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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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Set7 has no effect on mRNA level, protein level and nuclear translocation of HIF-1α. (A) Under hypoxia condition, overexpression of Set7 had no effect on HIF-1α mRNA level in HEK293T cells. (B) In RCC4 cells, knockdown of Set7 by Set7-shRNA-1 has no effect on HIF-1α mRNA level. (C) Overexpression of Set7 had no obvious effect on HIF-1α protein level in HEK293T cells. (D) The protein level of HIF-1α was not affected by overexpression of Set7 in HEK293T cells in the presence of CHX (50 μg/ml). Short, shorter exposure; long, longer exposure. (E) Overexpression of Set7 did not affect HIF-1α nuclear translocation. α-tubulin detection was used to monitor cytoplasmic protein and Histone H3 detection was used to monitor nuclear protein.
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Figure 7: Set7 has no effect on mRNA level, protein level and nuclear translocation of HIF-1α. (A) Under hypoxia condition, overexpression of Set7 had no effect on HIF-1α mRNA level in HEK293T cells. (B) In RCC4 cells, knockdown of Set7 by Set7-shRNA-1 has no effect on HIF-1α mRNA level. (C) Overexpression of Set7 had no obvious effect on HIF-1α protein level in HEK293T cells. (D) The protein level of HIF-1α was not affected by overexpression of Set7 in HEK293T cells in the presence of CHX (50 μg/ml). Short, shorter exposure; long, longer exposure. (E) Overexpression of Set7 did not affect HIF-1α nuclear translocation. α-tubulin detection was used to monitor cytoplasmic protein and Histone H3 detection was used to monitor nuclear protein.

Mentions: Next, we examined whether the suppression of Set7 on HIF-1α transcriptional activity was due to the effect of Set7 on HIF-1α mRNA and protein levels. Overexpression of Set7 in HEK293T cells did not reduce HIF-1α mRNA level under hypoxic condition (Figure 7A). Consistently, knockdown of Set7 in RCC4 cells also did not enhance HIF-1α mRNA level (Figure 7B). In addition, co-expression of Set7 together with HIF-1α did not alter HIF-1α protein level (Figure 7C). Furthermore, in the presence of cycloheximide (CHX, 50 μg/ml), overexpression of Set7 had no obvious effect on HIF-1α protein stability (Figure 7D). To determine whether the suppression of Set7 on HIF-1α transcriptional activity was due to the effect of Set7 on HIF-1α nuclear translocation (59), we performed subcellular localization assay. After overexpression of HIF-1α together with Set7 or empty vector control, we separated nucleus and cytoplasm fraction and conducted western blot analysis. To ensure the efficiency of fraction separation, we used anti-α-tubulin antibody to monitor cytoplasmic proteins and used anti-Histone H3 antibody to monitor nuclear proteins. As shown in Figure 7E, overexpression of Set7 did not alter HIF-1α nuclear localization. Taken together, these data suggest that the suppression of Set7 on HIF-1α transcriptional activity was neither due to the effect of Set7 on HIF-1α mRNA and protein levels, nor due to its effect on HIF-1α nuclear localization.


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)

Set7 has no effect on mRNA level, protein level and nuclear translocation of HIF-1α. (A) Under hypoxia condition, overexpression of Set7 had no effect on HIF-1α mRNA level in HEK293T cells. (B) In RCC4 cells, knockdown of Set7 by Set7-shRNA-1 has no effect on HIF-1α mRNA level. (C) Overexpression of Set7 had no obvious effect on HIF-1α protein level in HEK293T cells. (D) The protein level of HIF-1α was not affected by overexpression of Set7 in HEK293T cells in the presence of CHX (50 μg/ml). Short, shorter exposure; long, longer exposure. (E) Overexpression of Set7 did not affect HIF-1α nuclear translocation. α-tubulin detection was used to monitor cytoplasmic protein and Histone H3 detection was used to monitor nuclear protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4446437&req=5

Figure 7: Set7 has no effect on mRNA level, protein level and nuclear translocation of HIF-1α. (A) Under hypoxia condition, overexpression of Set7 had no effect on HIF-1α mRNA level in HEK293T cells. (B) In RCC4 cells, knockdown of Set7 by Set7-shRNA-1 has no effect on HIF-1α mRNA level. (C) Overexpression of Set7 had no obvious effect on HIF-1α protein level in HEK293T cells. (D) The protein level of HIF-1α was not affected by overexpression of Set7 in HEK293T cells in the presence of CHX (50 μg/ml). Short, shorter exposure; long, longer exposure. (E) Overexpression of Set7 did not affect HIF-1α nuclear translocation. α-tubulin detection was used to monitor cytoplasmic protein and Histone H3 detection was used to monitor nuclear protein.
Mentions: Next, we examined whether the suppression of Set7 on HIF-1α transcriptional activity was due to the effect of Set7 on HIF-1α mRNA and protein levels. Overexpression of Set7 in HEK293T cells did not reduce HIF-1α mRNA level under hypoxic condition (Figure 7A). Consistently, knockdown of Set7 in RCC4 cells also did not enhance HIF-1α mRNA level (Figure 7B). In addition, co-expression of Set7 together with HIF-1α did not alter HIF-1α protein level (Figure 7C). Furthermore, in the presence of cycloheximide (CHX, 50 μg/ml), overexpression of Set7 had no obvious effect on HIF-1α protein stability (Figure 7D). To determine whether the suppression of Set7 on HIF-1α transcriptional activity was due to the effect of Set7 on HIF-1α nuclear translocation (59), we performed subcellular localization assay. After overexpression of HIF-1α together with Set7 or empty vector control, we separated nucleus and cytoplasm fraction and conducted western blot analysis. To ensure the efficiency of fraction separation, we used anti-α-tubulin antibody to monitor cytoplasmic proteins and used anti-Histone H3 antibody to monitor nuclear proteins. As shown in Figure 7E, overexpression of Set7 did not alter HIF-1α nuclear localization. Taken together, these data suggest that the suppression of Set7 on HIF-1α transcriptional activity was neither due to the effect of Set7 on HIF-1α mRNA and protein levels, nor due to its effect on HIF-1α nuclear localization.

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