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Zinc-induced Dnmt1 expression involves antagonism between MTF-1 and nuclear receptor SHP.

Zhang Y, Andrews GK, Wang L - Nucleic Acids Res. (2012)

Bottom Line: SHP in turn represses MTF-1 expression and abolishes zinc-mediated changes in the chromatin configuration of the Dnmt1 promoter.In human hepatocellular carcinoma (HCC), increased DNMT1 expression is negatively correlated with SHP levels.Our study provides a molecular explanation for increased Dnmt1 expression in HCC and highlights SHP as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Dnmt1 is frequently overexpressed in cancers, which contributes significantly to cancer-associated epigenetic silencing of tumor suppressor genes. However, the mechanism of Dnmt1 overexpression remains elusive. Herein, we elucidate a pathway through which nuclear receptor SHP inhibits zinc-dependent induction of Dnmt1 by antagonizing metal-responsive transcription factor-1 (MTF-1). Zinc treatment induces Dnmt1 transcription by increasing the occupancy of MTF-1 on the Dnmt1 promoter while decreasing SHP expression. SHP in turn represses MTF-1 expression and abolishes zinc-mediated changes in the chromatin configuration of the Dnmt1 promoter. Dnmt1 expression is increased in SHP-knockout (sko) mice but decreased in SHP-transgenic (stg) mice. In human hepatocellular carcinoma (HCC), increased DNMT1 expression is negatively correlated with SHP levels. Our study provides a molecular explanation for increased Dnmt1 expression in HCC and highlights SHP as a potential therapeutic target.

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Zinc activation of Dnmt1 expression requires MTF-1. (A) qPCR analysis of MTF-1, SHP and Dnmt1 mRNA in MTF-1+/+ and MTF-1−/− MEF cells without (−Zn) or with 100 µM ZnSO4 treatment for 6 hr (+Zn). *P < 0.01, +Zn group versus −Zn group. (B) WB of Dnmt1 protein in MTF-1+/+ and MTF-1−/− MEF cells in the without (−) or with 100 µM ZnSO4 treatment for 6 hr (+). (C) Left: Transient transfection assays in MTF-1+/+ and MTF-1−/− MEF cells to determine the activation of Dnmt1 promoter-luciferase reporter (Luc) in response to zinc treatment. Right: Transient transfection assays of the Dnmt1 promoter-luciferase (Luc) reporter in HEK293 cells treated with zinc. Cells were cultured in medium containing 100 µM ZnSO4 for 6 hr. *P < 0.01, +Zn group versus −Zn group; §P < 0.01, −Zn group in MTF-1−/− versus −Zn group in MTF-1+/+ cells; †P < 0.01, +Zn group versus −Zn group in MTF-1−/− cells. (A and C) Statistical results represent mean ± SD of triplicate assays. (D) Left: diagram showing putative MREs in the Dnmt1 promoter and the location of primers used for ChIP assays. Middle: ChIP assays to monitor the association of MTF-1 to the endogenous Dnmt1 promoter in MTF-1−/− and M42 cells in the absence of zinc (upper panel), or in M42 cells treated with 100 µM ZnSO4 for 6 hr (lower panel). Right: Quantification of the binding signals in ChIP assays. (E) Left: qPCR analysis of MTF-1 and Dnmt1 mRNA in MEF cells (WT and M42). Right: WB of MTF-1 and Dnmt1 protein in MEF cells (WT and M42).
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gks159-F4: Zinc activation of Dnmt1 expression requires MTF-1. (A) qPCR analysis of MTF-1, SHP and Dnmt1 mRNA in MTF-1+/+ and MTF-1−/− MEF cells without (−Zn) or with 100 µM ZnSO4 treatment for 6 hr (+Zn). *P < 0.01, +Zn group versus −Zn group. (B) WB of Dnmt1 protein in MTF-1+/+ and MTF-1−/− MEF cells in the without (−) or with 100 µM ZnSO4 treatment for 6 hr (+). (C) Left: Transient transfection assays in MTF-1+/+ and MTF-1−/− MEF cells to determine the activation of Dnmt1 promoter-luciferase reporter (Luc) in response to zinc treatment. Right: Transient transfection assays of the Dnmt1 promoter-luciferase (Luc) reporter in HEK293 cells treated with zinc. Cells were cultured in medium containing 100 µM ZnSO4 for 6 hr. *P < 0.01, +Zn group versus −Zn group; §P < 0.01, −Zn group in MTF-1−/− versus −Zn group in MTF-1+/+ cells; †P < 0.01, +Zn group versus −Zn group in MTF-1−/− cells. (A and C) Statistical results represent mean ± SD of triplicate assays. (D) Left: diagram showing putative MREs in the Dnmt1 promoter and the location of primers used for ChIP assays. Middle: ChIP assays to monitor the association of MTF-1 to the endogenous Dnmt1 promoter in MTF-1−/− and M42 cells in the absence of zinc (upper panel), or in M42 cells treated with 100 µM ZnSO4 for 6 hr (lower panel). Right: Quantification of the binding signals in ChIP assays. (E) Left: qPCR analysis of MTF-1 and Dnmt1 mRNA in MEF cells (WT and M42). Right: WB of MTF-1 and Dnmt1 protein in MEF cells (WT and M42).

