Effects Of Transcription Factor AP-2? Nuclear Localization Signal Domain Mutations On PDA Related Genes

The patent ductus arteriosus(PDA)is a common congenital cardiovascular developmental defect in infants,and the incidence is up to 0.5‰ in term infants.At the same time,it is one of the major congenital diseases,which have seriously threatened human life and health.TFAP2B is a neural crest-derived transcription factor,which can regulate the expression of myocardial cell structure proteins or cardiac gene proteins by promoting the transcription activity of downstream target genes.It can also participate in cell cycle regulation and neural crest cell differentiation,finally promoting the closure of ductus arteriosus.In recent years,domestic and oversea scholars' research on TFAP2B and PDA were mainly focusing on the transcription activation domain,DNA binding domain and dimerization domain.However,mutations in nuclear localization signal(NLS)domain were less analyzed.In this paper,we first analyzed the mutations of TFAP2B in NLS domain.We analyzed and screened single nucleotide polymorphism(SNP)sites of TFAP2B,and obtained three point mutations,A275D(rs80338914),R285Q(rs80338915),R300C(rs80338917).Luciferase reporter assay showed that the mutations of A275D,R285Q and R300C significantly had reduced the transcriptional activity of TFAP2B.By analyzing the homology of TFAP2B and online prediction,we successfully obtained the sequence of NLS domain and discovered the mutations of A275D,R285Q,R300C were located in NLS.We further confirmed that the three mutations altered the distribution of TFAP2B protein in cells by immunofluorescence staining and western blotting,indicating that the three mutations actually located in the NLS.Luciferase reporter assay showed that with the increase of CITED2,the transcriptional activity of A275D was not changed and was far lower than WT group,while the transcriptional activities of R285Q and R300C were increased.It was indicated that CITED2 could recover the transcriptional activity of TFAP2B in a certain extent.Luciferase reporter assay also showed that the increasing dosage of the three mutations would alter the transcriptional activity of WT TFAP2B,suggesting that A275D,R285Q and R300C were likely to have a dominant negative effect on WT TFAP2B.Next we detected some TFAP2B downstream genes,CACNA1G,CACNB2,KCNA2,BMP2,BMP4,Et-1 and ERBB3,all of which are related to PDA.RT-qPCR results showed that the mRNA levels of these genes were altered,when TFAP2B mutation was introduced into cells.A275D,R285Q and R300C had made these genes upregulation or downregulation,which may eventually lead to PDA.All of the above results can provide some theoretical basis for elucidating the mechanism of PDA.