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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 activity of firefly luciferase driven by the Bax promoter.Bax promoter-driven reporter gene (pBa-luc) was co-transfected into Raw264.7 cells with increasing amounts (0.1-1µg ) of pcDNA3.1-ZNF667, or 1µg of ZNF667-pEGFP or truncated ZNF667(ZF-pEGFP and KRAB-pEGFP), or pcDNA3.1 empty vector. Site-mutated reporter gene (pBM-luc) was also co-transfected into Raw264.7 cells with 0.1 or 1µg of pcDNA3.1-ZNF667 plasmid. Resultant luciferase activities are expressed as relative luciferase activities normalized to the pRL-TK activity. **P<0.01 vs. pcDNA3.1; ##P<0.01 vs. pEGFP. Luc, luciferase.
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pone-0111653-g006: ZNF667 represses the activity of firefly luciferase driven by the Bax promoter.Bax promoter-driven reporter gene (pBa-luc) was co-transfected into Raw264.7 cells with increasing amounts (0.1-1µg ) of pcDNA3.1-ZNF667, or 1µg of ZNF667-pEGFP or truncated ZNF667(ZF-pEGFP and KRAB-pEGFP), or pcDNA3.1 empty vector. Site-mutated reporter gene (pBM-luc) was also co-transfected into Raw264.7 cells with 0.1 or 1µg of pcDNA3.1-ZNF667 plasmid. Resultant luciferase activities are expressed as relative luciferase activities normalized to the pRL-TK activity. **P<0.01 vs. pcDNA3.1; ##P<0.01 vs. pEGFP. Luc, luciferase.

Mentions: In order to quantitatively analyze the effect of ZNF667 overexpression on the Bax promoter, we constructed a luciferase reporter gene vector (pBa-luc) by inserting the Bax promoter into the pGL3 basic vector, in which the Bax promoter was located upstream of the firefly luciferase gene driving expression of firefly luciferase. We then constructed a mutation vector (pBM-luc), in which all the core sequences were mutated by nucleotide substitution from 5′-CTTA-3′ to 5′-GCGC-3′, as reported previously [3]. We performed studies to determine whether ZNF667 was transcriptionally repressive in a DNA binding-dependent manner. We examined the effects of ZNF667 expression on the activity of the Bax promoter containing either the ZNF667 DNA or the mutant DNA binding sequence. The construction scheme of the Bax promoter luciferase reporter vector used in these assays is shown in Fig. 1. As seen in Fig. 6, the co-transfection of RAW264.7 cells with a ZNF667 expressing plasmid (pcDNA3.1-ZNF667 or pEGFP-ZNF667) and the Bax promoter construct, which contained six ZNF667 core sequences (pBa-luc) could repress the promoter activity in a dose-dependent manner (Fig. 6, bars 2-5), whereas the co-transfection of the cells with pcDNA3.1 empty vector and the same reporter could not repress the promoter activity (Fig. 6, bar 1 vs. 4). Co-transfection of RAW264.7 cells with either pEGFP or pEGFP-KRAB and the same reporter construct failed to reduce the activity from the reporter gene promoter construct (Fig. 6, bars 7 and 8 vs. 6), suggesting that ZNF667 inhibited the activity of the firefly luciferase gene by inhibiting the Bax promoter, and this inhibition requires its intact structure. However, co-transfection of RAW264.7 cells with pcDNA3.1-ZNF667 and an all-binding-site-mutant ZNF667 binding sequence-reporter construct (pBM-luc) also failed to reduce the activity from the reporter gene promoter construct in both high and low doses (Fig. 6, bar 9 vs.2 and bar 10 vs.5), suggesting that ZNF667 inhibited the activity of the firefly luciferase gene by direct binding to the Bax promoter through its binding sequence. These results suggest that ZNF667 has transcription repressor activity on the Bax promoter, and this repression is dependent upon its binding to the promoter via the specific DNA sequence.


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 activity of firefly luciferase driven by the Bax promoter.Bax promoter-driven reporter gene (pBa-luc) was co-transfected into Raw264.7 cells with increasing amounts (0.1-1µg ) of pcDNA3.1-ZNF667, or 1µg of ZNF667-pEGFP or truncated ZNF667(ZF-pEGFP and KRAB-pEGFP), or pcDNA3.1 empty vector. Site-mutated reporter gene (pBM-luc) was also co-transfected into Raw264.7 cells with 0.1 or 1µg of pcDNA3.1-ZNF667 plasmid. Resultant luciferase activities are expressed as relative luciferase activities normalized to the pRL-TK activity. **P<0.01 vs. pcDNA3.1; ##P<0.01 vs. pEGFP. Luc, luciferase.
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Related In: Results  -  Collection

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pone-0111653-g006: ZNF667 represses the activity of firefly luciferase driven by the Bax promoter.Bax promoter-driven reporter gene (pBa-luc) was co-transfected into Raw264.7 cells with increasing amounts (0.1-1µg ) of pcDNA3.1-ZNF667, or 1µg of ZNF667-pEGFP or truncated ZNF667(ZF-pEGFP and KRAB-pEGFP), or pcDNA3.1 empty vector. Site-mutated reporter gene (pBM-luc) was also co-transfected into Raw264.7 cells with 0.1 or 1µg of pcDNA3.1-ZNF667 plasmid. Resultant luciferase activities are expressed as relative luciferase activities normalized to the pRL-TK activity. **P<0.01 vs. pcDNA3.1; ##P<0.01 vs. pEGFP. Luc, luciferase.
Mentions: In order to quantitatively analyze the effect of ZNF667 overexpression on the Bax promoter, we constructed a luciferase reporter gene vector (pBa-luc) by inserting the Bax promoter into the pGL3 basic vector, in which the Bax promoter was located upstream of the firefly luciferase gene driving expression of firefly luciferase. We then constructed a mutation vector (pBM-luc), in which all the core sequences were mutated by nucleotide substitution from 5′-CTTA-3′ to 5′-GCGC-3′, as reported previously [3]. We performed studies to determine whether ZNF667 was transcriptionally repressive in a DNA binding-dependent manner. We examined the effects of ZNF667 expression on the activity of the Bax promoter containing either the ZNF667 DNA or the mutant DNA binding sequence. The construction scheme of the Bax promoter luciferase reporter vector used in these assays is shown in Fig. 1. As seen in Fig. 6, the co-transfection of RAW264.7 cells with a ZNF667 expressing plasmid (pcDNA3.1-ZNF667 or pEGFP-ZNF667) and the Bax promoter construct, which contained six ZNF667 core sequences (pBa-luc) could repress the promoter activity in a dose-dependent manner (Fig. 6, bars 2-5), whereas the co-transfection of the cells with pcDNA3.1 empty vector and the same reporter could not repress the promoter activity (Fig. 6, bar 1 vs. 4). Co-transfection of RAW264.7 cells with either pEGFP or pEGFP-KRAB and the same reporter construct failed to reduce the activity from the reporter gene promoter construct (Fig. 6, bars 7 and 8 vs. 6), suggesting that ZNF667 inhibited the activity of the firefly luciferase gene by inhibiting the Bax promoter, and this inhibition requires its intact structure. However, co-transfection of RAW264.7 cells with pcDNA3.1-ZNF667 and an all-binding-site-mutant ZNF667 binding sequence-reporter construct (pBM-luc) also failed to reduce the activity from the reporter gene promoter construct in both high and low doses (Fig. 6, bar 9 vs.2 and bar 10 vs.5), suggesting that ZNF667 inhibited the activity of the firefly luciferase gene by direct binding to the Bax promoter through its binding sequence. These results suggest that ZNF667 has transcription repressor activity on the Bax promoter, and this repression is dependent upon its binding to the promoter via the specific DNA sequence.

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