Mentions: We tested the effect of zinc on the expression of MTF-1, SHP and Dnmt1 in MTF-1+/+ and MTF-1−/− MEF cells. Zinc (Zn) exposure resulted in a marked induction of MTF-1 mRNA in MTF-1+/+ MEFs, which was accompanied by a decreased SHP expression and increased Dnmt1 expression (Figure 4A, left). Zn treatment in MTF-1 deficient (MTF-1−/−) MEFs induced SHP expression, which corresponded with the attenuated expression of Dnmt1 in response to Zn (Figure 4A, right). SHP mRNA was induced by zinc in MTF-1−/− MEFs, suggesting an alternative mechanism that activates SHP in the absence of MTF-1. The increased expression of SHP may be responsible for the inhibition of Dnmt1 expression.Figure 4.


Zinc-induced Dnmt1 expression involves antagonism between MTF-1 and nuclear receptor SHP.

Zhang Y, Andrews GK, Wang L - Nucleic Acids Res. (2012)

Zinc activation of Dnmt1 expression requires MTF-1. (A) qPCR analysis of MTF-1, SHP and Dnmt1 mRNA in MTF-1+/+ and MTF-1−/− MEF cells without (−Zn) or with 100 µM ZnSO4 treatment for 6 hr (+Zn). *P < 0.01, +Zn group versus −Zn group. (B) WB of Dnmt1 protein in MTF-1+/+ and MTF-1−/− MEF cells in the without (−) or with 100 µM ZnSO4 treatment for 6 hr (+). (C) Left: Transient transfection assays in MTF-1+/+ and MTF-1−/− MEF cells to determine the activation of Dnmt1 promoter-luciferase reporter (Luc) in response to zinc treatment. Right: Transient transfection assays of the Dnmt1 promoter-luciferase (Luc) reporter in HEK293 cells treated with zinc. Cells were cultured in medium containing 100 µM ZnSO4 for 6 hr. *P < 0.01, +Zn group versus −Zn group; §P < 0.01, −Zn group in MTF-1−/− versus −Zn group in MTF-1+/+ cells; †P < 0.01, +Zn group versus −Zn group in MTF-1−/− cells. (A and C) Statistical results represent mean ± SD of triplicate assays. (D) Left: diagram showing putative MREs in the Dnmt1 promoter and the location of primers used for ChIP assays. Middle: ChIP assays to monitor the association of MTF-1 to the endogenous Dnmt1 promoter in MTF-1−/− and M42 cells in the absence of zinc (upper panel), or in M42 cells treated with 100 µM ZnSO4 for 6 hr (lower panel). Right: Quantification of the binding signals in ChIP assays. (E) Left: qPCR analysis of MTF-1 and Dnmt1 mRNA in MEF cells (WT and M42). Right: WB of MTF-1 and Dnmt1 protein in MEF cells (WT and M42).
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gks159-F4: Zinc activation of Dnmt1 expression requires MTF-1. (A) qPCR analysis of MTF-1, SHP and Dnmt1 mRNA in MTF-1+/+ and MTF-1−/− MEF cells without (−Zn) or with 100 µM ZnSO4 treatment for 6 hr (+Zn). *P < 0.01, +Zn group versus −Zn group. (B) WB of Dnmt1 protein in MTF-1+/+ and MTF-1−/− MEF cells in the without (−) or with 100 µM ZnSO4 treatment for 6 hr (+). (C) Left: Transient transfection assays in MTF-1+/+ and MTF-1−/− MEF cells to determine the activation of Dnmt1 promoter-luciferase reporter (Luc) in response to zinc treatment. Right: Transient transfection assays of the Dnmt1 promoter-luciferase (Luc) reporter in HEK293 cells treated with zinc. Cells were cultured in medium containing 100 µM ZnSO4 for 6 hr. *P < 0.01, +Zn group versus −Zn group; §P < 0.01, −Zn group in MTF-1−/− versus −Zn group in MTF-1+/+ cells; †P < 0.01, +Zn group versus −Zn group in MTF-1−/− cells. (A and C) Statistical results represent mean ± SD of triplicate assays. (D) Left: diagram showing putative MREs in the Dnmt1 promoter and the location of primers used for ChIP assays. Middle: ChIP assays to monitor the association of MTF-1 to the endogenous Dnmt1 promoter in MTF-1−/− and M42 cells in the absence of zinc (upper panel), or in M42 cells treated with 100 µM ZnSO4 for 6 hr (lower panel). Right: Quantification of the binding signals in ChIP assays. (E) Left: qPCR analysis of MTF-1 and Dnmt1 mRNA in MEF cells (WT and M42). Right: WB of MTF-1 and Dnmt1 protein in MEF cells (WT and M42).
Mentions: We tested the effect of zinc on the expression of MTF-1, SHP and Dnmt1 in MTF-1+/+ and MTF-1−/− MEF cells. Zinc (Zn) exposure resulted in a marked induction of MTF-1 mRNA in MTF-1+/+ MEFs, which was accompanied by a decreased SHP expression and increased Dnmt1 expression (Figure 4A, left). Zn treatment in MTF-1 deficient (MTF-1−/−) MEFs induced SHP expression, which corresponded with the attenuated expression of Dnmt1 in response to Zn (Figure 4A, right). SHP mRNA was induced by zinc in MTF-1−/− MEFs, suggesting an alternative mechanism that activates SHP in the absence of MTF-1. The increased expression of SHP may be responsible for the inhibition of Dnmt1 expression.Figure 4.

Bottom Line: SHP in turn represses MTF-1 expression and abolishes zinc-mediated changes in the chromatin configuration of the Dnmt1 promoter.In human hepatocellular carcinoma (HCC), increased DNMT1 expression is negatively correlated with SHP levels.Our study provides a molecular explanation for increased Dnmt1 expression in HCC and highlights SHP as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Dnmt1 is frequently overexpressed in cancers, which contributes significantly to cancer-associated epigenetic silencing of tumor suppressor genes. However, the mechanism of Dnmt1 overexpression remains elusive. Herein, we elucidate a pathway through which nuclear receptor SHP inhibits zinc-dependent induction of Dnmt1 by antagonizing metal-responsive transcription factor-1 (MTF-1). Zinc treatment induces Dnmt1 transcription by increasing the occupancy of MTF-1 on the Dnmt1 promoter while decreasing SHP expression. SHP in turn represses MTF-1 expression and abolishes zinc-mediated changes in the chromatin configuration of the Dnmt1 promoter. Dnmt1 expression is increased in SHP-knockout (sko) mice but decreased in SHP-transgenic (stg) mice. In human hepatocellular carcinoma (HCC), increased DNMT1 expression is negatively correlated with SHP levels. Our study provides a molecular explanation for increased Dnmt1 expression in HCC and highlights SHP as a potential therapeutic target.

Show MeSH
Related in: MedlinePlus