Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence
Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence
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DOI：
10.1093/nar/30.4.1038
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发表时间：
2002-02-15
影响因子：
14.9
通讯作者：
Lan, MS
中科院分区：
文献类型：
--
作者：
Breslin, MB;Zhu, M;Lan, MS
从人胰岛素瘤消减文库中分离到一个新的cDNA，胰岛素瘤相关抗原-1（IA-1），它含有5个锌指DNA结合基序。IA-1表达仅限于胎儿胰腺和脑组织以及神经内分泌来源的肿瘤，而不限于成人胰腺和脑组织。使用各种GAL 4 DNA结合结构域（DBD）/1A-1融合蛋白构建体，我们证明IA-1具有转录抑制因子的功能，并且氨基酸168和263之间的区域包含大部分抑制因子活性。使用选择的和扩增的随机寡核苷酸结合测定和细菌表达的GST-IA-1DBD融合蛋白（257-510 a.a.），我们鉴定了共有IA-1结合序列T-G/（C）（T）/（C）（T）/（T）（T）/（A）GGGG（G）/C-T（G）/（A）。进一步的实验表明，IA-1的锌指2和3足以证明使用含有报告构建体的IA-1共有位点的转录活性。用IA-1结合序列的数据库搜索揭示了与其受限表达模式一致的许多胰腺和脑特异性基因中的靶位点。最显著的匹配是IA-1和NeuroD/β 2基因的5 '侧翼区。用全长IA-1或hEgr-1AD/IA-1DBD构建体和IA-1或NeuroD/β 2启动子/CAT构建体共转染细胞调节CAT活性。这些发现表明IA-1蛋白可能是自动调节的，并在胰腺和神经元发育中发挥作用，特别是在NeuroD/beta2基因的调节中。
A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/1A-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the majority of the repressor activity. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST-IA-1DBD fusion protein (257-510 a.a.), we identified the consensus IA-1 binding sequence, T-G/(C)(T)/(C)(T)/(T)(T)/(A)GGGG(G)/C-T(G)/(A). Further experiments showed that zinc-fingers 2 and 3 of IA-1 are sufficient to demonstrate transcriptional activity using an IA-1 consensus site containing a reporter construct. A database search with the consensus IA-1 binding sequence revealed target sites in a number of pancreas- and brain-specific genes consistent with its restricted expression pattern. The most significant matches were for the 5'-flanking regions of IA-1 and NeuroD/beta2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/beta2 promoter/CAT construct modulated CAT activity. These findings suggest that the IA-1 protein may be auto-regulated and play a role in pancreas and neuronal development, specifically in the regulation of the NeuroD/beta2 gene.