha and AIB1/SRC-3, is important in activating the transcription of estrogen-responsive genes, including cyclin D1 and c-myc, to result in the enhanced proliferation of breast cancer cells. Estrogen treatment facilitated the association of IKKalpha, ERalpha, and AIB1/SRC-3 to estrogen-responsive promoters and increased IKKalpha phosphorylation of ERalpha, AIB1/SRC-3, and histone H3. These results suggest that IKKalpha plays a major role in regulating the biological effects of estrogen via its promoter association and modification of components of the transcription complex.

Selective hormone-dependent repression of estrogen receptor beta by a p38-activated ErbB2/ErbB3 pathway

Deregulated signaling of ErbB2 receptor tyrosine kinase is often associated with hormone resistance in estrogen receptor alpha (ERα)-positive breast cancers, establishing a relationship between ErbB2 and ERα pathways. Although ERα and ERβ are expressed in many breast cancer cells, the response of ERβ to ErbB2 signaling is less well defined. In the present study, we demonstrate that ERβ activity can be modulated by ErbB2 signaling in ER-expressing breast cancer cells. The estrogen-dependent transcriptional activity of ERβ was altered in a manner similar to ERα by either activation of ErbB2/ErbB3 signaling by growth factor heregulin β or expression of a constitutively active mutant of ErbB2. However, as opposed to ERα, the p38 MAPK pathway was found to be involved in liganded ERβ repression activity by ErbB2 signaling and in regulating estrogen-responsive promoter occupancy by ERβ. The repression in ERβ response to hormone was dependent upon its AF-1 domain which includes serines 106 and 124, two phosphorylation target sites for Erk that we previously showed to be involved in SRC-1 recruitment to ERβ. Substitution of these two serines by aspartic acid residues abolished the repression of ERβ by activated ErbB2/ErbB3. Moreover, expression of SRC-1 also relieved the inhibition of ERβ in heregulin-treated cells. Our study demonstrates a functional coupling between ERβ and ErbB receptors and outlines the differential role of the AF-1 region in the regulation of the estrogen-dependent cell growth and activity of both estrogen receptors in response to growth factor signaling.

新しいアッセイ用ほ乳類細胞（ＣＨＯＯＳＥＲ）を用いた簡便，高感度で再現性の良いエストロゲン様活性測定方法の開発

Several in vitro assays have been developed to assess the activity of estrogenic substances. Recently, a yeast two-hybrid screen or a MCF-7 (human breast cancer cell) screen has been adopted mainly for screening chemicals and environmental estrogenic activity. However a yeast two-hybrid screen does not assay using an animal cell. MCF-7 screen is a complicated assay and the cell used proliferates depending on estrogen, thus obstructing a precise assay for estrogenic activity. In addition both screens need long incubation period (4-6 days) for a highly sensitive assay. The CHOOSER is a Chinese hamster ovary (CHO) cell transformed with the gene encoding the human estrogen receptor (ER α) and an estrogen responsive promoter linked to a reporter gene which was developed by Suzuki et. al. at Osaka prefectural institute of public health. When the reporter gene is activated by estrogenic substances, is expressed, producing alkaline phosphatase which is secreted into the medium. The CHOOSER can assay high sensitively (under 10-11M as 17 β-estradiol) at any time in a short incubation period (2 days). The number of the CHOOSER can be counted precisely because a CHO is dissociated the aggregates easily with trypsin and form a single cell suspension. And the CHOOSER can proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more proliferate in the medium without estrogen, therefore it is possible to assay for estrogenic activity more precisely. The CHOOSER assay will be a useful prescreening method for estrogenic activity before in vivo assay.

BRG1/BRM and prohibitin are required for growth suppression by estrogen antagonists.

