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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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MBD2 modulates the estrogen response of pS2 gene.(A) Transcriptional expression of pS2 was analyzed using relative RT-PCR in HeLa cells expressing ERα and/or depleted in MBD2. Mock, mock-treated HeLa cells. ER, HeLa cells 24 h after transfection with a human ERα expression vector, HEG0. MBD2 siRNA, HeLa cells pretreated for 72 h with MBD2 siRNA and again for 24 h. MBD2 siRNA + ER, HeLa cells pretreated for 72 h with MBD2 siRNA, then cotransfected with MBD2 siRNA and HEG0 for 24 h. OHT, 24 h treatment with 4-hydroxytamoxifen. MCF7, MCF7 cells. (B) Bar chart showing the fold change of pS2 expression in HeLa cells expressing ERα and/or depleted in MBD2. pS2 transcripts were quantified by real time RT-PCR. The fold change was calculated from the relative pS2 mRNA in treated compared to mock-treated cells. Each bar represents the mean ± standard deviation of three analyses. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).
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pone-0009665-g008: MBD2 modulates the estrogen response of pS2 gene.(A) Transcriptional expression of pS2 was analyzed using relative RT-PCR in HeLa cells expressing ERα and/or depleted in MBD2. Mock, mock-treated HeLa cells. ER, HeLa cells 24 h after transfection with a human ERα expression vector, HEG0. MBD2 siRNA, HeLa cells pretreated for 72 h with MBD2 siRNA and again for 24 h. MBD2 siRNA + ER, HeLa cells pretreated for 72 h with MBD2 siRNA, then cotransfected with MBD2 siRNA and HEG0 for 24 h. OHT, 24 h treatment with 4-hydroxytamoxifen. MCF7, MCF7 cells. (B) Bar chart showing the fold change of pS2 expression in HeLa cells expressing ERα and/or depleted in MBD2. pS2 transcripts were quantified by real time RT-PCR. The fold change was calculated from the relative pS2 mRNA in treated compared to mock-treated cells. Each bar represents the mean ± standard deviation of three analyses. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).

Mentions: The opposite effects of MBD2 and ERα proteins on pS2 expression suggest an antagonistic action between these two transcriptional regulators, in HeLa cells. To investigate this possibility, the concentrations of MBD2 and ERα proteins were artificially manipulated in these cells. After MBD2 depletion mediated by RNA interference, ectopic ERα expression resulted in a dramatic (approximately 31-fold) enhancement of pS2 mRNA concentration, approaching the level to that observed in MCF7 (Figure 8A, B). Thus, pS2 responses to ERα activation (4-fold increase) and MBD2 depletion (3-fold increase) are not additive and suggest a cross-talk between these two transcriptional regulators. Concomitant exposure to OHT knocked down pS2 expression to the level observed in HeLa cells transfected by MBD2 siRNA alone, (about 3-fold) when compared with control HeLa cells (Figure 8A, B). It should be noted that pS2 transcription cannot be induced by concomitant ectopic expression of ERα and MBD2 depletion when its 5′ end is fully methylated, as observed in MDA MB231 cells (data not shown). In cells exhibiting a bimodal methylation profile as HeLa cells, a synergic activation was observed. Thus, the binding of MBD2 to the methylated TATA-box of pS2 reduces but does not abolish pS2 response to ERα, suggesting that as a result of regional demethylation, pS2 is poised for transcription.


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)

MBD2 modulates the estrogen response of pS2 gene.(A) Transcriptional expression of pS2 was analyzed using relative RT-PCR in HeLa cells expressing ERα and/or depleted in MBD2. Mock, mock-treated HeLa cells. ER, HeLa cells 24 h after transfection with a human ERα expression vector, HEG0. MBD2 siRNA, HeLa cells pretreated for 72 h with MBD2 siRNA and again for 24 h. MBD2 siRNA + ER, HeLa cells pretreated for 72 h with MBD2 siRNA, then cotransfected with MBD2 siRNA and HEG0 for 24 h. OHT, 24 h treatment with 4-hydroxytamoxifen. MCF7, MCF7 cells. (B) Bar chart showing the fold change of pS2 expression in HeLa cells expressing ERα and/or depleted in MBD2. pS2 transcripts were quantified by real time RT-PCR. The fold change was calculated from the relative pS2 mRNA in treated compared to mock-treated cells. Each bar represents the mean ± standard deviation of three analyses. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837351&req=5

pone-0009665-g008: MBD2 modulates the estrogen response of pS2 gene.(A) Transcriptional expression of pS2 was analyzed using relative RT-PCR in HeLa cells expressing ERα and/or depleted in MBD2. Mock, mock-treated HeLa cells. ER, HeLa cells 24 h after transfection with a human ERα expression vector, HEG0. MBD2 siRNA, HeLa cells pretreated for 72 h with MBD2 siRNA and again for 24 h. MBD2 siRNA + ER, HeLa cells pretreated for 72 h with MBD2 siRNA, then cotransfected with MBD2 siRNA and HEG0 for 24 h. OHT, 24 h treatment with 4-hydroxytamoxifen. MCF7, MCF7 cells. (B) Bar chart showing the fold change of pS2 expression in HeLa cells expressing ERα and/or depleted in MBD2. pS2 transcripts were quantified by real time RT-PCR. The fold change was calculated from the relative pS2 mRNA in treated compared to mock-treated cells. Each bar represents the mean ± standard deviation of three analyses. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).
Mentions: The opposite effects of MBD2 and ERα proteins on pS2 expression suggest an antagonistic action between these two transcriptional regulators, in HeLa cells. To investigate this possibility, the concentrations of MBD2 and ERα proteins were artificially manipulated in these cells. After MBD2 depletion mediated by RNA interference, ectopic ERα expression resulted in a dramatic (approximately 31-fold) enhancement of pS2 mRNA concentration, approaching the level to that observed in MCF7 (Figure 8A, B). Thus, pS2 responses to ERα activation (4-fold increase) and MBD2 depletion (3-fold increase) are not additive and suggest a cross-talk between these two transcriptional regulators. Concomitant exposure to OHT knocked down pS2 expression to the level observed in HeLa cells transfected by MBD2 siRNA alone, (about 3-fold) when compared with control HeLa cells (Figure 8A, B). It should be noted that pS2 transcription cannot be induced by concomitant ectopic expression of ERα and MBD2 depletion when its 5′ end is fully methylated, as observed in MDA MB231 cells (data not shown). In cells exhibiting a bimodal methylation profile as HeLa cells, a synergic activation was observed. Thus, the binding of MBD2 to the methylated TATA-box of pS2 reduces but does not abolish pS2 response to ERα, suggesting that as a result of regional demethylation, pS2 is poised for transcription.

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