儿童孤独症相关基因NRXN1β启动子功能分析

摘要

Neurexins are neuron-specific synaptic proteins, and abnormal structure of Neurexin1β is closely associ-ated with autism. To characterize the minimal promoter of autism-associatedNRXN1β gene and identify functional elements regulating its transcription, luciferase reporter plasmids containing different regulatory regions upstream of NRXN1β gene were constructed. After transfecting HEK293 cells with these plasmids, the minimal promoter region of NRXN1β gene was determined by detecting the transcriptional activity of luciferase reporter genes while the corre-sponding functional elements that significantly enhance or inhibit the activity of reporter genes were further screened out. To identifycis-acting elements, continuous nucleotide mutation within the functional regions and adjacent DNA sequences were generated using site-directed mutagenesis techniques and then transcriptional regulatory elements in corresponding regions were analyzed using transcription factor binding prediction tool. Our results showed for the first time that the minimal promoter region of humanNRXN1β gene is located between positions‒88 and +156 (−88/+156); two regions−88/−73 and +156/+149 enhance while the region +229/+419 inhibits promoter activity. The region−84/−63 significantly enhances promoter activity ascis-acting elements, suggesting the presence of DBP and ABF1 transcription factor binding sites in this region.%Neurexins 是神经特异性突触蛋白，Neurexin1β结构的异常与孤独症密切相关。为分析孤独症相关基因NRXN1β最小启动子和调节基因转录的功能元件，本文构建了含NRXN1β基因上游调控区不同区域的荧光素酶报告基因质粒，转染HEK293细胞后，利用检测双荧光素酶报告基因的转录活性以确定NRXN1β基因最小启动子区，进而筛选出相应的显著增强或抑制报告基因活性的功能区；同时，为鉴定顺式作用元件，利用基因定点突变技术对基因功能区内和临近DNA序列进行连续的碱基突变；最后，采用转录因子预测工具对启动子功能区内的转录调控元件进行分析。结果首次发现NRXN1β最小启动子区位于−88~+156 bp，−88~−73 bp和+156~+149 bp可增强启动子活性，+229~+419 bp可抑制启动子活性，且−84~−63 bp为能够显著性增强启动子活性的顺式作用元件，该区域可能存在DBP(Albumin D-site-binding protein，DBP)和ABF1(Autonomously replicating sequence-binding factor 1，ABF1)两个转录因子结合位点。