Promoter Structrue In-Depth Analysis Of NRXN3β Gene Associated Wite Autism Spectrum Disorder

Objective:To determine the promoter activity region of NRXN3β gene and define the minimal promoter region, search for the cis-acting region that is critical for transcription of NRXN3fβ gene, identify the core bases of the cis-acting region which play the leading role in the promoter activity.Methods:According to the Genbank database, two luciferase reporter gene vectors were constructed that contain the forward sequence or reverse sequence of NRXN3β gene promoter region. The insert fragment covers the 1500bp upstream and 300bp downstream of the transcriptional start site of NRXN3β gene. Human embryo kidney cells (HEK293) was cultured by the conventional methods. With the transfection reagent Lipofectamine 2000 (Invitrogen), the two luciferase reporter gene vectors were transfected in order to express the luciferase in HEK293 cells that was cultured in 48-well plates. Luciferase assay was performed with the dual luciferase assay reagent (E1910 Promega) after lysis of the transfected cells with the passive lysis buffer. The final activity ratios reflect the activity of NRXN3fβ gene promoter after the calibration with the internal control pGL4.10 luciferase activity that was transfected in the same batch, the result showed the sequence NRXN3β-1445/+302E in NRXN3β gene has the promoter activity. Continue to construct the luciferase reporter gene vectors based on the deletion of 5’end and 3’end from the NRXN3p-1445/+302E and compare the luciferase activity of different plasmids. Discover the two minimal promoter region NRXN3p-543/-121E and NRXN3β-50/+117E in NRXN3β gene. Confirm the sequence NRXN3β-110/-80 which contains the cis-acting region that is critical for promoter activity. To investigate core bases of the cis-acting region, the Mutations in region NRXN3β-110/-80 of reporter plasmids were constructed with p4NRXN3β-543/+117E p4NRXN3β-100/+117E and p4NRXN3p-110/+117E as template and compared the luciferase activity among them.Results:We successfully constructed a series of luciferase reporter gene vectors that contained different NRXN3β gene promoter region. The longest insert fragment is 1747bp, contains the 1445bp upstream and 302bp downstream of the transcriptional start site of NRXN3β gene. The results of 51 end and 3’end deletion from the NRXN3β-1445/+302E indicate that there is some activating region and inhibiting region in NRXN3β gene which affects the promoter activity. Moreover, we discovered the two minimal promoter region NRXN3β-543/-121E and NRXN3p-50/+117E in NRXN3β gene. The deletion of the promoter NRXN3β-50/+117E 5’ end reveals that there is a cis-acting element which regulates the transcription of the promoter in the region from-110 to-80 of NRXN3β gene. Mutations in the region from-110 to -90 shown that bases of-107 to-105>-101 to-99 and-98 to 95 are the core bases of the cis-acting element. The sequence of NRXN3β-110/-80 is GC-rich content and has several candidate transcriptional factor binding sites.Conclusion:We have successfully found two minimal promoter region NRXN3β-543/-121E and NRXN3β-50/+117E of NRXN3β gene, and identified the cis-acting element which is critical for transcription of NRXN3β gene existing in the sequence of NRXN3β-110/-80. We also identify the core bases of the cis-acting element which play the leading role in the promoter activity.