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ZNF667/Mipu1 is a novel anti-apoptotic factor that directly regulates the expression of the rat Bax gene in H9c2 cells.

Jiang L, Wang H, Shi C, Liu K, Liu M, Wang N, Wang K, Zhang H, Wang G, Xiao X - PLoS ONE (2014)

Bottom Line: We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox.Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter.Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, P. R. China.

ABSTRACT
ZNF667/Mipu1, a C2H2-type zinc finger transcription factor, was suggested to play an important role in oxidative stress. However, none of the target genes or potential roles of ZNF667 in cardiomyocytes have been elucidated. Here, we investigated the functional role of ZNF667 in H9c2 cell lines focusing on its molecular mechanism by which it protects the cells from apoptosis. We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox. Western immunoblotting analysis revealed that ZNF667 also inhibited Bax protein expression, accompanied by attenuation of the mitochondrial translocation of Bax protein, induced by H2O2. EMSA and target detection assay showed that the purified ZNF667 fusion proteins could interact with the Bax promoter sequence in vitro, and this interaction was dependent upon the ZNF667 DNA binding sequences or its core sequence in the promoter. Furthermore, ChIP assay demonstrated that a stimulus H2O2 could enhance the ability of ZNF667 protein binding to the promoter. Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter. Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment.

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ZNF667 represses the Bax gene expression in H9c2 cardiomyocytes.Cells were transfected with plasmid pcDNA3.1 or pcDNA3.1-ZNF667, and transfected with pRNA-U6.1 or pRNA-U6.1-ZNF667 (shRNA), as indicated. Immunoblotting showing the effect of ZNF667 gene transfection (A) or ZNF667 siRNA (B) on expression of ZNF667 protein using the anti-ZNF667 and/or anti-myc tag antibodies (n = 3). Upper panel showing densitometry analysis of ZNF667 band against β-actin band. *P<0.05 vs. pcDNA3.1, #P<0.05 vs. control siRNA. After transfected with the indicated vector, or co-transfected with the indicated vector and the reporter construct pBa-luc plus pRL-TK, and serum starved in DMEM overnight, the cells were treated with 0.5 mM H2O2 for 12 h or untreated. qRT-PCR was performed to quantify Bax mRNA expression levels, and shown as the relative difference from the pcDNA3.1 or control siRNA normalized to GAPDH expression levels (n = 4, triplicate for each sample) (C). The reporter activity is shown as the relative luciferase activity normalized to the pRL-TK activity (n = 8) (D). *P<0.05 vs. pcDNA3.1; #P<0.05 vs. ctrl siRNA; $P<0.05 vs. pcDNA3.1+ H2O2; &P<0.05 vs. ctrl siRNA + H2O2. IB, immunoblot; P, pcDNA3.1; Z, pcDNA3.1-ZNF667; Cs, control siRNA; Zs, ZNF667 siRNA; Luc, luciferase.
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pone-0111653-g002: ZNF667 represses the Bax gene expression in H9c2 cardiomyocytes.Cells were transfected with plasmid pcDNA3.1 or pcDNA3.1-ZNF667, and transfected with pRNA-U6.1 or pRNA-U6.1-ZNF667 (shRNA), as indicated. Immunoblotting showing the effect of ZNF667 gene transfection (A) or ZNF667 siRNA (B) on expression of ZNF667 protein using the anti-ZNF667 and/or anti-myc tag antibodies (n = 3). Upper panel showing densitometry analysis of ZNF667 band against β-actin band. *P<0.05 vs. pcDNA3.1, #P<0.05 vs. control siRNA. After transfected with the indicated vector, or co-transfected with the indicated vector and the reporter construct pBa-luc plus pRL-TK, and serum starved in DMEM overnight, the cells were treated with 0.5 mM H2O2 for 12 h or untreated. qRT-PCR was performed to quantify Bax mRNA expression levels, and shown as the relative difference from the pcDNA3.1 or control siRNA normalized to GAPDH expression levels (n = 4, triplicate for each sample) (C). The reporter activity is shown as the relative luciferase activity normalized to the pRL-TK activity (n = 8) (D). *P<0.05 vs. pcDNA3.1; #P<0.05 vs. ctrl siRNA; $P<0.05 vs. pcDNA3.1+ H2O2; &P<0.05 vs. ctrl siRNA + H2O2. IB, immunoblot; P, pcDNA3.1; Z, pcDNA3.1-ZNF667; Cs, control siRNA; Zs, ZNF667 siRNA; Luc, luciferase.

