Search

The focus of this study is on the expression and regulation of the estrogen-regulated breast cancer and salivary gland expression (BASE) gene that may function as a breast cancer marker. In MCF7 cells, BASE is repressed by estrogen in an estrogen receptor α (ERα)-dependent manner. Promoter analysis of the BASE gene led to the identification of a 2-kb upstream enhancer that harbors binding sites for ERα and FoxA1. The recruitment of both ERα and FoxA1 to this region was shown by chromatin immunoprecipitation analysis. Furthermore, mutation studies and knockdown experiments show a clear separation between gene expression mediated by FoxA1 and ERα-dependent gene regulation. Additionally, we provide information on BASE expression in human breast tumor samples. [Cancer Res 2008;68(1):106–14]

Supplementary Figure 1, Tables 2-5 from Estrogen Induces Repression of the Breast Cancer and Salivary Gland Expression Gene in an Estrogen Receptor α–Dependent Manner

Estrogen receptor alpha (ERalpha) is a ligand dependent transcription factor that regulates the expression of target genes through interacting with cis-acting estrogen response elements (EREs). However, only a minority of ERalpha binding sites are located within the proximal promoter regions of responsive genes. Here we report the characterization of an ERE located 9kbp upstream of the TSS of the cathepsin D gene (CTSD) that up-regulates CTSD expression upon estrogen stimulation in MCF-7 cells. Using ChIP, we show recruitment of ERalpha and phosphorylated PolII at the CTSD distal enhancer region. Moreover, we determine the kinetics of transient CpG methylation on the promoter region of CTSD and for the first time, at a distal enhancer element. We show that ERalpha is crucial for long-distance regulation of CTSD expression involving a looping mechanism.

A series of nitrofuran-based compounds were identified as inhibitors of estrogen signaling in a cell-based, high-throughput screen of a diverse library of small molecules. These highly related compounds were subsequently found to inhibit topoisomerase II in vitro at concentrations similar to that required for the inhibition of estrogen signaling in cells. The most potent nitrofuran discovered is ∼10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with ∼20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G0-G1 phase of the cell cycle, do not induce double-stranded DNA breaks, are not substrates for multidrug resistance protein-1 export from the cell, and are amenable to synthetic development. In addition, the nitrofurans synergize with etoposide phosphate in cell killing. Clonogenic assays done on a panel of human tumors maintained ex vivo in nude mice show that the most active compound identified in the screen is selective against tumors compared with normal hematopoietic stem cells. However, this compound had only moderate activity in a mouse xenograft model. This novel class of topoisomerase II inhibitor may provide additional chemotherapeutic strategies for the development of cytotoxic agents with proven clinical utility. [Cancer Res 2007;67(9):4451–8]

The human estrogen receptor α (ERα ) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERα F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C α (PKCα ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERα resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERα signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERα in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-alpha (ERalpha), resulting in subsequent clearance of ERalpha protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERalpha positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERalpha mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERalpha expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERalpha from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERalpha, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.

Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.

It is by now well established that the estrogen receptor alpha (ER alpha) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5'-untranslated regions (5'-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5'-UTRs of ER alpha contain between one and six upstream open reading frames. In this study the effect of the 5'-UTRs of major human and mouse ER alpha mRNA variants on translation was evaluated. Some of the 5'-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5'-UTRs of ER alpha mRNAs have the potential to regulate estrogen receptor alpha expression.

The role of estrogens in the development and physiology of the male reproductive tract remains provocative, with a growing body of evidence suggesting that estrogens are able to influence normal testis development and physiology, through their classical receptors, estrogen receptor (ER)-alpha and ER-beta. We describe the identification and characterization of a new promoter that is involved in the expression of ER-alpha in the epididymis and in testis. This promoter lies on chromosome 6q25.1, approximately 16 kb upstream of the first coding exon of ER-alpha. Sequence analysis indicates that this promoter has a conventional TATA box and GC box but no upstream CAAT sequence. Alternative splicing results in at least two species of mRNA encoding ER-alpha being synthesized from this promoter. Transcription profiling of human tissues shows that, among those tested, this promoter is predominantly active only in testis and epididymal tissues. Transient transfection assays using this new promoter in a number of cell lines indicate that the region we have identified functions as a promoter and that tissue-specific regulation is likely to be dependent on inhibitory sequences greater than 1 kb upstream of the transcription start site.

