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FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

Gong C, Fujino K, Monteiro LJ, Gomes AR, Drost R, Davidson-Smith H, Takeda S, Khoo US, Jonkers J, Sproul D, Lam EW - Oncogene (2014)

Bottom Line: Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity.We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter.These associations were validated in a familial breast cancer patient cohort.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.

ABSTRACT
FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.

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Knockdown BRCA1 in MFC-7 cells induces FOXA1 promoter methylation. (a) Schematic representation, as presented on UCSC genome web browser, of the location of CpG islands in the human FOXA1 gene and the position of primers used for pyrosequencing analysis. (b) MCF-7 cells were either non-transfected (Mock) or transfected with NSC siRNA or with FOXA1 siRNA-specific pool. Forty-eight hours post transfection, DNA was extracted, bisulphite converted and analyzed by pyrosequencing. Average FOXA1 methylation values of the two analyzed regions within the CpG island 143, located in the FOXA1 promoter region, are shown. (c) Western blot analysis was performed to determine protein expression of BRCA1, FOXA1 and β-Tubulin (arrows indicate specific protein band) in MCF-7 cells. Results are presented as the mean±s.d. from two independent experiments in triplicates. *P<0.05, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
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fig5: Knockdown BRCA1 in MFC-7 cells induces FOXA1 promoter methylation. (a) Schematic representation, as presented on UCSC genome web browser, of the location of CpG islands in the human FOXA1 gene and the position of primers used for pyrosequencing analysis. (b) MCF-7 cells were either non-transfected (Mock) or transfected with NSC siRNA or with FOXA1 siRNA-specific pool. Forty-eight hours post transfection, DNA was extracted, bisulphite converted and analyzed by pyrosequencing. Average FOXA1 methylation values of the two analyzed regions within the CpG island 143, located in the FOXA1 promoter region, are shown. (c) Western blot analysis was performed to determine protein expression of BRCA1, FOXA1 and β-Tubulin (arrows indicate specific protein band) in MCF-7 cells. Results are presented as the mean±s.d. from two independent experiments in triplicates. *P<0.05, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.

Mentions: In the human FOXA1 gene, five CpG islands (CpG 110, CpG 52, CpG 99, CpG 143 and CpG 123) were identified. We focused on CpG island 143 as it corresponds to CpG island 86 located at the promoter region of the mouse Foxa1 gene (Figure 5a). MCF-7 was transiently transfected with either NSC or BRCA1 siRNA, and FOXA1 methylation levels analyzed. Methylated and unmethylated DNAs were also used in the pyrosequencing experiment. In the MCF-7 cells, the FOXA1 promoter was hypomethylated. Knockdown of BRCA1 led to significantly higher levels of FOXA1 methylation compared with the mock (non-transfected) and the NSC siRNA-transfected controls (Figure 5b). Western blot analysis confirmed the knockdown of BRCA1 in MCF-7 cell lines and FOXA1 expression was reduced by BRCA1 knockdown (Figure 5c). Collectively, these data suggest that BRCA1 positively regulates FOXA1 expression, in part, through suppressing its promoter methylation. However, the relatively low levels of DNA methylation before and after 5'-aza-dC treatment suggested that the FOXA1 promoter in the MCF-7 cell line is undermethylated.


FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

Gong C, Fujino K, Monteiro LJ, Gomes AR, Drost R, Davidson-Smith H, Takeda S, Khoo US, Jonkers J, Sproul D, Lam EW - Oncogene (2014)

Knockdown BRCA1 in MFC-7 cells induces FOXA1 promoter methylation. (a) Schematic representation, as presented on UCSC genome web browser, of the location of CpG islands in the human FOXA1 gene and the position of primers used for pyrosequencing analysis. (b) MCF-7 cells were either non-transfected (Mock) or transfected with NSC siRNA or with FOXA1 siRNA-specific pool. Forty-eight hours post transfection, DNA was extracted, bisulphite converted and analyzed by pyrosequencing. Average FOXA1 methylation values of the two analyzed regions within the CpG island 143, located in the FOXA1 promoter region, are shown. (c) Western blot analysis was performed to determine protein expression of BRCA1, FOXA1 and β-Tubulin (arrows indicate specific protein band) in MCF-7 cells. Results are presented as the mean±s.d. from two independent experiments in triplicates. *P<0.05, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430311&req=5

fig5: Knockdown BRCA1 in MFC-7 cells induces FOXA1 promoter methylation. (a) Schematic representation, as presented on UCSC genome web browser, of the location of CpG islands in the human FOXA1 gene and the position of primers used for pyrosequencing analysis. (b) MCF-7 cells were either non-transfected (Mock) or transfected with NSC siRNA or with FOXA1 siRNA-specific pool. Forty-eight hours post transfection, DNA was extracted, bisulphite converted and analyzed by pyrosequencing. Average FOXA1 methylation values of the two analyzed regions within the CpG island 143, located in the FOXA1 promoter region, are shown. (c) Western blot analysis was performed to determine protein expression of BRCA1, FOXA1 and β-Tubulin (arrows indicate specific protein band) in MCF-7 cells. Results are presented as the mean±s.d. from two independent experiments in triplicates. *P<0.05, ***P⩽0.001 by Student's t-test. TSS, Transcription start site.
Mentions: In the human FOXA1 gene, five CpG islands (CpG 110, CpG 52, CpG 99, CpG 143 and CpG 123) were identified. We focused on CpG island 143 as it corresponds to CpG island 86 located at the promoter region of the mouse Foxa1 gene (Figure 5a). MCF-7 was transiently transfected with either NSC or BRCA1 siRNA, and FOXA1 methylation levels analyzed. Methylated and unmethylated DNAs were also used in the pyrosequencing experiment. In the MCF-7 cells, the FOXA1 promoter was hypomethylated. Knockdown of BRCA1 led to significantly higher levels of FOXA1 methylation compared with the mock (non-transfected) and the NSC siRNA-transfected controls (Figure 5b). Western blot analysis confirmed the knockdown of BRCA1 in MCF-7 cell lines and FOXA1 expression was reduced by BRCA1 knockdown (Figure 5c). Collectively, these data suggest that BRCA1 positively regulates FOXA1 expression, in part, through suppressing its promoter methylation. However, the relatively low levels of DNA methylation before and after 5'-aza-dC treatment suggested that the FOXA1 promoter in the MCF-7 cell line is undermethylated.

Bottom Line: Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity.We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter.These associations were validated in a familial breast cancer patient cohort.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, UK.

ABSTRACT
FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.

Show MeSH
Related in: MedlinePlus