Mentions: Our previous studies have reported that H2O2 could increase the Bax expression [17], [19], [20]. In this study, we performed qRT-PCR with ZNF667-overexpressed or –knockdowned cells to determine if ZNF667 has any effect on the Bax gene expression. As shown in Fig. 2A and B, there was a significant increase or a significant reduction in ZNF667 protein levels after ZNF667 gene transfection or transfection with ZNF667 siRNA (#1) compared to the respective control. In cells transfected with ZNF667 plasmid (pcDNA3.1-ZNF667) or ZNF667 siRNA (pRNA-U6.1-ZNF667), there was a significant decrease or increase in Bax mRNA levels compared to control plasmid (pcDNA3.1) (Fig. 2C, bar 1 vs.2) or control siRNA (pRNA-U6.1) cells (Fig. 2C, bar 3 vs.4), respectively, which turns out contrary to ZNF667 levels. H2O2 largely induced Bax mRNA expression (Fig. 2C, bar1 vs. 5). However, the expression of Bax in H2O2-treated cells transfected with pcDNA3.1-ZNF667 was lower than that in the treated cells transfected with pcDNA3.1 (Fig. 2C, bar 5 vs.6). In contrast, Bax in the H2O2-treated cells transfected with ZNF667 siRNA (#1) was further induced to near 200% of that in the treated cells transfected with control siRNA (Fig. 2C, bar 7 vs.8). These results indicate that ZNF667 could inhibit the Bax gene expression induced with or without H2O2 in H9c2 cells.


ZNF667/Mipu1 is a novel anti-apoptotic factor that directly regulates the expression of the rat Bax gene in H9c2 cells.

Jiang L, Wang H, Shi C, Liu K, Liu M, Wang N, Wang K, Zhang H, Wang G, Xiao X - PLoS ONE (2014)

