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

ERα only associates hypomethylated ERE region of pS2.Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2. The position of the “pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2837351&req=5

pone-0009665-g005: ERα only associates hypomethylated ERE region of pS2.Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2. The position of the “pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.

Mentions: ChIP assays, using anti-ERα antibodies, were performed from, HeLa and MDA MB231 cells transiently transfected with the vector HEG0 encoding ERα. ERα-rich MCF7 cells were used as a positive control and untransfected HeLa and MDA MB231 cells as negative controls. As expected, ChIP assays performed from MCF7 chromatin indicated that the amount of pS2 DNA per ng of total DNA in immunoprecipitated fraction (Figure 5, “bound”) is higher (about 8-fold, Q-PCR assays) than in input, or non-retained fractions (Figure 5, “input”, “unbound”, and “IgG”) while no enrichment in pS2 sequence was observed in immunoprecipitated fraction obtained from untransfected HeLa and MDA MB231 cells (Figure 5). All together, these data indicate that anti-ERα antibodies specifically precipitated chromatin bound by ERα.


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)

ERα only associates hypomethylated ERE region of pS2.Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2. The position of the “pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009665-g005: ERα only associates hypomethylated ERE region of pS2.Representative experiments of ERα ChIP assays in ERα-rich MCF7 cells, in ERα-negative HeLa and MDA MB231 cells, and in HeLa and MDA MB231 expressing the vector HEG0 encoding ERα (HeLa::ERα, and MDA MB231::ERα). ChIP assays were performed as described in Figure 2. The position of the “pS2 ERE fragment” analyzed by PCR are represented on the pS2 5′ end schema.
Mentions: ChIP assays, using anti-ERα antibodies, were performed from, HeLa and MDA MB231 cells transiently transfected with the vector HEG0 encoding ERα. ERα-rich MCF7 cells were used as a positive control and untransfected HeLa and MDA MB231 cells as negative controls. As expected, ChIP assays performed from MCF7 chromatin indicated that the amount of pS2 DNA per ng of total DNA in immunoprecipitated fraction (Figure 5, “bound”) is higher (about 8-fold, Q-PCR assays) than in input, or non-retained fractions (Figure 5, “input”, “unbound”, and “IgG”) while no enrichment in pS2 sequence was observed in immunoprecipitated fraction obtained from untransfected HeLa and MDA MB231 cells (Figure 5). All together, these data indicate that anti-ERα antibodies specifically precipitated chromatin bound by ERα.

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