Functional Research Of Negative Regulatory Elements In Human Sodium Channel SCN1A Gene Promoter

IntroductionVoltage-gated sodium channel α subunit type I (Nav1.1) is mainly expressed in thecentral nervous system and plays a critical role in the initiation of action potential inneurons. The ion channel disease that results from the Nav1.1dysfuction is the mostcommon human disease by far. It has been reported that Nav1.1dysfuction isassociated with epilepsy and several other neurological disorders, such as familialhemiplegic migraine (FHM), familial autism (FA). In the past study found SCN1A(encoding Nav1.1) expression under strict regulation, has the characteristics ofdevelopment of dependence and tissue specificity, and in all ion channel than expressin centure nervous system, only when Nav1.1decreased expression is lack ofcompensatory mechanism. The accuracy of SCN1A expression directly affects thenormal physiological function of the central nervous system. In recent study foundthat there are multiple promoters of SCN1A respectively in different5′-untranslatedexons upstream. It has not been reported that the functional differences between thesepromoters and its contribution to the SCN1A expression of time and space. Our previous studies have found that there are at least three functional promoters ofSCN1A and confirmed that they exist in different activity in different cell types of theculture cell. This study will be more in-depth research the characteristics of thepromoters and its functional differences and their impact on SCN1A expression.ObjectiveThe purpose of this study is to identify the minimum functional promoter, the corepromoter elements, the5′-untranslated exons upstream regulatory elements and theircorresponding transcription factors of SCN1A, also compare each promoter of SCN1Aexpression differences in different cell lines to explore SCN1A transcriptionalregulation mechanism in the central nervous system. This study may offer a newavenue for revealing nervous system diseases such as epilepsy pathogenesis andtreatment.Methods1. By PCR amplification, with SCN1A5’ upstream transcription start site4Kbfragment DNA as a template, through stepwise truncating at the5′end andextending at the3′end, amplification segments of different lengths, cloning tofluorescent expression vector PGL4.10so construct the recombinant plasmid.Mutation recombinant plasmid is established using site-directed mutagenesismethod.2. Dual luciferase reporter gene system was adopted to detect promoter activity ofdifferent length in order to find the minimum functional promoter and the corepromoter element.3. Synthesize identified significant regulatory elements with biotin labeled probe,prepare the cytoplasmic extract and nuclear extract of NT-2cell, then the EMSAexperiments to verify whether there is a target protein in vitro combined withexpected transcriptional regulatory elements.4. With Biotin-Pull Down and SDS-PAGE electrophoresis separation combinedwith expected transcriptional regulatory elements of the NT-2cells nuclear extract, using liquid chromatography tandem mass spectrometry (LC-MS/MS)analysis of the binding protein ingredients, and then use the correspondingantibody for Super-shift experiment further validation of the combination oftranscription factors.5. ChIP experiments further in vivo validates the specific transcription factors andexpected the combination of transcriptional regulatory elements.6. Use the shRNA interference experiments further validates the transcriptionalregulation function of transcription factor.Results1. Human SCN1A P1c promoter has a high activity in NT2cells.2. The region nt+53to+62was a transcriptional silencer downstream of the P1cpromoters.3. EMSA and ChIP analysis found that the role of transcriptional regulationthrough RACK1protein combined with transcriptional silencer.4. Knockdown of RACK1expression increases the activity of the P1c promoterand SCN1A transcription.5. The promoter silencer is critical for the increased SCN1A transcription in theneuronal-differentiated NT2cells. Neuronal differentiation decreases thecapability of RACK1’s binding to the silencer downstream of the SCN1Apromoter.ConclutionThis study shows an important role of a scaffolding protein, RACK1, in regulatinghuman SCN1A expression. Our results demonstrate that RACK1plays an importantrole in repressing SCN1A expression by interacting with a silencer elementdownstream of SCN1A promoter in undifferentiated NT2cells. We furtherdemonstrate that upregulation of SCN1A expression in RA-induced differentiatedNT2cells is mediated by the decrease of nuclear proteins binding to the silencer viaRACK1. These findings suggest a novel mechanism of SCN1A expression regulated by RACK1in neuronal and nonneuronal cells.