PROMOTER REGION OF THE TRANSCRIPTIONAL UNIT FOR HUMAN ALPHA-1-CHIMAERIN, A NEURON-SPECIFIC GTPASE-ACTIVATING PROTEIN FOR P21RAC
PROMOTER REGION OF THE TRANSCRIPTIONAL UNIT FOR HUMAN ALPHA-1-CHIMAERIN, A NEURON-SPECIFIC GTPASE-ACTIVATING PROTEIN FOR P21RAC
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DOI：
10.1111/j.1432-1033.1995.tb20183.x
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发表时间：
1995-02-01
期刊：
影响因子：
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通讯作者：
LIM, L
中科院分区：
文献类型：
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作者：
DONG, JM;SMITH, P;LIM, L
α 1-嵌合蛋白是p21 rac的神经元特异性GTP酶激活蛋白，p21 rac是一种参与形态学事件的蛋白质。mRNA在某些大脑区域高度表达。在培养的神经元细胞中也检测到，但在非神经元细胞中未检测到。作为理解这种调控机制的第一步，分离并表征了含有人α 1-嵌合蛋白转录单位的5 '侧翼区的基因组克隆。通过引物延伸和S1-作图分析，检测到α 1-嵌合蛋白mRNA的多个转录起始位点簇。将该簇定位于基因组DNA中的核苷酸-463至-434（相对于起始密码子中的核苷酸A）。5 '端不含TATA盒、起始基序和Sp1结合位点。在使用杂合氯霉素乙酰转移酶报告基因构建体和瞬时转染分析后，具有约110 bp 5 '侧翼序列的210 bp片段可以作为最小启动子起作用。内部缺失和点突变实验表明，位于核苷酸-519至-512的GGCCAATC序列是α 1-嵌合蛋白启动子活性所必需的。迁移率变化分析显示核因子与该区域的特异性结合，其被对应于野生型而非突变型形式的寡核苷酸竞争。数据还表明可能存在与α 1-嵌合蛋白CCAAT盒结合位点相互作用的新型CCAAT结合因子。发现位于5 '远端区域的细胞类型偏好抑制因子可能在控制α 1-嵌合蛋白mRNA的神经元特异性表达中发挥作用。这些发现的特异性启动子的α 1-chimaerin转录将有助于进一步研究其神经元特异性表达和功能。
alpha 1-chimaerin is a neuron-specific GTPase-activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5'-flanking region of the human alpha 1-chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of alpha 1-chimaerin mRNAs was detected by primer-extension and S1-mapping analyses. The cluster was mapped to nucleotides -463 to -434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5'-proximal region contained no TATA box, initiator motif and Sp1-binding site. A 210-bp fragment with approximately 110 bp 5'-flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point-mutation experiments revealed that a GGCCAATC sequence located at nucleotides -519 to -512 was essential for alpha 1-chimaerin promoter activity. Mobility-shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild-type but not mutant forms. The data also suggest the existence of possible novel CCAAT-binding factor(s) interacting with the alpha 1-chimaerin CCAAT box binding site. A cell-type-preferred suppressor located in the 5'-distal region was found which may play a role in controlling neuron-specific expression of alpha 1-chimaerin mRNA. These findings of a specific promoter for alpha 1-chimaerin transcription will facilitate further studies on its neuronal specific expression and function.