Limits...
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.

Show MeSH

Related in: MedlinePlus

The transactivators ERα and the methylation-dependant repressor MBD2 can simultaneously bound the pS2 promoter in HeLa cells.(A) MBD2 ChIP assays in HeLa cells expressing ERα (HeLa::ERα). Relative amounts of immunoprecipitaded pS2 promoter measured by quantitative PCR from HeLa or HeLa::ERα cells. Each bar represents the mean ± standard deviation of at least three independent experiments. (B) Serial ERα-MBD2 ChIP assays to pS2 promoter. Chromatin prepared from HeLa cells transfected with a human ERα expression vector was subjected to the ChIP procedure with the anti-ERα antibody, and again immunoprecipitated using antibodies as indicated at the top of the figure (Non-specific antibody, IgG; anti-ERα antibody, ER; anti-MBD2 antibody, MBD2).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2837351&req=5

pone-0009665-g007: The transactivators ERα and the methylation-dependant repressor MBD2 can simultaneously bound the pS2 promoter in HeLa cells.(A) MBD2 ChIP assays in HeLa cells expressing ERα (HeLa::ERα). Relative amounts of immunoprecipitaded pS2 promoter measured by quantitative PCR from HeLa or HeLa::ERα cells. Each bar represents the mean ± standard deviation of at least three independent experiments. (B) Serial ERα-MBD2 ChIP assays to pS2 promoter. Chromatin prepared from HeLa cells transfected with a human ERα expression vector was subjected to the ChIP procedure with the anti-ERα antibody, and again immunoprecipitated using antibodies as indicated at the top of the figure (Non-specific antibody, IgG; anti-ERα antibody, ER; anti-MBD2 antibody, MBD2).

Mentions: HeLa cells expressing the vector encoding ERα were used to identify the proteins bound to the 5′ end of pS2. Dose-dependent and quantitative PCR amplifications of each fraction obtained from the ChIP procedure were performed. These assays showed that MBD2 was still present on the methylated region of pS2 promoter (Figure 7A), and the enrichment in the bound fraction was not modified by the presence of ERα on the ERE (Figure 7A). These results provide evidence that ERα can overcome, at least partially, the inhibitory effect of MBD2 binding to pS2 promoter and imply that both proteins can occupy the 5′ end of the pS2 gene.


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)

The transactivators ERα and the methylation-dependant repressor MBD2 can simultaneously bound the pS2 promoter in HeLa cells.(A) MBD2 ChIP assays in HeLa cells expressing ERα (HeLa::ERα). Relative amounts of immunoprecipitaded pS2 promoter measured by quantitative PCR from HeLa or HeLa::ERα cells. Each bar represents the mean ± standard deviation of at least three independent experiments. (B) Serial ERα-MBD2 ChIP assays to pS2 promoter. Chromatin prepared from HeLa cells transfected with a human ERα expression vector was subjected to the ChIP procedure with the anti-ERα antibody, and again immunoprecipitated using antibodies as indicated at the top of the figure (Non-specific antibody, IgG; anti-ERα antibody, ER; anti-MBD2 antibody, MBD2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009665-g007: The transactivators ERα and the methylation-dependant repressor MBD2 can simultaneously bound the pS2 promoter in HeLa cells.(A) MBD2 ChIP assays in HeLa cells expressing ERα (HeLa::ERα). Relative amounts of immunoprecipitaded pS2 promoter measured by quantitative PCR from HeLa or HeLa::ERα cells. Each bar represents the mean ± standard deviation of at least three independent experiments. (B) Serial ERα-MBD2 ChIP assays to pS2 promoter. Chromatin prepared from HeLa cells transfected with a human ERα expression vector was subjected to the ChIP procedure with the anti-ERα antibody, and again immunoprecipitated using antibodies as indicated at the top of the figure (Non-specific antibody, IgG; anti-ERα antibody, ER; anti-MBD2 antibody, MBD2).
Mentions: HeLa cells expressing the vector encoding ERα were used to identify the proteins bound to the 5′ end of pS2. Dose-dependent and quantitative PCR amplifications of each fraction obtained from the ChIP procedure were performed. These assays showed that MBD2 was still present on the methylated region of pS2 promoter (Figure 7A), and the enrichment in the bound fraction was not modified by the presence of ERα on the ERE (Figure 7A). These results provide evidence that ERα can overcome, at least partially, the inhibitory effect of MBD2 binding to pS2 promoter and imply that both proteins can occupy the 5′ end of the pS2 gene.

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