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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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Related in: MedlinePlus

ChIP-on-chip analysis of MBD2-binding sites on pS2 5′ end regionally methylated in HeLa cells.(A) Array peaks on pS2 5′ end of MBD2 log2 signal ratio (MBD2 / Input) values are shown below the Affymetrix' Integrated Genome Browser (IGB) window. Red bars, MBD2 binding sites; yellow bars, MBD2 free sites. Genes are transcribed from right to left. pS2 methylation status from nt positions −464 to +314 is shown by a diagram. “pS2 ERE fragment” and “pS2 promoter fragment” analyzed by PCR following ChIP are represented by a white box. (B) BRCA1 5′ end viewed as a MBD2 free control.
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pone-0009665-g003: ChIP-on-chip analysis of MBD2-binding sites on pS2 5′ end regionally methylated in HeLa cells.(A) Array peaks on pS2 5′ end of MBD2 log2 signal ratio (MBD2 / Input) values are shown below the Affymetrix' Integrated Genome Browser (IGB) window. Red bars, MBD2 binding sites; yellow bars, MBD2 free sites. Genes are transcribed from right to left. pS2 methylation status from nt positions −464 to +314 is shown by a diagram. “pS2 ERE fragment” and “pS2 promoter fragment” analyzed by PCR following ChIP are represented by a white box. (B) BRCA1 5′ end viewed as a MBD2 free control.

Mentions: ChIP-on-chip experiments indicated that MBD2 associated specifically the region containing the methylated pS2 TATA-box, where a strong positive signal (red bars) is observed, while the region containing the unmethylated ERE is devoid of MBD2. Thus, the positive signals for MBD2 binding parallels the methylation status of the pS2 5′ end and indicates that MBD2 does not spread outside the methylated region on pS2 promoter (Figure 3A). As a control, results obtained for a previously identified MBD2 free promoter [26], BRCA1, are also shown on Figure 3B. Consistent with previous findings, no MBD2 positive signal was observed in the region spanning the nucleotides −1000 to +1000 of BRCA1 (Figure 3B).


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)

ChIP-on-chip analysis of MBD2-binding sites on pS2 5′ end regionally methylated in HeLa cells.(A) Array peaks on pS2 5′ end of MBD2 log2 signal ratio (MBD2 / Input) values are shown below the Affymetrix' Integrated Genome Browser (IGB) window. Red bars, MBD2 binding sites; yellow bars, MBD2 free sites. Genes are transcribed from right to left. pS2 methylation status from nt positions −464 to +314 is shown by a diagram. “pS2 ERE fragment” and “pS2 promoter fragment” analyzed by PCR following ChIP are represented by a white box. (B) BRCA1 5′ end viewed as a MBD2 free control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009665-g003: ChIP-on-chip analysis of MBD2-binding sites on pS2 5′ end regionally methylated in HeLa cells.(A) Array peaks on pS2 5′ end of MBD2 log2 signal ratio (MBD2 / Input) values are shown below the Affymetrix' Integrated Genome Browser (IGB) window. Red bars, MBD2 binding sites; yellow bars, MBD2 free sites. Genes are transcribed from right to left. pS2 methylation status from nt positions −464 to +314 is shown by a diagram. “pS2 ERE fragment” and “pS2 promoter fragment” analyzed by PCR following ChIP are represented by a white box. (B) BRCA1 5′ end viewed as a MBD2 free control.
Mentions: ChIP-on-chip experiments indicated that MBD2 associated specifically the region containing the methylated pS2 TATA-box, where a strong positive signal (red bars) is observed, while the region containing the unmethylated ERE is devoid of MBD2. Thus, the positive signals for MBD2 binding parallels the methylation status of the pS2 5′ end and indicates that MBD2 does not spread outside the methylated region on pS2 promoter (Figure 3A). As a control, results obtained for a previously identified MBD2 free promoter [26], BRCA1, are also shown on Figure 3B. Consistent with previous findings, no MBD2 positive signal was observed in the region spanning the nucleotides −1000 to +1000 of BRCA1 (Figure 3B).

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