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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 methylates HIF-1α. (A) (A1) Sequence alignment of partial HIF-1α proteins (1–39 amino acids) of human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by Set7; the blue arrow indicates the conserved lysine (K32) mutated to arginine in methylation assays. The amino acid is numbered based on human HIF-1α amino acid sequence. (A2) Dot-blot assay for HIF-1α peptides containing various degrees of methylation. Equal amounts of unmodified (K32), mono-(K32-me1), di-(K32-me2) and tri-(K32-me3) methylated HIF-1α were immunoblotted with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (B) (B1) Western blot analysis of HIF-1α methylated by the wild-type Set7 or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. Anti-HA agarose beads were used for immunoprecipitation. (B2) Western blot analysis of mutant HIF-1α (K32R) methylated by the wild-type or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (C) (C1) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines overexpressing the wild-type or mutant Set7 (H297A). Methylated HIF-1α was detected by western blot analysis with anti-HIF-1αK32me antibody [anti-HIF-1α(CH3)-K32] (top). Western blot signals obtained with anti-HIF-1α antibody as loading controls. Anti-HIF-1α conjugated agarose beads were used for immunoprecipitation. (C2) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines with knockdown of endogenous Set7 by Set7-shRNA-1 and Set7-shRNA-2. (D) (D1) Western blot analysis of recombinant partial HIF-1α peptide (1–80 amino acids) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32] (top). Coomassie-blue staining of the peptide (1–80 amino acids) as loading control (bottom). (D2) Western blot analysis of the synthesized HIF-1α peptide (ARSRRSKESEVF) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32].
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Figure 1: Set7 methylates HIF-1α. (A) (A1) Sequence alignment of partial HIF-1α proteins (1–39 amino acids) of human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by Set7; the blue arrow indicates the conserved lysine (K32) mutated to arginine in methylation assays. The amino acid is numbered based on human HIF-1α amino acid sequence. (A2) Dot-blot assay for HIF-1α peptides containing various degrees of methylation. Equal amounts of unmodified (K32), mono-(K32-me1), di-(K32-me2) and tri-(K32-me3) methylated HIF-1α were immunoblotted with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (B) (B1) Western blot analysis of HIF-1α methylated by the wild-type Set7 or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. Anti-HA agarose beads were used for immunoprecipitation. (B2) Western blot analysis of mutant HIF-1α (K32R) methylated by the wild-type or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (C) (C1) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines overexpressing the wild-type or mutant Set7 (H297A). Methylated HIF-1α was detected by western blot analysis with anti-HIF-1αK32me antibody [anti-HIF-1α(CH3)-K32] (top). Western blot signals obtained with anti-HIF-1α antibody as loading controls. Anti-HIF-1α conjugated agarose beads were used for immunoprecipitation. (C2) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines with knockdown of endogenous Set7 by Set7-shRNA-1 and Set7-shRNA-2. (D) (D1) Western blot analysis of recombinant partial HIF-1α peptide (1–80 amino acids) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32] (top). Coomassie-blue staining of the peptide (1–80 amino acids) as loading control (bottom). (D2) Western blot analysis of the synthesized HIF-1α peptide (ARSRRSKESEVF) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32].

Mentions: To determine the involvement of Set7 in the hypoxia signaling pathway, we analyzed whether the HIF-1α protein sequence harbors a conserved domain. The three ‘RSK’ residues in the bHLH domain (amino acids 30–32) of HIF-1α (Figure 1A1) are identical to the core local recognition sequence of Set7, namely, (K/R) (S/T) K. This core sequence is identified in Set7-targeted proteins, such as histone H3, p53, E2F1, estrogen receptor-α and LIN28A (9,11,17,19,20,67,68). These three residues are also evolutionarily conserved among human, macaque, cow, pig, dog, mouse and rat (Figure 1A1). Thus, HIF-1α is a potential substrate of Set7.


