儿童孤独症相关基因NRXN1β启动子功能分析

doi:10.16288/j.yczz.15-105

遗传 ›› 2015, Vol. 37 ›› Issue (8): 801-810.doi: 10.16288/j.yczz.15-105

儿童孤独症相关基因NRXN1β启动子功能分析

刘静茹, 孟莎莎, 周卫辉

重庆医科大学附属儿童医院,儿童发育疾病研究教育部重点实验室,认知发育与学习记忆障碍转化医学重庆市重点实验室,重庆市儿童发育重大疾病诊治与预防国际科技合作基地,重庆 400014

收稿日期:2015-03-13 修回日期:2015-06-16 出版日期:2015-08-20 发布日期:2015-08-20
通讯作者: 周卫辉,博士,研究员,研究方向：分子生物学。E-mail: zhouweihui@hotmail.com
作者简介:刘静茹,硕士研究生,专业方向：孤独症致病机制。Tel: 023-63633751;E-mail: yanse.429093913@163.com
基金资助:
国家重点基础研究发展计划项目(973计划)(编号：2012CB517903)资助

Functional analysis of autism-associated NRXN1β gene promoter

Jingru Liu, Shasha Meng, Weihui Zhou

Ministry of Education Key Laboratory of Child Development and Disorders;
Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorder;
Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’
s Hospital of Chongqing Medical University, Chongqing 400014, China

Received:2015-03-13 Revised:2015-06-16 Online:2015-08-20 Published:2015-08-20

摘要/Abstract

摘要： 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。

关键词: NRXN1β, 启动子, DBP, ABF1, 转录因子

Abstract: Neurexins are neuron-specific synaptic proteins, and abnormal structure of Neurexin1β is closely associated with autism. To characterize the minimal promoter of autism-associated NRXN1β 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 corresponding functional elements that significantly enhance or inhibit the activity of reporter genes were further screened out. To identify cis-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 human NRXN1β 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 as cis-acting elements, suggesting the presence of DBP and ABF1 transcription factor binding sites in this region.

Key words: NRXN1β, promoter, ABF1, DBP, transcription factor

刘静茹, 孟莎莎, 周卫辉. 儿童孤独症相关基因NRXN1β启动子功能分析[J]. 遗传, 2015, 37(8): 801-810.

Jingru Liu, Shasha Meng, Weihui Zhou. Functional analysis of autism-associated NRXN1β gene promoter[J]. HEREDITAS(Beijing), 2015, 37(8): 801-810.

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儿童孤独症相关基因NRXN1β启动子功能分析

Functional analysis of autism-associated NRXN1β gene promoter

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