BAV-associated Regulatory Regions Reveal The Regulation And Function Of GATA4 During EndoMT

Background:Bicuspid aortic valve(BAV)is an inheritable disease,whereas the mutations identified to cause BAV only explain a small portion of cases.Our previous genome-wide association study identified a non-coding segment enriched with BAV-associated risk variants.So far,non-coding risk variants contributing to BAV susceptibility have been unexplored.The endothelial-to-mesenchymal cell transition(EndoMT)is a fundamental process for heart valve formation.Objective:This study aims to explore the function of BAV-associated non-coding genetic variants in EndoMT and the underlying mechanism.Methods(1)Human peripheral blood mononuclear cells PBMC were transfected with Yamanaka factors and reprogrammed into human pluripotent stem cells(hiPSC).Immunofluorescence staining was conducted to dectect hiPSC biomarkers.(2)The 81 kb non-coding region enriched with BAV-associated variants were deleted using CRISPR/Cas9 technology.PCR,T7 endonuclease assay,and Sanger sequencing were ulitlized to analyse cell genotypes.(3)According to the cell differentiation process during heart valve development,wild type(WT)and BAV-associated non-coding region knockout(KO)hiPSCs were differentiated into cardiovascular progenitor cells(CPC)and then differentiated into endothelial cells(EC),and at last,EndoMT induction was conducted and cells were differentiated into mesenchymal stem cells(MSC).Cell biomarkers and cell function were detected to investigate the influence of BAV-associated non-coding segment KO on EndoMT process.(4)Genes(GATA4,NEIL2,CTSB,FDFT1,DEFB134,DEFB135,DEFB136)flanking the BAV-associated non-coding region were tested with q PCR and immunofluorescence staining to explore the target gene/genes of this non-coding segment.(5)CRISPR inhibition(CRISPRi)technology was utilized to inhibit transcriptional activity of each variants(rs117157630,rs6601627,rs118065347,rs75747817,rs117430032).MSC biomarkers and target gene of the BAV-associated non-coding region was dedected with q PCR to explore the potential causal variant in the BAV-associated non-coding region.(6)Luciferase reporter assay was conducted to investigate the molecular mechanism of how target gene was regulated by non-coding variant.(7)Single-cell sequencing was used to explore the downstream mechanism of target gene regulating EndoMT process.Results:(1)Immunofluorescence results showed high expression level of hiPSC markers,indicating hiPSC cell line was succeesully generated.(2)PCR,T7 endonuclease assay and Sanger sequencing showed BAVassociated non-coding region was conpmletely deleted from hiPSC.(3)Knockout of BAV-associated non-coding region led to impaired EndoMT process,demonstrated as significantly higher EC biomarker levels,significantly lower MSC biomarker levels,and reduced cell migration ability in KO cells than WT cells at the end of EndoMT induction.Also,KO cells showed significantly higher cell apoptosis level than WT cells during EndoMT process.(4)During EndoMT process,KO cells expressed significantly lower GATA4 than WT,whereas other genes flanking BAV-associated noncoding region showed no significant difference in KO and WT cells.(5)Inhibition of rs117430032 specifically led to reduced GATA4 expression and impaired EndoMT,whereas no significant change was observed by inhibiting the other four variants.(6)Results of luciferase reporter assay indicated that transcription factor TWIST1 specifically bound to this enhancer motif and thereby promoting GATA4 expression,the process of which was interrupted by rs117430032 variant.(7)Single-cell sequencing results indicated GATA4 played an important role in EndoMT process by regulating the expression of genes like MYOCD,GPC6,LTBP,VCAN.Conclusions:This study discovered that BAV-associated variant rs117430032 tags an E-box enhancer motif.By interfering TWIST1 binding to E-box,rs11743003 led to decreased GATA4 expression,and hence caused impaired EndoMT,cell migration and cell survival during EndoMT.This study suggests that epigenetic factors may play a potential regulatory role in EndoMT and BAV,and also suggests that the pathogenesis of BAV may involve complex genetic and epigenetic interactions,providing new research targets and important insights for the future study of genetic pathogenic factors of BAV.