Stefanie Denger

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.

Multiple mechanisms induce transcriptional silencing of a subset of genes, including oestrogen receptor alpha, in response to deacetylase inhibition by valproic acid and trichostatin A.

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-α (ERα), resulting in subsequent clearance of ERα 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 ERα positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERα 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 ERα expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERα from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERα, 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.

Upstream Open Reading Frames Regulate the Translation of the Multiple mRNA Variants of the Estrogen Receptor α

It is by now well established that the estrogen receptor α (ERα) 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 ERα contain between one and six upstream open reading frames. In this study the effect of the 5′-UTRs of major human and mouse ERα 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 ERα mRNAs have the potential to regulate estrogen receptor α expression.

E2-mediated cathepsin D (CTSD) activation involves looping of distal enhancer elements

Estrogen receptor alpha (ERα) 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 ERα 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 ERα 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 ERα is crucial for long‐distance regulation of CTSD expression involving a looping mechanism.

Estrogen Induces Repression of the Breast Cancer and Salivary Gland Expression Gene in an Estrogen Receptor α–Dependent Manner

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 alpha (ER alpha)-dependent manner. Promoter analysis of the BASE gene led to the identification of a 2-kb upstream enhancer that harbors binding sites for ER alpha and FoxA1. The recruitment of both ER alpha 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 ER alpha-dependent gene regulation. Additionally, we provide information on BASE expression in human breast tumor samples.

Thanatop: A Novel 5-Nitrofuran that Is a Highly Active, Cell-Permeable Inhibitor of Topoisomerase II

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 approximately 10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with approximately 20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G(0)-G(1) 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.