The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ERα in osteoblasts clearly demonstrated that the well characterized 66-kDa ERα was only one of the ERα isoforms present. Here we describe a 46-kDa isoform of ERα, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ERα gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ERα isoform is absent. Functional analysis revealed that human (h)ERα46 is able to heterodimerize with the full-length ERα and also with ERβ. Further, a DNA-binding complex that corresponds to hERα46 is detectable in human osteoblasts. We have shown that hERα46 is a strong inhibitor of hERα66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hERα46 are cotransfected with hERα66. In addition to human bone, the expression of the alternatively spliced ERα mRNA variant is also detectable in bone of ERα knockout mice. These data suggest that, in osteoblasts, E2 can act in part through an ERα isoform that is markedly different from the 66-kDa receptor. The expression of two ERα protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ERα protein isoforms in bone and perhaps in other tissues.

The important role of estrogens in women in physiological and pathological processes is well accepted, but recently it has become evident that estrogens are also important in male physiology, in particular, within bone metabolism and reproduction. Consequently, it is necessary to identify and to characterize the molecular mechanisms of estrogen action in order to evaluate how the pleiotropic effects of estrogens are mediated in a variety of tissues. We have recently shown that human estrogen receptor α (ERα) mRNA is transcribed from at least six different promoters (1A–1F). Transcription of ERα in bone is exclusively dependent on the F-promoter. To study the regulation of ER expression in this tissue, we examined 1 kbp of the F-promoter region of human ERα, which is located more than 70 kbp upstream of the transcription start site of the ERα gene. Transient transfection experiments demonstrated a basal activity from the F-promoter, which was further increased when ERα was cotransfected. We have shown recently that the F-promoter can give rise to at least two ERα isoforms in bone. On the contrary, ERβ expression in primary osteoblasts is extremely low, indicating that this ER isoform plays only a minor role in these cells. In contrast to bone, we have demonstrated that both ERα and ERβ transcripts are readily detected in testis. Here, we report that besides ERα, ERβ transcripts can give rise to two protein isoforms and that this complex situation could have important functional consequences for the signalling of estrogens and their analogs.

The focus of this study is on the expression and regulation of the estrogen-regulated breast cancer and salivary gland expression (BASE) gene that may function as a breast cancer marker. In MCF7 cells, BASE is repressed by estrogen in an estrogen receptor α (ERα)-dependent manner. Promoter analysis of the BASE gene led to the identification of a 2-kb upstream enhancer that harbors binding sites for ERα and FoxA1. The recruitment of both ERα and FoxA1 to this region was shown by chromatin immunoprecipitation analysis. Furthermore, mutation studies and knockdown experiments show a clear separation between gene expression mediated by FoxA1 and ERα-dependent gene regulation. Additionally, we provide information on BASE expression in human breast tumor samples. [Cancer Res 2008;68(1):106–14]

Estradiol (E2) is believed to modulate physiological functions relevant to osteoblast biology through the actions of estrogen receptors (ERs) that in turn regulate the expression of target genes. The molecular effects of estrogen action in bone remain to be fully elucidated. This study reports a genome-wide molecular and computational analysis of the interaction between ER and regulatory elements on the DNA of target genes in human primary osteoblasts. Of approximately 54,000 gene probes surveyed in this study, a total of 375 genes were up-regulated and 418 genes were down-regulated on exposure to E2, with only 46 of these being direct target genes after 24 h, as determined by concomitant cycloheximide treatment. Computational analysis discovered several pathways where E2 co-regulates multiple functionally linked components. Examination of the genomic sequence of IGF binding protein 4 located ER response elements within the first intron. Using by chromatin immunoprecipitation, we show a site- and cell-specific recruitment of transcription factors to this newly identified regulatory region. Transient transfection studies revealed that this intronic region acts as a functional promoter in human osteoblasts. Taken together, this analysis provides a comprehensive gene transcription profile and identifies several genes of potential physiological importance in controlling estrogen-mediated signaling in primary osteoblasts.

Estrogens play a key role in bone structural integrity, which is maintained by the opposing activity of bone forming osteoblasts and bone resorbing osteoclasts. The cellular effects of estrogens are mediated by estrogen receptors, however, the detailed molecular mechanism of ER regulation in osteoclasts has not yet been elucidated. We provide here a detailed analysis of the expression profile and functionality of ER during osteoclast differentiation. We employed a human primary osteoclast cell culture model to evaluate the regulation of estrogen receptor (ER) variant expression. We characterized the expression profile of estrogen receptors and studied the regulation of the predominant estrogen receptor-alpha (ER-alpha) during differentiation into osteoclasts. In addition to the full-length ER-alpha, a shorter ER-alpha mRNA variant is expressed and both ER-alpha variants are regulated during osteoclastogenesis. Furthermore, we show that the pS2 gene is an estrogen-regulated gene in osteoclasts. Analysis of the activity of the pS2 gene throughout differentiation, using chromatin immunoprecipitation (ChIP), revealed the functionality of ER-alpha during differentiation and shows that the occupancy of ER-alpha and activated polymerase II on the pS2 promoter decrease with time and can be blocked by the ER antagonist ICI 182780. These results help to dissect the molecular events relevant to estrogen signaling and provide a better understanding of the role of ER-alpha regulation during bone resorption mediated by osteoclasts.

