George Reid

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.

Transient cyclical methylation of promoter DNA.

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor α (ERα) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERα and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERα and of pS2 transcripts.

Identification of a new isoform of the human estrogen receptor‐alpha (hER‐α) that is encoded by distinct transcripts and that is able to repress hER‐α activation function 1

A new isoform of the human estrogen receptor‐alpha (hER‐α) has been identified and characterized. This 46 kDa isoform (hERα46) lacks the N‐terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERα66). hERα46 is encoded by a new class of hER‐α transcript that lacks the first coding exon (exon 1A) of the ER‐α gene. We demonstrated that these Δ1A hER‐α transcripts originate from the E and F hER‐α promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERα46 showed that, in a cell context sensitive to the transactivation function AF‐2, this receptor is an effective ligand‐inducible transcription factor. In contrast, hERα46 is a powerful inhibitor of hERα66 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‐α DNA‐binding site. hERα66/hERα46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERα46 in cellular proliferation.

Transcription in four dimensions: nuclear receptor-directed initiation of gene expression

Regulated gene expression, achieved through the coordinated assembly of transcription factors, co‐regulators and the basal transcription machinery on promoters, is an initial step in accomplishing cell specificity and homeostasis. Traditional models of transcriptional regulation tend to be static, although gene expression profiles change with time to adapt to developmental and environmental cues. Furthermore, biochemical and structural studies have determined that initiation of transcription progresses through a series of ordered events. By integrating time into the analysis of transcription, chromatin immunoprecipitation assays and live‐cell imaging techniques have revealed the dynamic, cooperative, functionally redundant and cyclical nature of gene expression. In this review, we present a dynamic model of gene transcription that integrates data obtained by these two techniques.

Human estrogen receptor-α: regulation by synthesis, modification and degradation

Abstract. This review aims to evaluate the impact that human estrogen receptor-α (ER-α) 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-α that lack structural features of full-length ER-α are discussed. The influence of differential promoter usage on the amount and isoform of ER-α within individual cell types is also reviewed. Moreover, the potential role of phosphorylation, ubiquitination and acetylation in the function and dynamic turnover of ER-α is presented.

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.

A Dynamic Structural Model for Estrogen Receptor-α Activation by Ligands, Emphasizing the Role of Interactions between Distant A and E Domains

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.

Transcriptional complexes engaged by apo‐estrogen receptor‐α isoforms have divergent outcomes

Unliganded (apo‐) estrogen receptor α (ERα, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo‐human ERα (ERα66) and its N‐terminally truncated isoform (ERα46) 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‐ERα66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo‐ERα46 recruits corepressors, following engagement of cofactors identical to those recruited by apo‐ERα66. Together, these data describe differential activities of ERα 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.

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.

Marking time: the dynamic role of chromatin and covalent modification in transcription.

The expression of genes subject to strict regulation can be a highly dynamic, cyclical process that sequentially achieves and then limits transcription. Kinetic investigations of the estrogen responsive pS2 (TFF1) promoter, to determine the occupancy of factors or the occurrence of covalent marks on chromatin, have provided the most comprehensive picture of the complexity of transcriptional cycling to date. Cycles are initiated by the assembly of intermediate transcription factors that in turn provoke conscription of the basal transcription machinery. These events then achieve activation of the polymerase II complex, which is subsequently followed by limitation of productivity through the action of repressive complexes. This latter phase resets the target promoter, through acting on chromatin structure, such that a subsequent cycle can be initiated. In consequence, transcription is dependent upon cis-acting elements (DNA and nucleosomes) that either interact with or are modified by trans-acting factors. Induced local structural changes to chromatin encompassing regulatory elements of gene promoters include alteration of the positional phasing of nucleosomes, substitution by variant histones, post-translational modification of nucleosomes, changes in the methylation of CpG dinucleotides and breaks in the sugar-phosphate backbone of DNA. A primary function of covalent modification of chromatin may be to drive a sequential progression of reversible interactions that achieve and regulate gene expression.