ZNF667 represses the Bax gene expression in H9c2 cardiomyocytes.Cells were transfected with plasmid pcDNA3.1 or pcDNA3.1-ZNF667, and transfected with pRNA-U6.1 or pRNA-U6.1-ZNF667 (shRNA), as indicated. Immunoblotting showing the effect of ZNF667 gene transfection (A) or ZNF667 siRNA (B) on expression of ZNF667 protein using the anti-ZNF667 and/or anti-myc tag antibodies (n = 3). Upper panel showing densitometry analysis of ZNF667 band against β-actin band. *P<0.05 vs. pcDNA3.1, #P<0.05 vs. control siRNA. After transfected with the indicated vector, or co-transfected with the indicated vector and the reporter construct pBa-luc plus pRL-TK, and serum starved in DMEM overnight, the cells were treated with 0.5 mM H2O2 for 12 h or untreated. qRT-PCR was performed to quantify Bax mRNA expression levels, and shown as the relative difference from the pcDNA3.1 or control siRNA normalized to GAPDH expression levels (n = 4, triplicate for each sample) (C). The reporter activity is shown as the relative luciferase activity normalized to the pRL-TK activity (n = 8) (D). *P<0.05 vs. pcDNA3.1; #P<0.05 vs. ctrl siRNA; $P<0.05 vs. pcDNA3.1+ H2O2; &P<0.05 vs. ctrl siRNA + H2O2. IB, immunoblot; P, pcDNA3.1; Z, pcDNA3.1-ZNF667; Cs, control siRNA; Zs, ZNF667 siRNA; Luc, luciferase.
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pone-0111653-g002: ZNF667 represses the Bax gene expression in H9c2 cardiomyocytes.Cells were transfected with plasmid pcDNA3.1 or pcDNA3.1-ZNF667, and transfected with pRNA-U6.1 or pRNA-U6.1-ZNF667 (shRNA), as indicated. Immunoblotting showing the effect of ZNF667 gene transfection (A) or ZNF667 siRNA (B) on expression of ZNF667 protein using the anti-ZNF667 and/or anti-myc tag antibodies (n = 3). Upper panel showing densitometry analysis of ZNF667 band against β-actin band. *P<0.05 vs. pcDNA3.1, #P<0.05 vs. control siRNA. After transfected with the indicated vector, or co-transfected with the indicated vector and the reporter construct pBa-luc plus pRL-TK, and serum starved in DMEM overnight, the cells were treated with 0.5 mM H2O2 for 12 h or untreated. qRT-PCR was performed to quantify Bax mRNA expression levels, and shown as the relative difference from the pcDNA3.1 or control siRNA normalized to GAPDH expression levels (n = 4, triplicate for each sample) (C). The reporter activity is shown as the relative luciferase activity normalized to the pRL-TK activity (n = 8) (D). *P<0.05 vs. pcDNA3.1; #P<0.05 vs. ctrl siRNA; $P<0.05 vs. pcDNA3.1+ H2O2; &P<0.05 vs. ctrl siRNA + H2O2. IB, immunoblot; P, pcDNA3.1; Z, pcDNA3.1-ZNF667; Cs, control siRNA; Zs, ZNF667 siRNA; Luc, luciferase.
Mentions: Our previous studies have reported that H2O2 could increase the Bax expression [17], [19], [20]. In this study, we performed qRT-PCR with ZNF667-overexpressed or –knockdowned cells to determine if ZNF667 has any effect on the Bax gene expression. As shown in Fig. 2A and B, there was a significant increase or a significant reduction in ZNF667 protein levels after ZNF667 gene transfection or transfection with ZNF667 siRNA (#1) compared to the respective control. In cells transfected with ZNF667 plasmid (pcDNA3.1-ZNF667) or ZNF667 siRNA (pRNA-U6.1-ZNF667), there was a significant decrease or increase in Bax mRNA levels compared to control plasmid (pcDNA3.1) (Fig. 2C, bar 1 vs.2) or control siRNA (pRNA-U6.1) cells (Fig. 2C, bar 3 vs.4), respectively, which turns out contrary to ZNF667 levels. H2O2 largely induced Bax mRNA expression (Fig. 2C, bar1 vs. 5). However, the expression of Bax in H2O2-treated cells transfected with pcDNA3.1-ZNF667 was lower than that in the treated cells transfected with pcDNA3.1 (Fig. 2C, bar 5 vs.6). In contrast, Bax in the H2O2-treated cells transfected with ZNF667 siRNA (#1) was further induced to near 200% of that in the treated cells transfected with control siRNA (Fig. 2C, bar 7 vs.8). These results indicate that ZNF667 could inhibit the Bax gene expression induced with or without H2O2 in H9c2 cells.

Bottom Line: We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox.Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter.Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, P. R. China.

ABSTRACT
ZNF667/Mipu1, a C2H2-type zinc finger transcription factor, was suggested to play an important role in oxidative stress. However, none of the target genes or potential roles of ZNF667 in cardiomyocytes have been elucidated. Here, we investigated the functional role of ZNF667 in H9c2 cell lines focusing on its molecular mechanism by which it protects the cells from apoptosis. We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox. Western immunoblotting analysis revealed that ZNF667 also inhibited Bax protein expression, accompanied by attenuation of the mitochondrial translocation of Bax protein, induced by H2O2. EMSA and target detection assay showed that the purified ZNF667 fusion proteins could interact with the Bax promoter sequence in vitro, and this interaction was dependent upon the ZNF667 DNA binding sequences or its core sequence in the promoter. Furthermore, ChIP assay demonstrated that a stimulus H2O2 could enhance the ability of ZNF667 protein binding to the promoter. Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter. Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment.

Show MeSH