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 methylates HIF-1α. (A) (A1) Sequence alignment of partial HIF-1α proteins (1–39 amino acids) of human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by Set7; the blue arrow indicates the conserved lysine (K32) mutated to arginine in methylation assays. The amino acid is numbered based on human HIF-1α amino acid sequence. (A2) Dot-blot assay for HIF-1α peptides containing various degrees of methylation. Equal amounts of unmodified (K32), mono-(K32-me1), di-(K32-me2) and tri-(K32-me3) methylated HIF-1α were immunoblotted with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (B) (B1) Western blot analysis of HIF-1α methylated by the wild-type Set7 or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. Anti-HA agarose beads were used for immunoprecipitation. (B2) Western blot analysis of mutant HIF-1α (K32R) methylated by the wild-type or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (C) (C1) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines overexpressing the wild-type or mutant Set7 (H297A). Methylated HIF-1α was detected by western blot analysis with anti-HIF-1αK32me antibody [anti-HIF-1α(CH3)-K32] (top). Western blot signals obtained with anti-HIF-1α antibody as loading controls. Anti-HIF-1α conjugated agarose beads were used for immunoprecipitation. (C2) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines with knockdown of endogenous Set7 by Set7-shRNA-1 and Set7-shRNA-2. (D) (D1) Western blot analysis of recombinant partial HIF-1α peptide (1–80 amino acids) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32] (top). Coomassie-blue staining of the peptide (1–80 amino acids) as loading control (bottom). (D2) Western blot analysis of the synthesized HIF-1α peptide (ARSRRSKESEVF) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32].
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Figure 1: Set7 methylates HIF-1α. (A) (A1) Sequence alignment of partial HIF-1α proteins (1–39 amino acids) of human, macaque, cow, pig, dog, mouse and rat. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by Set7; the blue arrow indicates the conserved lysine (K32) mutated to arginine in methylation assays. The amino acid is numbered based on human HIF-1α amino acid sequence. (A2) Dot-blot assay for HIF-1α peptides containing various degrees of methylation. Equal amounts of unmodified (K32), mono-(K32-me1), di-(K32-me2) and tri-(K32-me3) methylated HIF-1α were immunoblotted with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (B) (B1) Western blot analysis of HIF-1α methylated by the wild-type Set7 or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. Anti-HA agarose beads were used for immunoprecipitation. (B2) Western blot analysis of mutant HIF-1α (K32R) methylated by the wild-type or mutant Set7 (H297A) in transfected cells with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32]. (C) (C1) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines overexpressing the wild-type or mutant Set7 (H297A). Methylated HIF-1α was detected by western blot analysis with anti-HIF-1αK32me antibody [anti-HIF-1α(CH3)-K32] (top). Western blot signals obtained with anti-HIF-1α antibody as loading controls. Anti-HIF-1α conjugated agarose beads were used for immunoprecipitation. (C2) Detection of endogenous methylated HIF-1α from whole-cell extracts of RCC4 stable cell lines with knockdown of endogenous Set7 by Set7-shRNA-1 and Set7-shRNA-2. (D) (D1) Western blot analysis of recombinant partial HIF-1α peptide (1–80 amino acids) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32] (top). Coomassie-blue staining of the peptide (1–80 amino acids) as loading control (bottom). (D2) Western blot analysis of the synthesized HIF-1α peptide (ARSRRSKESEVF) methylated by Set7 (Millipore) in vitro with monomethyl HIF-1α antibody [anti-HIF-1α(CH3)-K32].
Mentions: To determine the involvement of Set7 in the hypoxia signaling pathway, we analyzed whether the HIF-1α protein sequence harbors a conserved domain. The three ‘RSK’ residues in the bHLH domain (amino acids 30–32) of HIF-1α (Figure 1A1) are identical to the core local recognition sequence of Set7, namely, (K/R) (S/T) K. This core sequence is identified in Set7-targeted proteins, such as histone H3, p53, E2F1, estrogen receptor-α and LIN28A (9,11,17,19,20,67,68). These three residues are also evolutionarily conserved among human, macaque, cow, pig, dog, mouse and rat (Figure 1A1). Thus, HIF-1α is a potential substrate of Set7.

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