Estrogen antagonists are universally employed in the breast cancer therapy, although antagonist therapy is limited by the inevitable development of cellular resistance. The molecular mechanisms by which these agents inhibit cellular proliferation in breast cancer cells are not fully defined. Recent studies have shown the involvement of the E2F pathway in tamoxifen-induced growth arrest. We show that an E2F repressor, prohibitin, and the chromatin modifiers Brg1/Brm are required for estrogen antagonist-mediated growth suppression through the estrogen receptor, and that their recruitment to native promoter-bound E2F is induced via a JNK1 pathway. In addition, we demonstrate major mechanistic differences among the signaling pathways initiated by estrogen, estrogen deprivation, and estrogen antagonists. Collectively, these findings suggest that the prohibitin/Brg1/Brm node is a major cellular target for estrogen antagonists, and thereby also implicate prohibitin/Brg1/Brm as potentially important targets for breast cancer therapy.

Interplay between Estrogen Response Element Sequence and Ligands Controls in Vivo Binding of Estrogen Receptor to Regulated Genes

To examine the role of the estrogen response element (ERE) sequence in binding of liganded estrogen receptor (ER) to promoters, we analyzed in vivo interaction of liganded ER with the imperfect ERE in the pS2 gene and the composite estrogen-responsive unit (ERU) in the proteinase inhibitor 9 (PI-9) gene. In transient transfections of ER-positive HepG2-ER7 cells, PI-9 was strongly induced by estrogen, moxestrol (MOX), and 4-hydroxytamoxifen (OHT). PI-9 was not induced by raloxifene or ICI 182,780. Quantitative reverse transcriptase-PCR showed that moxestrol strongly induced cellular PI-9 and pS2 mRNAs, whereas OHT moderately induced PI-9 mRNA and weakly induced pS2 mRNA. Chromatin immunoprecipitation experiments demonstrated strong and similar association of 17beta-estradiol-hERalpha and MOX-hERalpha with the PI-9 ERU and with the pS2 ERE. Binding of MOX-hERalpha to the PI-9 ERU and the pS2 ERE was rapid and continuous. Although MOX-hERalpha bound strongly to the PI-9 ERU and less well to the pS2 ERE in chromatin immunoprecipitation, gel shift assays showed that estrogen-hERalpha binds with higher affinity to the deproteinized pS2 ERE than to the PI-9 ERU. Across a broad range of OHT concentrations, OHT-hERalpha associated strongly with the pS2 ERE and weakly with the PI-9 ERU. ICI-hERalpha bound poorly to the PI-9 ERU and effectively to the pS2 ERE. Raloxifene-hERalpha and MOX-hERalpha exhibited similar binding to the PI-9 ERU and the pS2 ERE. These studies demonstrate that ER ligand and ERE sequence work together to regulate in vivo binding of ER to estrogen-responsive promoters.

Cyclic, Proteasome-Mediated Turnover of Unliganded and Liganded ERα on Responsive Promoters Is an Integral Feature of Estrogen Signaling

We present an integrated model of hERalpha-mediated transcription where both unliganded and liganded receptors cycle on estrogen-responsive promoters. Using ChIP, FRAP, and biochemical analysis we evaluate hERalpha at several points in these cycles, establishing the ubiquitination status and subnuclear distribution of hERalpha, its mobility, the kinetics of transcriptional activation, and the cyclic recruitment of E3 ligases and the 19S regulatory component of the proteasome. These experiments, together with an evaluation of the inhibition of transcription and proteasome action, demonstrate that proteasome-mediated degradation and hERalpha-mediated transactivation are inherently linked and act to continuously turn over hERalpha on responsive promoters. Cyclic turnover of hERalpha permits continuous responses to changes in the concentration of estradiol.

