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.
Highlights
Zinc finger proteins are a superfamily of transcription factors
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)
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)
Summary
The rat zinc finger protein 667, ZNF667, provisionally named myocardial ischemic preconditioning upregulated protein 1 (Mipu1) in our lab due to its upregulation during myocardial ischemia/reperfusion, belongs to the KRAB/C2H2 zinc finger proteins that contains a KRAB domain at its N-terminus and 14 zinc fingers at its C-terminus. Both the ZNF667 mRNA and protein are expressed abundantly and predominantly in the brain and heart [1,2]. We hypothesized that ZNF667 might be involved in Bax regulation and interfere with the apoptotic pathway in rat heart-derived H9c2 cells in response to oxidative stress
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