Functional Analysis Of The Transcriptional Regulatory Element C-Fos In The Promoter Region Of Human Sodium Channel SCN3A Gene

Background：The expression of voltage-gated sodium channel α-subunit III codinggene SCN3A is spatially and temporally regulated in nervous system. SCN3A productNav1.3mainly appears in embryonic and early postnatal brain, with significantlydecreasing in adult brain. Several neurological disorders such as epilepsy, mentalretardation, autism and neuropathic pain after spinal cord injury and other nervous systemdisease are related to the abnormal expression of SCN3A. Abnormal of the promoterregion and its regulatory elements of SCN3A might be associated with these diseases.C-Fos is a transcriptional factor which takes part in the early stage of regulating theexpression of gene in eukaryotes and plays an important role in the development of humanand mammalian nervous system. However, the expression regulation of SCN3A by c-Foshas not be reported so far.Objective：This study aims to demonostrate the transcriptional regulatory role ofc-Fos on sodium channel SCN3A gene in vitro through identifying the SCN3A minimalfunctional promoter and upstream transcriptional regulatory regions using dual luciferase reporter system and bioinformatics analysis. Our study will help to explore thespatio-temporal regulatory mechanism of SCN3A and its relationship with the occurrenceand development of epilepsy.Methods：Our research group has successfully constructed the chimeric plasmidsconsisting of hSCN3A gene promoter and luciferase reporter gene PGL4.10previously.Now we are using a stepwise5′truncation method to construct chimeric plasmidsconsisting of different length of hSCN3A gene promoter and luciferase reporter genePGL4.10. By transfecting the PC12cells induced by Never Growth Factor (NGF), we canproceed statistical analysis to the function of promoter and upstream regulating elementsof the hSCN3A gene according to the relative fluorescence value derived from thebi-fluorescence reporter system. Use bioinformatics to analyze the characteristics of thetranscriptional initiator and upstream sequence and predict related regulating elements, weselect predicted transcriptionally regulatory element c-Fos for the functional research.Using site-directed mutagenesis method to delete the regulatory element c-Fos, weconstruct mutant luciferase report gene expression vector (PGL4-F1.1-c-Fos-del), aftertransfection of PC12induced by NGF or not, we can take statistical analysis to thefunction of regulating elements of the hSCN3A gene according to the relative fluorescencevalue derived from the bi-fluorescence reporter system. In order to study the transcriptionfactor binding to the regulatory element, we construct the plasmid expressing thetranscription factor, insert sequence coding the V5label into the front of the sequenceencoding the transcription factor, and take the Western Blot Assay to show the expressionof the fusion protein. Synthesis the biotin labeled DNA probe which contains theregulatory element, and analyze the binding of probe and protein by the gel retardation ofthe electrophoretic mobility experiment.Results:1. The identification of the minimal functional promoter and its upstreamtranscriptional regulatory regions of the hSCN3A gene The activity analysis of hSCN3A gene with different promoter region length(F0.9,F0.7, F0.5, F0.3, F0.2, F0.1) shows that, F0.9and F0.7compared with F1.1respectively,the relative luciferase activity were decreased about30%and80%, these two area are thepositive regulatory regions and there may be some positive regulatory elements; theactivity of the area which is located in the upstream-548bp of the transcriptional initiatorto downstream+174bp are not changed, considered as the minimal functional promoter.2. Potential transcriptionally regulatory elements in the promoter region of thehSCN3A geneUsing software to predict the transcriptionally regulatory element within the positiveregulatory region at the upstream of the transcriptional initiator of hSCN3A gene, we finda large number of transcriptionally regulatory elements, and at the same time selectpredicted c-Fos transcriptionally regulatory element for followed function research.3. The transcriptionally regulatory element c-Fos in the promoter region of hSCN3Agene can up-regulate the expression of the reporter gene and it needs the participation ofNGF.Based on the activity analysis of c-Fos transcriptionally regulatory element deletedplasmid, we find the activity of the wild type plasmid has decreased40%in the PC12cellscompared with in the PC12cells induced by NGF, and the c-Fos deleted plasmid has nodifference; In the PC12cells induced by NGF, the c-Fos deleted plasmid has decreased50%compared with the wild type plasmid, and the activity of the c-Fos deleted plasmidand the wild type plasmid has no difference. The result show that, in PC12cells, only inthe condition of induced by NGF, c-Fos element can significantly up-regulate the activityof the promoter of hSCN3A gene.4. NGF up-regulate the expression of endogenous Scn3a gene of the PC12cellUsing RT-PCR and fluorescence quantitative PCR method to analyze the expressionof endogenous Scn3a gene, both with the consistency of internal reference, RT-PCR showthat, in case of PC12cells induced by NGF, the endogenous Scn3a gene PCR bandconcentration was significantly higher that PC12cells without NGF. At the same timefluorescent quantitative PCR also shows that in case of PC12cells induced by NGF, the endogenous Scn3a gene transcript was significantly higher that PC12cells whitout NGF.The results above suggest NGF can up-regulate the expression of endogenous Scn3a genein PC12cells.5. The c-Fos protein do not directly bind to the transcriptional regulatory elementTo study whether c-Fos directly binding to the transcriptionally regulatory element,our research first constructs the plasmid expressing the fusion protein V5-c-Fos, and usesthe Western Blot assay to demonstrate that the heterologously expressed V5-c-Fos fusionprotein is transported to the cellular nucleus. Take the gel retardation of the electrophoreticmobility experiment to observe the combination of V5-c-Fos protein with DNA fragmentwhich contains the transcriptionally regulatory element. The results showed that V5-c-Fosprotein did not bind to the DNA fragment which contains the regulatory element. Itsuggests that c-Fos protein may combine with transcriptionally regulatory element inindirect ways.Conclusion:Our study has identified the minimal functional promoter and itsupstream regulatory region of hSCN3A gene and found that a functional c-Fos regulatoryelement in the upstream region of559bp to549bp increased the promoter activity of theSCN3A gene only in the presence of NGF. These results implicate that NGF might byessential for the expressional regulation of SCN3A gene by c-Fos in vivo.