Regulation Of WNK4 Gene Expression By GATA-1 Acetylation

IntroductionWNK4(with no lysine[K]kinase-4),which is belonged to a unique protein kinase family,acts as a multifunctional regulator of diverse ion transporters and therefore plays a crucial role in regulating blood pressure.We have now examined the effects of acetylation on WNK4 expression and explored the possibility that acetylation of GATA-1 may enhance its DNA binding affinity and thus the transcription activity of the WNK4 gene.The treatment of human embryo kidney(HEK)293 cells with histone deacetylase (HDAC)inhibitor,trichostatin A(TSA),gave rise to a significant mRNA and protein increase of human WNK4(hWNK4)gene by real-time PCR and Westem blot assays. Luciferase assay revealed that the region-484～-337 within the WNK4 promoter was important for its transcription activity and responded to TSA,where a potential GATA-1 binding site was found.RT-PCR verified the presence of the mRNA expression of GA TA-1 in HEK293 cells and human fetal kidney tissues.Electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation(CHIP)assay further demonstrated that the site(-426～-416)was a GATA-1 protein responsive element and its DNA-binding affinity was increased with TSA treatment in vitro and in vivo. Immunoprecipitation combined with Western blot showed that GATA-1 may be acetylated by TSA and the enhancement of acetylation was in accordance with its DNA-binding affinity.Moreover,results of luciferase assay showed GATA-1 responsive element acted as a powerful positive regulation element.The findings suggest that GATA-1 acetylation takes an important part in the TSA-induced WNK4 expression. Materials and MethodsMaterials1.Human Embryonic Kidney cell line HEK2932.Human fetal kidney tissues from autopsy specimen of fetuses at 22 wk of gestation3.Reagents for real-time RT-PCR and Western blot4.Reagents for gene cloning and luciferase assay5.Reagents for EMSA,ChIP and immunoprecipitation Methods1.Real-time RT-PCR was used to test the changes of WNK4 mRNA level with TSA stimulation.2.Western blot was performed to test the influence of TSA on WNK4 protein level.3.Softwares and luciferase assay were used to analyze WNK4 promoter.4.RT-PCR tested the presence of the mRNA expression of GATA-1 in HEK293 cells and human fetal kidney tissues.5.EMSA and ChIP assay were carried out to identify GATA-1 responsive element within WNK4 promoter and the effect of TSA on GATA-1 binding to this site.6.Immunoprecipitaion using specific antibody of GATA-1 combined with Western blot using monoclonal antibody against acetylated lysine as primary antibody was used to test the effect of TSA on GATA-1 acetylation level.7.Luciferase assay was performed to detect the role of GATA-1 element in the effect of TSA on WNK4 promoter activity.Results1.Real-time RT-PCR results showed TSA may upregulate WNK4 mRNA in a concentration dependent manner.2.Western blot indicated TSA-induced actylation may increase WNK4 protein expression in HEK293 cells.3.Software analysis and luciferase assay suggested some putative responsive elements of transcription factors that were subject to acetylation.4.RT-PCR verified the presence of the mRNA expression of GATA-1 in HEK293 cells and human fetal kidney tissues. 5.EMSA and ChIP assay demonstrated the region-426～-416 of WNK4 promoter was a GATA-1 responsive element under in vitro and in vivo conditions and TSA may enhance the binding of GATA-1 to this site.6.Immunoprecipitation combined with Western blot demonstrated TSA may directly acetylate GATA-1.7.Results of luciferase assay showed GATA-1 responsive element acted a powerful positive regulation element and took an important part in TSA-induced activation of WNK4 promoter.Conclusion1.The position-426～-416 of WNK4 promoter was a GATA-1 responsive element.2.TSA may directly acetylate GATA-1 and thus increases its DNA binding affinity and WNK4 promoter activity.This may be one of the mechanisms by which TSA-induced acetylation upregulate WNK4 gene expression.