Phytoestrogen regulation of a Vitamin D 3 receptor promoter and 1,25-dihydroxyvitamin D 3 actions in human breast cancer cells

1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3), a steroid hormone derived from Vitamin D 3, is a negative growth regulator of breast cancer cells, and Vitamin D 3 analogs represent a novel treatment approach for human cancer. Elucidation of Vitamin D 3 receptor (VDR) regulation may reveal strategies to sensitize cancer cells to the effects of 1,25-dihydroxyvitamin D 3 and Vitamin D 3 analogs. We have previously characterized an estrogen responsive promoter region (800 bp upstream of exon 1c) in the human VDR gene, and the present studies examined regulation of this VDR promoter region by two phytoestrogens, resveratrol (present in red wine) and genistein (present in soy). We transiently transfected a VDR promoter luciferase construct into the estrogen receptor (ER) positive human breast cancer cell lines T47D and MCF-7, and treated with 0.4–4 μM resveratrol or 5–500 nM genistein. Both phytoestrogens up-regulated the transcription of the VDR promoter, as measured by reporter gene activity, approximately two-fold compared to vehicle treated cells. Co-treatment with the anti-estrogen tamoxifen (TAM) in T47D cells and transfection in an estrogen receptor negative breast cancer cell line demonstrated that the effects of phytoestrogens on the VDR promoter are dependent on estrogen receptor. Resveratrol and genistein also increased VDR protein expression as detected by Western blotting. Treatment with resveratrol had no effect on cell number or cell cycle profile, while treatment with genistein increased cell number. Because resveratrol could up-regulate VDR without increasing breast cancer cell growth, we hypothesized that resveratrol mediated increase in VDR expression would sensitize breast cancer cells to the effects of 1,25-dihydroxyvitamin D 3 and Vitamin D 3 analogs. In support of this hypothesis, both T47D and MCF-7 cells pre-treated with resveratrol exhibited increased VDR mediated transactivation of a Vitamin D 3 responsive promoter compared to cells pre-treated with vehicle. In addition, co-treatment with resveratrol enhanced the growth inhibitory effects of 1,25-dihydroxyvitamin D 3 and the Vitamin D 3 analog EB1089. These data support the concept that dietary factors, such as phytoestrogens, may impact on breast cancer cell sensitivity to Vitamin D 3 analogs through regulation of the VDR promoter.

Analysis of the Molecular Mechanisms of Human Estrogen Receptors α and β Reveals Differential Specificity in Target Promoter Regulation by Xenoestrogens

Most of the currently available information on the transcriptional activities of endocrine-disrupting chemicals (xenoestrogens) through estrogen receptors alpha (ERalpha) and beta (ERbeta) has been derived from transactivation studies on synthetic estrogen-responsive reporters. Thus, the ability of the xenoestrogen-liganded ERs to regulate endogenous estrogen-responsive gene expression has not been well characterized. Here, we have evaluated the activities of xenoestrogens through ERalpha and ERbeta on the vitellogenin A2 estrogen-response element (ERE) and the human pS2, lactoferrin, and complement 3 physiological target gene promoters. Using mammalian cell transient transfection assays, we found that the activities of xenoestrogens were mediated in a promoter-specific manner. For example, when bound to all ligands examined, ERalpha displayed high levels of transcription on the vitellogenin ERE and the lactoferrin promoter, but substantially lower activity on the complement 3 and pS2 promoters. However, one of the most important observations was that there were significant differences in the relative transcriptional activities of xenoestrogen-bound ERalpha and ERbeta on different promoters, suggesting that ERalpha and ERbeta make unique contributions to xenoestrogen action in target cells. When probing the molecular mechanism of the promoter-specific activities observed, we found that the transcriptional activity of the ERs correlated with the ability of each receptor to assume an active conformation on specific promoters. Taken together, the results indicate that the transcriptional activities of xenoestrogens are mediated in a promoter-specific manner and that estrogen-responsive promoters communicate differently with ERalpha and ERbeta by influencing their structures in a distinct manner that leads to diversity in their transcriptional responses.

17beta-estradiol is a hormonal regulator of mirex tumor promotion sensitivity in mice.

Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-beta estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ERalpha and ERbeta, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ERalpha or ERbeta and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ERalpha/beta. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.

Targeting of SWI/SNF chromatin remodelling complexes to estrogen-responsive genes.

SWI/SNF complexes are ATP-dependent chromatin remodelling enzymes that have been implicated in the regulation of gene expression in yeast and higher eukaryotes. BRG1, a catalytic subunit in the mammalian SWI/SNF complex, is required for transcriptional activation by the estrogen receptor, but the mechanisms by which the complex is recruited to estrogen target genes are unknown. Here, we have identified an interaction between the estrogen receptor and BAF57, a subunit present only in mammalian SWI/SNF complexes, which is stimulated by estrogen and requires both a functional hormone-binding domain and the DNA-binding region of the receptor. We also found an additional interaction between the p160 family of coactivators and BAF57 and demonstrate that the ability of p160 coactivators to potentiate transcription by the estrogen receptor is dependent on BAF57 in transfected cells. Moreover, chromatin immunoprecipitation assays demonstrated that BAF57 is recruited to the estrogen-responsive promoter, pS2, in a ligand-dependent manner. These results suggest that one of the mechanisms for recruiting SWI/SNF complexes to estrogen target genes is by means of BAF57.

Sugar-Coating Gene Expression

Two groups report glycosylation of transcriptional regulators. Yang et al. provide evidence that the enzyme that catalyzes the addition of O -linked N -acetylglucosamine ( O -GlcNAc) is recruited to promoters that are repressed. O -GlcNAc transferase (OGT) interacted with mammalian Sin3 (mSin3), a scaffold for corepressor complex formation. Using reporter genes, Yang et al. found that targeting OGT to a promoter inhibited transcription. OGT with and without the catalytic domain retained the ability to inhibit transcription, suggesting that there may be two mechanisms by which OGT contributes to transcriptional repression. OGT, mSin3, and the histone deacetylase HDAC1 coprecipitated, suggesting an interaction between OGT and the corepressor complex in vivo. In the presence of an inhibitor of HDAC activity, mSin3 interaction with OGT appeared to contribute to mSin3 transcriptional repression, because transcriptional activity was higher if the interaction domain between mSin3 and OGT was removed. Finally, mSin3 and HDAC1 are modified by the addition of O -GlcNAc. In breast cancer cells, depletion of estrogen caused increased association of O -GlcNAc-modified proteins with estrogen-responsive promoters. Kane et al. provide evidence that the transcription factor NFIC is modified by N -glycosylation during mammary gland involution in mice. As seen from electrophoretic mobility shift assays, NFIC that is bound to DNA from lactating mammary gland migrated with a smaller apparent size (49 kD), whereas NFIC that is associated with DNA during early stages of involution appeared to be a larger protein (74 kD). The 74-kD NFIC bound conconavalin A, a lectin that recognizes α-mannose in the core of N -linked glycans; whereas the 49-kD form did not. The apparent size of the 74-kD NFIC associated with involution was decreased following treatment with a specific N -glycanase. Furthermore, the larger form of the protein was not formed in mammary glands or transfected cells in which N -glycosylation was inhibited by tunicamycin. Thus, N -glycosylation appears to be the posttranslational modification responsible for the change in size of NFIC. What remains unknown is how this posttranslational modification might affect the transcriptional activity of NFIC. X. Yang, F. Zhang, J. E. Kudlow, Recruitment of O -GlcNAc transferase to promoters by corepressor mSin3A: Coupling protein O -GlcNAcylation to transcriptional repression. Cell 110 , 69-80 (2002). [Online Journal] R. Kane, J. Murtagh, D. Finaly, A. Marti, R. Jaggi, D. Blatchford, C. Wilde, F. Martin, Transcription factor NFIC undergoes N -glycosylation during early mammary gland involution. J. Biol. Chem. 277 , 25893-25903 (2002). [Abstract] [Full Text]