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Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation.

Liu X, Chen Z, Xu C, Leng X, Cao H, Ouyang G, Xiao W - Nucleic Acids Res. (2015)

Bottom Line: Set7- fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes.Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression.These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China.

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Depletion of Set7 enhances HIF-1α transactivity. (A) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition (P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions (P = 0.0125 or P = 0.0057, respectively). (B) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (C) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t-test).
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Figure 4: Depletion of Set7 enhances HIF-1α transactivity. (A) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition (P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions (P = 0.0125 or P = 0.0057, respectively). (B) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (C) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t-test).

Mentions: To assess the functional importance of HIF-1α methylation by Set7 on HIF-1 transcriptional activity, we examined the effect of Set7 on the activation of five well-defined luciferase reporters, including BNIP3-luciferase (BNIP3-luc), EPO-luciferase (EPO-luc), VEGF-luciferase (VEGF-luc), p2.1-reporter and multiple HRE repeat reporters (70–74). The overexpression of Set7 in HEK293T cells inhibited the activity of these reporters under normoxic and hypoxic conditions (Figure 3A–E). The knockdown of Set7 in HEK293T cells by Set7-shRNA-1 and Set7-shRNA-2 significantly increased the activity of p2.1 and EPO-luc reporters (Figure 4A1 and A2).


Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation.

Liu X, Chen Z, Xu C, Leng X, Cao H, Ouyang G, Xiao W - Nucleic Acids Res. (2015)

Depletion of Set7 enhances HIF-1α transactivity. (A) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition (P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions (P = 0.0125 or P = 0.0057, respectively). (B) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (C) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t-test).
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Figure 4: Depletion of Set7 enhances HIF-1α transactivity. (A) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition (P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions (P = 0.0125 or P = 0.0057, respectively). (B) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7- MEF cells (Set7−/−) compared with that in the wild-type Set7 MEF cells (Set+/+) under hypoxic conditions. (C) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7- MEF cells (Set7−/−) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t-test).
Mentions: To assess the functional importance of HIF-1α methylation by Set7 on HIF-1 transcriptional activity, we examined the effect of Set7 on the activation of five well-defined luciferase reporters, including BNIP3-luciferase (BNIP3-luc), EPO-luciferase (EPO-luc), VEGF-luciferase (VEGF-luc), p2.1-reporter and multiple HRE repeat reporters (70–74). The overexpression of Set7 in HEK293T cells inhibited the activity of these reporters under normoxic and hypoxic conditions (Figure 3A–E). The knockdown of Set7 in HEK293T cells by Set7-shRNA-1 and Set7-shRNA-2 significantly increased the activity of p2.1 and EPO-luc reporters (Figure 4A1 and A2).

Bottom Line: Set7- fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes.Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression.These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China.

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Related in: MedlinePlus