Das folgende Kapitel beschaftigt sich mit der Frage, wie die im Zellkern gespeicherte Information die Funktion einer Zelle steuern und regulieren kann.

The mouse knockout of the estrogen receptor alpha (ERalpha) gene, known as alphaERKO, has been extensively used for several years to study the role and function of ERalpha. Residual estradiol binding capacity in uterine tissue of 5-10% raised doubts if this knockout is a genuine null mutation of ERalpha. Although alternatively spliced ERalpha mRNA variants in the alphaERKO mouse were reported previously, the corresponding protein isoforms have not been detected to date. Here we show that a variant ERalpha protein, 61 kDa in size, is expressed in the uterine tissue of alphaERKO mice as a result of an alternative splicing. The transactivation capability of this protein is cell dependent and can be as high as 75% of the wild type ERalpha.

The functional interplay between different domains of estrogen receptor-alpha (ERalpha, NR3A1) is responsible for the overall properties of the full-length protein. We previously identified an interaction between the N-terminal A and C-terminal domains, which we demonstrate here to repress ligand-independent transactivation and transrepression abilities of ERalpha. Using targeted mutations based on ERalpha structural models, we determine the basis for this interaction that defines a regulatory interplay between ERalpha A domain, corepressors, and ERalpha Helix 12 for binding to the same C-terminal surface. We propose a dynamic model where binding of different ligands influences the A/D-F domain interaction and results in specific functional outcomes. This model gives insights into the dynamic properties of full-length ERalpha and into the structure of unliganded ERalpha.

This review aims to evaluate the impact that human estrogen receptor-alpha (ER-alpha) synthesis, modification and degradation has on estrogen-dependant physiological and pathological processes within the body. Estrogen signaling is transduced through estrogen receptors, which act as ligand-inducible transcription factors. The significance of different isoforms of ER-alpha that lack structural features of full-length ER-alpha are discussed. The influence of differential promoter usage on the amount and isoform of ER-alpha within individual cell types is also reviewed. Moreover, the potential role of phosphorylation, ubiquitination and acetylation in the function and dynamic turnover of ER-alpha is presented.

The ERalpha gene has been intensively studied for more than a decade. During this long time, multiple promoters used in ERalpha expression have been discovered in several species. Although an already large body of literature describing various aspects of the regulation of ERalpha expression and utilization of different promoters is constantly growing, the inconsistent terminology used by individual authors makes the interpretation and comparison of data very difficult. Furthermore, completion of the human genome project now allows all known human ERalpha promoters to be placed on a physical map. This review describes promoters used in the generation of ERalpha transcripts in human and in other species and suggests a consistent nomenclature. The possible role of multiple promoters in the differential expression of ERalpha in tissues and during development is also discussed.

A new isoform of the human estrogen receptor-alpha (hER-alpha) has been identified and characterized. This 46 kDa isoform (hERalpha46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERalpha66). hERalpha46 is encoded by a new class of hER-alpha transcript that lacks the first coding exon (exon 1A) of the ER-alpha gene. We demonstrated that these Delta1A hER-alpha transcripts originate from the E and F hER-alpha promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERalpha46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERalpha46 is a powerful inhibitor of hERalpha66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominant-negative action is exerted may involve heterodimeri zation of the two receptor isoforms and/or direct competition for the ER-alpha DNA-binding site. hERalpha66/hERalpha46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERalpha46 in cellular proliferation.

„Vernetzung“ und „Kooperation” sind Begriffe, die die zehnjahrige Arbeit des Dortmunder Musikerinnenprojekts von „rocksie!“ treffend kennzeichnen: Neben die Forderung und Beratung von Musikerinnen trat in den letzten Jahren die stadteubergreifende Organisation von Workshops, Festivals und Symposien. Seit 1997 arbeitet rocksie! daruber hinaus an der Vernetzung verschiedener europaischer Lander und Stadte. Obwohl rocksie! immer noch abhangig ist von jahrlich zu bewilligenden Zuschussen und unter einer standigen personellen Unterbesetzung zu leiden hat, haben die Initiatorinnen eine gut organisierte Initiative fur Frauen in Deutschland geschaffen und Verbindungen fur ein europaisches Musikerinnennetzwerk hergestellt.