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A role for methyl-CpG binding domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.

Chatagnon A, Ballestar E, Esteller M, Dante R - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U590, Lyon, France.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Histone H3 marks on pS2 promoter in presence or absence of MBD2 and/or ERα.HeLa cells, wild type (HeLa), expressing ectopically ERα (HeLa::ERα), and depleted in MBD2 and expressing ectopically ERα (MBD2 KD HeLa::ERα), were subjected to ChIP analysis using anti-histone H3 acetylation (H3Ac) or an anti-histone H3 lysine 9 trimethylation (H3K9me3) antibodies. The pS2 promoter was amplified by real-time PCR from an equal amount (0,5 ng) of total DNA immunoprecipitated by the different antibodies. Relative amounts of H3Ac or H3K9me3 marks were measured by comparing fractions immunoprecipitated by the anti-H3Ac or anti-H3K9me3 antibodies to fractions immunoprecipitated by the anti-histone H3 pan antibody. Each bar represents the mean ± standard deviation.
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pone-0009665-g009: Histone H3 marks on pS2 promoter in presence or absence of MBD2 and/or ERα.HeLa cells, wild type (HeLa), expressing ectopically ERα (HeLa::ERα), and depleted in MBD2 and expressing ectopically ERα (MBD2 KD HeLa::ERα), were subjected to ChIP analysis using anti-histone H3 acetylation (H3Ac) or an anti-histone H3 lysine 9 trimethylation (H3K9me3) antibodies. The pS2 promoter was amplified by real-time PCR from an equal amount (0,5 ng) of total DNA immunoprecipitated by the different antibodies. Relative amounts of H3Ac or H3K9me3 marks were measured by comparing fractions immunoprecipitated by the anti-H3Ac or anti-H3K9me3 antibodies to fractions immunoprecipitated by the anti-histone H3 pan antibody. Each bar represents the mean ± standard deviation.

Mentions: In our study, ChIP assays indicated that ERα pS2 stimulation was associated with increase in histone H3 acetylation (∼2.5 fold) and enhanced the demethylation of H3K9 (∼500 fold) at pS2 promoter, when compared with wild type HeLa cells (Figure 9). Moreover, in HeLa cells, the synergic activity of MBD2 depletion and ectopic ERα expression on pS2 transcription led to a stronger induction of histone H3 acetylation (∼28 fold) at pS2 promoter, while H3K9 methylation was still lowered (∼10 fold), (Figure 9). From these findings we conclude that the transcriptional MBD2 repressor and ERα transactivator co-participate to the regulation of pS2 expression by mediating a balance between repressive histone H3 lysine 9 trimethylation and active histone H3 acetylation marks at pS2 promoter. Thus, the repressive effect of MBD2 on the transactivation of pS2 mediated by ERα is linked to histone modifications.


A role for methyl-CpG binding domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.

Chatagnon A, Ballestar E, Esteller M, Dante R - PLoS ONE (2010)

Histone H3 marks on pS2 promoter in presence or absence of MBD2 and/or ERα.HeLa cells, wild type (HeLa), expressing ectopically ERα (HeLa::ERα), and depleted in MBD2 and expressing ectopically ERα (MBD2 KD HeLa::ERα), were subjected to ChIP analysis using anti-histone H3 acetylation (H3Ac) or an anti-histone H3 lysine 9 trimethylation (H3K9me3) antibodies. The pS2 promoter was amplified by real-time PCR from an equal amount (0,5 ng) of total DNA immunoprecipitated by the different antibodies. Relative amounts of H3Ac or H3K9me3 marks were measured by comparing fractions immunoprecipitated by the anti-H3Ac or anti-H3K9me3 antibodies to fractions immunoprecipitated by the anti-histone H3 pan antibody. Each bar represents the mean ± standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837351&req=5

pone-0009665-g009: Histone H3 marks on pS2 promoter in presence or absence of MBD2 and/or ERα.HeLa cells, wild type (HeLa), expressing ectopically ERα (HeLa::ERα), and depleted in MBD2 and expressing ectopically ERα (MBD2 KD HeLa::ERα), were subjected to ChIP analysis using anti-histone H3 acetylation (H3Ac) or an anti-histone H3 lysine 9 trimethylation (H3K9me3) antibodies. The pS2 promoter was amplified by real-time PCR from an equal amount (0,5 ng) of total DNA immunoprecipitated by the different antibodies. Relative amounts of H3Ac or H3K9me3 marks were measured by comparing fractions immunoprecipitated by the anti-H3Ac or anti-H3K9me3 antibodies to fractions immunoprecipitated by the anti-histone H3 pan antibody. Each bar represents the mean ± standard deviation.
Mentions: In our study, ChIP assays indicated that ERα pS2 stimulation was associated with increase in histone H3 acetylation (∼2.5 fold) and enhanced the demethylation of H3K9 (∼500 fold) at pS2 promoter, when compared with wild type HeLa cells (Figure 9). Moreover, in HeLa cells, the synergic activity of MBD2 depletion and ectopic ERα expression on pS2 transcription led to a stronger induction of histone H3 acetylation (∼28 fold) at pS2 promoter, while H3K9 methylation was still lowered (∼10 fold), (Figure 9). From these findings we conclude that the transcriptional MBD2 repressor and ERα transactivator co-participate to the regulation of pS2 expression by mediating a balance between repressive histone H3 lysine 9 trimethylation and active histone H3 acetylation marks at pS2 promoter. Thus, the repressive effect of MBD2 on the transactivation of pS2 mediated by ERα is linked to histone modifications.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U590, Lyon, France.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus