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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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Purified GST-ZNF667 fusion proteins interact with the Bax gene promoter in vitro.(A) Purified GST-ZF, GST-ZF2 were separated on 10% SDS-PAGE and stained with Coomasie blue (left panel). A target detection assay was performed after the same proteins in A were transfected onto a nitrocellulose membrane. Biotin-labeled DNAs were hybridized to ZF and ZF2 of ZNF667. Arrows represent ZF-DNA complex (upper) and ZF2-DNA complex (lower), respectively (right panel). (B) Purified GST-ZF was added into the binding buffer containing the Bax promoter sequence or the mutant Bax sequence. After incubated at 30°C for 30 min, the mixtures were loaded onto a non-denatured electrophoresis. For both A and B, the biotin-labeled DNA-protein complexes were detected by a lightshift chemiluminescent EMSA kit. EMSA, electrophoretic mobility shift assay.
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pone-0111653-g004: Purified GST-ZNF667 fusion proteins interact with the Bax gene promoter in vitro.(A) Purified GST-ZF, GST-ZF2 were separated on 10% SDS-PAGE and stained with Coomasie blue (left panel). A target detection assay was performed after the same proteins in A were transfected onto a nitrocellulose membrane. Biotin-labeled DNAs were hybridized to ZF and ZF2 of ZNF667. Arrows represent ZF-DNA complex (upper) and ZF2-DNA complex (lower), respectively (right panel). (B) Purified GST-ZF was added into the binding buffer containing the Bax promoter sequence or the mutant Bax sequence. After incubated at 30°C for 30 min, the mixtures were loaded onto a non-denatured electrophoresis. For both A and B, the biotin-labeled DNA-protein complexes were detected by a lightshift chemiluminescent EMSA kit. EMSA, electrophoretic mobility shift assay.

Mentions: Being a transcriptional repressor, ZNF667 could inhibit the expression of the rat Bax gene and protein, which enticed us to investigate whether the Bax gene is one of its downstream genes and regulated directly. Based on bioinformatic analysis, we found that the promoter sequence of the Bax pro-apoptotic gene contains up to six putative ZNF667 binding sites or its core sequence CTTA, and the transcription factor ZNF667 regulates the expression of the Bax gene perhaps by directly binding to the promoter. In order to test this possibility, we first investigated if the ZNF667 protein can bind to the Bax promoter. We expressed and purified ZNF667 truncated fusion proteins. Using the purified ZF and ZF2 fusion proteins, the target detection assay was performed to test whether or not they could bind the Bax promoter. After the membrane was visualized with Pierce Lightshift chemiluminescent EMSA kit (Pierce, cat#20148), we were able to compare the membrane and the SDS-PAGE gel stained with Coomassie blue. As shown in Fig. 4A, there was a single distinct binding band present in either the lane containing the ZF fusion protein corresponding to 80 kDa or the lane containing the ZF2 fusion protein corresponding to 46 kDa, suggesting that both ZF and ZF2 fusions could form a protein-DNA complex with the Bax promoter in vitro, indicating that ZNF667 could bind to the promoter perhaps through its binding 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)

Purified GST-ZNF667 fusion proteins interact with the Bax gene promoter in vitro.(A) Purified GST-ZF, GST-ZF2 were separated on 10% SDS-PAGE and stained with Coomasie blue (left panel). A target detection assay was performed after the same proteins in A were transfected onto a nitrocellulose membrane. Biotin-labeled DNAs were hybridized to ZF and ZF2 of ZNF667. Arrows represent ZF-DNA complex (upper) and ZF2-DNA complex (lower), respectively (right panel). (B) Purified GST-ZF was added into the binding buffer containing the Bax promoter sequence or the mutant Bax sequence. After incubated at 30°C for 30 min, the mixtures were loaded onto a non-denatured electrophoresis. For both A and B, the biotin-labeled DNA-protein complexes were detected by a lightshift chemiluminescent EMSA kit. EMSA, electrophoretic mobility shift assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232351&req=5

pone-0111653-g004: Purified GST-ZNF667 fusion proteins interact with the Bax gene promoter in vitro.(A) Purified GST-ZF, GST-ZF2 were separated on 10% SDS-PAGE and stained with Coomasie blue (left panel). A target detection assay was performed after the same proteins in A were transfected onto a nitrocellulose membrane. Biotin-labeled DNAs were hybridized to ZF and ZF2 of ZNF667. Arrows represent ZF-DNA complex (upper) and ZF2-DNA complex (lower), respectively (right panel). (B) Purified GST-ZF was added into the binding buffer containing the Bax promoter sequence or the mutant Bax sequence. After incubated at 30°C for 30 min, the mixtures were loaded onto a non-denatured electrophoresis. For both A and B, the biotin-labeled DNA-protein complexes were detected by a lightshift chemiluminescent EMSA kit. EMSA, electrophoretic mobility shift assay.
Mentions: Being a transcriptional repressor, ZNF667 could inhibit the expression of the rat Bax gene and protein, which enticed us to investigate whether the Bax gene is one of its downstream genes and regulated directly. Based on bioinformatic analysis, we found that the promoter sequence of the Bax pro-apoptotic gene contains up to six putative ZNF667 binding sites or its core sequence CTTA, and the transcription factor ZNF667 regulates the expression of the Bax gene perhaps by directly binding to the promoter. In order to test this possibility, we first investigated if the ZNF667 protein can bind to the Bax promoter. We expressed and purified ZNF667 truncated fusion proteins. Using the purified ZF and ZF2 fusion proteins, the target detection assay was performed to test whether or not they could bind the Bax promoter. After the membrane was visualized with Pierce Lightshift chemiluminescent EMSA kit (Pierce, cat#20148), we were able to compare the membrane and the SDS-PAGE gel stained with Coomassie blue. As shown in Fig. 4A, there was a single distinct binding band present in either the lane containing the ZF fusion protein corresponding to 80 kDa or the lane containing the ZF2 fusion protein corresponding to 46 kDa, suggesting that both ZF and ZF2 fusions could form a protein-DNA complex with the Bax promoter in vitro, indicating that ZNF667 could bind to the promoter perhaps through its binding 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