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.

A new isoform of the human estrogen receptor-alpha (hER-a) has been identi®ed and characterized. This 46 kDa isoform (hERa46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERa66). hERa46 is encoded by a new class of hER-a transcript that lacks the ®rst coding exon (exon 1A) of the ER-a gene. We demonstrated that these D1A hER-a transcripts originate from the E and F hER-a promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERa46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERa46 is a powerful inhibitor of hERa66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominantnegative action is exerted may involve heterodimerization of the two receptor isoforms and/or direct competition for the ER-a DNA-binding site. hERa66/ hERa46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERa46 in cellular proliferation.

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

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.