A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

Background: In human Estrogen Receptor alpha (ER alpha)-positive breast cancers, 59 end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ER alpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. Methodology/Principal Findings: We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ER alpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 59 end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ER alpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ER alpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ER alpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. Conclusions/Significance: MBD2 and ER alpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ER alpha and MBD2

A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation.We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated.MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2

Specific association between the methyl-CpG-binding domain protein 2 and the hypermethylated region of the human telomerase reverse transcriptase promoter in cancer cells

Human telomerase reverse transcriptase (hTERT) is expressed in most cancer cells. Paradoxically, its promoter is embedded in a hypermethylated CpG island. A short region escapes to this alteration, allowing a basal level of transcription. However, the methylation of adjacent regions may play a role in the maintenance of low hTERT expression. It is now well established that methyl-CpG binding domain proteins mediate the transcriptional silencing of hypermethylated genes. The potential involvement of these proteins in the control of hTERT expression was firstly investigated in HeLa cells. Chromatin immunoprecipitation assays showed that only methyl-CpG-binding domain protein 2 (MBD2) associated the hypermethylated hTERT promoter. In MBD2 knockdown HeLa cells, constitutively depleted in MBD2, neither methyl CpG binding protein 2 (MeCP2) nor MBD1 acted as substitutes for MBD2. MBD2 depletion by transient or constitutive RNA interference led to an upregulation of hTERT transcription that can be downregulated by expressing mouse Mbd2 protein. Our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT in HeLa cells. This specific transcriptional repression was also observed in breast, liver and neuroblastoma cancer cell lines. Thus, MBD2 seems to be a general repressor of hTERT in hTERT-methylated telomerase-positive cell

The Methyl-CpG-Binding Domain Protein 2 (MBD2) : a specific interpret of methylated loci in cancer cells

De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l’expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l’expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l’analyse globale des profils de distribution de MBD2, de la méthylation de l’ADN, ainsi que de l’ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d’un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.In the past few years, several clinical trials have shown that targeting DNA methylation machinery might be of interest in cancer therapy to restore tumor suppressor genes expression and inhibit tumor growth. The Methyl-CpG-Binding Domain protein 2 (MBD2) is an important constituent of the DNA methylation machinery since this protein is directly involved in the mediation of the epigenetic signal. Moreover, MBD2 seems to show some gene specificity, its inhibition reactivate a limited number of genes. Taken together these data suggest that MBD2 represents potential new target in cancer therapy and, therefore, new insights on MBD2 specificities are, in this context, of importance. To this end, we have developed two different approaches: a candidate genes analysis and a genome-wide analysis, using ChIP-on-chip method, in order to map MBD2 binding sites. The candidate gene approaches are strongly in favour of the “one gene – one MBD” hypothesis, at least for the genes analyzed. Indeed, our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT and pS2/TFF1 genes. Furthermore, a new role of MBD2 in the fine-scale modulation of these genes was demonstrated, and the antagonist actions between MBD2 and the natural trans-activator of pS2 gene, the estrogen α, were explored. Genome wide distribution of MBD2 binding sites, DNA Methylation profiles, and silencing potential, showed that the MBD2 is a real methylation-dependant transcriptional repressor: 74% of the MBD2 binding promoters are methylated and 65% silenced

Spécificité de liaison et de répression de la « Methyl-CpG-Binding Domain protein 2 » (MBD2) : identification de gènes cibles impliqués dans les cancers

In the past few years, several clinical trials have shown that targeting DNA methylation machinery might be of interest in cancer therapy to restore tumor suppressor genes expression and inhibit tumor growth. The Methyl-CpG-Binding Domain protein 2 (MBD2) is an important constituent of the DNA methylation machinery since this protein is directly involved in the mediation of the epigenetic signal. Moreover, MBD2 seems to show some gene specificity, its inhibition reactivate a limited number of genes. Taken together these data suggest that MBD2 represents potential new target in cancer therapy and, therefore, new insights on MBD2 specificities are, in this context, of importance. To this end, we have developed two different approaches: a candidate genes analysis and a genome-wide analysis, using ChIP-on-chip method, in order to map MBD2 binding sites. The candidate gene approaches are strongly in favour of the “one gene – one MBD” hypothesis, at least for the genes analyzed. Indeed, our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT and pS2/TFF1 genes. Furthermore, a new role of MBD2 in the fine-scale modulation of these genes was demonstrated, and the antagonist actions between MBD2 and the natural trans-activator of pS2 gene, the estrogen α, were explored. Genome wide distribution of MBD2 binding sites, DNA Methylation profiles, and silencing potential, showed that the MBD2 is a real methylation-dependant transcriptional repressor: 74% of the MBD2 binding promoters are methylated and 65% silenced.De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l’expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l’expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l’analyse globale des profils de distribution de MBD2, de la méthylation de l’ADN, ainsi que de l’ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d’un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle

A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

Background: In human Estrogen Receptor alpha (ER alpha)-positive breast cancers, 59 end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ER alpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. Methodology/Principal Findings: We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ER alpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 59 end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ER alpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ER alpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ER alpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. Conclusions/Significance: MBD2 and ER alpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ER alpha and MBD2

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements

International audienceIn mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status

Genome-wide in Silico Identification of New Conserved and Functional Retinoic Acid Receptor Response Elements (Direct Repeats Separated by 5 bp)

International audienceThe nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located +/- 10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species

Retinoic acid receptors recognise the mouse genome through binding elements with diverse spacing and topology

peer reviewedRetinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2 or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8 and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half sites with DR2 and DR0 spacings. This specific half site organisation constitutes a previously unrecognised, but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, while DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5 and DR8 to mediate RA-dependent transcriptional activation indicates that half site spacing allosterically regulates RAR function