The Regulatory Role And Mechanism Of GATA2 On The Expression Of Transcription Factor ERG In Vascular Endothelial Cells

Background:The E26 transformation specific(ETS)-related gene(ERG)is a transcription factor in the ETS family and expression is clearly restricted to ECs,megakaryocytes,and chondrocytes.ERG drives transcription of genes specifically associated with endothelial lineage in endothelial cells.GATA Binding Protein 2(GATA2)is a member of the GATA transcription factor family,which is widely expressed in ECs and involved in the regulation of a variety of endothelialspecific genes.Vascular endothelial cells(ECs)line the inner layers of blood vessels and participate in the exchange of nutrients and metabolic waste products between blood and tissue fluid,sensing changes in the circulatory environment and participating in signal transduction.ECs secrete a variety of endothelium-derived cytokines,which are involved in the regulation of vascular homeostasis.The homeostasis of ECs is indispensable for blood vessels to perform normal physiological functions.ECs are activated under pathological conditions,resulting in disruption of the homeostasis of ECs,which in turn causes vascular dysfunction.As an endothelial cell transcription factor,ERG involved in a variety of biological processes in endothelial cells and plays an integral role in maintaining ECs homeostasis and vascular stability.However,the upstream regulatory mechanism of ERG gene is not clear.Based on the fact that the promoter region of ERG gene contains multiple GATA family binding sites,we investigated the regulatory role and mechanism of GATA2 on ERG in Human umbilical vein endothelial cells(HUVECs)in order to provide a new theoretical basis for the homeostasis of ECs and vascular stability.Objective:To investigate the regulatory effect and mechanism of transcription factor GATA2 on ERG gene in HUVECs.Methods:1.Knock down GATA2 in HUVECs to detect the expression levels of ERG mRNA and proteinGATA2 small interfering RNA(GATA2 siRNA)was synthesized,and GATA2 siRNA and negative control small interfering RNA(Ctrl siRNA)were transfected into HUVECs using Lipofectamine 2000 transfection reagent,respectively,and total cellular RNA and protein were extracted 48 hours later.The mRNA expression levels of GATA2 and ERG genes were detected using RT-qPCR technique in control and experimental groups.The protein expression levels of GATA2 and ERG genes in the control and experimental groups were detected using Western blot techniques.2.Overexpression of GATA2 in HUVECs to detect the expression levels of ERG mRNA and proteinGATA2 overexpression plasmid was constructed,and GATA2 overexpression plasmid and negative control plasmid(CON)were transfected into HUVECs with Lipofectamine 3000 transfection reagent,respectively,and total cellular RNA and protein were extracted 48 hours later.The mRNA expression levels of GATA2 and ERG genes were detected using RT-qPCR technique in control and experimental groups.The protein expression levels of GATA2 and ERG genes in the control and experimental groups were detected using Western blot techniques.3.To detect GATA2 binding to the ERG gene promoter regionAfter bioinformatics analysis,-359 and-1015 GATA binding sites in the promoter region of ERG gene were selected and corresponding primers were designed.Chromatin immunoprecipitation(ChIP)was used to detect GATA2 binding to positions-359 and-1015 in the promoter region of the ERG gene.4.Verify the regulation of ERG gene by GATA2 at positions-359 and-1015 in the promoter region of ERG genePGL3-basic was used as a vector to construct a wild-type fragment containing the ERG promoter as well as a mutant luciferase plasmid at the-359/-1015 GATA binding sites in the ERG promoter region.Luciferase plasmids were co-transfected into HUVECs cells in groups with GATA2 siRNA and GATA2 overexpression plasmids,respectively.The ERG gene promoter activity was detected by extracting cell lysates 48 hours later.Results:1.GATA2 siRNA and GATA2 overexpression plasmids were transfected into HUVECs,the total RNA and protein were extracted 48 hours later.RT-qPCR and Western blot results showed that the mRNA and protein of GATA2 gene were significantly decreased in GATA2 siRNA group,and the mRNA and protein of GATA2 gene were significantly increased in GATA2 overexpression group.2.GATA2 siRNA and GATA2 overexpression plasmids were transfected into HUVECs,the total RNA and protein were extracted 48 hours later.RT-qPCR and Western blot results showed that the mRNA and protein of ERG gene were significantly decreased in GATA2 siRNA group,and the mRNA and protein of ERG gene were significantly increased in GATA2 overexpression group.3.ChIP assay results showed that GATA2 bound to the ERG gene promoter region at site-359,and GATA2 did not bind obviously to the ERG gene promoter region at site-1015.4.The results of Sanger sequencing showed that we successfully constructed the luciferase plasmids containing the wild-type fragment of the ERG promoter and the mutated fragment of the-359/-1015 GATA binding site in the ERG promoter region.The base sequence at position-359 in the ERG promoter region was mutated from AGCTAAGG to CTAGCCTT,and the base sequence at position-1015 in the ERG promoter region was mutated from TGATTT to GTCGGG.5.The results of dual-luciferase activity assay showed that the promoter activity of ERG gene was significantly decreased in the GATA2 siRNA group;the promoter activity of ERG gene was significantly increased in the GATA2 overexpression group;GATA2 had no significant effect on the promoter activity of ERG gene after mutation at position-359 in the promoter region of ERG gene;GATA2 still had a significant effect on the promoter activity of ERG gene after mutation at position-1015 in the promoter region of ERG gene.Conclusions:1.GATA2 positively regulates ERG gene in HUVECs.2.GATA2 binds to the ERG gene promoter region.3.GATA2 positively regulates the ERG gene by binding to the ERG gene promoter region-359 GATA binding site.