Analysis Of NRXN3α Gene Promoter Region Related To Autism

Objective:To define the minimal promoter region of NRXN3a gene, identify the cis-acting region that essential to the transcription of NRXN3α.Methods:Various 5’upstream DNA fragments of the human NRXN3α gene were inserted into PGL4.10 vector to build the luciferase reporter plasmids of containing different NRXN3a promoter regions according to conventional molecular cloning techniques.The NRXN3a reporter plasmids and Renilla luciferase control reporter plasmid were co-transfected into U87 MG cells with the transfection reagent Lipofectamine 2000.Luciferase activity in U87 MG cell was measured with the dual luciferase assay reagent (Promega) 24 hours after transfection, and the firefly luciferase activity (Fluc) from NRXN3a reporter plasmid was normalized to Renilla luciferase ativity (Rluc) from control plasmid to reflect the Luciferase activity.To define the minimal promoter region,the Luciferase activity of various NRXN3α luciferase reporter plasmids containing different promoter regions was compared with each other.To identify the cis-acting region, plasmids containing site-directed nucleotide mutation were constructed with P4NRXN3a-443/-162 as template, and the mutation plasmids having the biggest impact on the Luciferase activity were detected by comparing luciferase activity of mutation plasmids to that of P4NRXN3a-443/-162.Results:The results revealed that the sequence from the first exon to ATG in mRNA sequence indeed exists. A series of the luciferase reporter plasmids containing NRXN3a different promoter regions were successfully constructed.The longest inserted fragment is 2359bp, which contains the sequence of 2308 nucleotides upstream first exon and 51 necleotids downstream of transcriptional start site, named as P4NRXN3α-2308/+51E. The results indicated that the region of +99/+109 inhibitsgene transcription. The region of -322/-162 is the minimal promoter of NRXN3a gene.Two cis-acting regions -318/-311 and -300/-295 were determined to play an important role in activating the transcription of NRXN3a.Conclusion:We have found a new promoter region of NRXN3a gene and identified the minimal promoter region and two cis-acting elements.