遗传
遺傳
유전
HEREDITAS(BEIJING)
2015年
8期
801-810
,共10页
NRXN1β%启动子%DBP%ABF1%转录因子
NRXN1β%啟動子%DBP%ABF1%轉錄因子
NRXN1β%계동자%DBP%ABF1%전록인자
NRXN1β%promoter%ABF1%DBP%transcription factor
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。
Neurexins 是神經特異性突觸蛋白,Neurexin1β結構的異常與孤獨癥密切相關。為分析孤獨癥相關基因NRXN1β最小啟動子和調節基因轉錄的功能元件,本文構建瞭含NRXN1β基因上遊調控區不同區域的熒光素酶報告基因質粒,轉染HEK293細胞後,利用檢測雙熒光素酶報告基因的轉錄活性以確定NRXN1β基因最小啟動子區,進而篩選齣相應的顯著增彊或抑製報告基因活性的功能區;同時,為鑒定順式作用元件,利用基因定點突變技術對基因功能區內和臨近DNA序列進行連續的堿基突變;最後,採用轉錄因子預測工具對啟動子功能區內的轉錄調控元件進行分析。結果首次髮現NRXN1β最小啟動子區位于?88~+156 bp,?88~?73 bp和+156~+149 bp可增彊啟動子活性,+229~+419 bp可抑製啟動子活性,且?84~?63 bp為能夠顯著性增彊啟動子活性的順式作用元件,該區域可能存在DBP(Albumin D-site-binding protein,DBP)和ABF1(Autonomously replicating sequence-binding factor 1,ABF1)兩箇轉錄因子結閤位點。
Neurexins 시신경특이성돌촉단백,Neurexin1β결구적이상여고독증밀절상관。위분석고독증상관기인NRXN1β최소계동자화조절기인전록적공능원건,본문구건료함NRXN1β기인상유조공구불동구역적형광소매보고기인질립,전염HEK293세포후,이용검측쌍형광소매보고기인적전록활성이학정NRXN1β기인최소계동자구,진이사선출상응적현저증강혹억제보고기인활성적공능구;동시,위감정순식작용원건,이용기인정점돌변기술대기인공능구내화림근DNA서렬진행련속적감기돌변;최후,채용전록인자예측공구대계동자공능구내적전록조공원건진행분석。결과수차발현NRXN1β최소계동자구위우?88~+156 bp,?88~?73 bp화+156~+149 bp가증강계동자활성,+229~+419 bp가억제계동자활성,차?84~?63 bp위능구현저성증강계동자활성적순식작용원건,해구역가능존재DBP(Albumin D-site-binding protein,DBP)화ABF1(Autonomously replicating sequence-binding factor 1,ABF1)량개전록인자결합위점。
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 <br> (?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.