Raphaël Métivier

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.

The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARγ and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARγ target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARγ coregulators that includes Med1 and PGC-1α, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

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.

Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.

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.

Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.

The Relative Contribution Exerted by AF-1 and AF-2 Transactivation Functions in Estrogen Receptor α Transcriptional Activity Depends upon the Differentiation Stage of the Cell

The activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor α (ERα) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERα AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERα activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERα transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERα signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERα in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERα-positive cell lines tested strongly suggest that the transcriptional activity of ERα relies on its AF-1 in most estrogen target cell types.

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.