Hunchback sequence binding protein suppresses mouse TGF-beta3 promoter in vitro.
Hunchback sequence binding protein suppresses mouse TGF-beta3 promoter in vitro.
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DOI：
10.1016/j.bbrc.2006.05.178
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发表时间：
2006-08
影响因子：
3.1
通讯作者：
K. Yamazaki;D. Crowe;C. Shuler
中科院分区：
文献类型：
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作者：
K. Yamazaki;D. Crowe;C. Shuler
转化生长因子-β3 (TGF-β3) 在体内胚胎和组织特异性基因表达模式中具有特定作用。我们克隆并测序了小鼠 TGF-β3 5'-侧翼区域，以研究该基因的转录调控。将启动子片段克隆到无启动子荧光素酶报告质粒中，以研究人皮肤黑色素瘤细胞系 A375 (A375) 的功能活性。包含-2297至-1003bp DNA序列的连续5'缺失在A375细胞中表现出高启动子活性，而在-742至104bp区域启动子活性降至最低，表明TGF-β3启动子中存在正向和负向转录调节。含有1.8kb的片段具有最高的荧光素酶活性。翻译起始位点上游这个 1.8kb 5' 侧翼区域的表征显示了假定的驼背结合位点共有序列。电泳迁移率变动分析 (EMSA) 和瞬时转染实验表明，假定的驼背结合位点具有功能并调节 TGF-β3 启动子转录活性。包括驼背序列结合蛋白 (HbSBP) 的 DNA 复合物对于抑制 A375 细胞中的启动子活性非常重要。驼背共有序列的突变导致启动子活性比野生型构建体高出 2 倍。通过 EMSA 和蛋白质印迹分析确定，其他测试的细胞系（包括 3T3 成纤维细胞和 B-16 小鼠皮肤黑色素瘤）中不存在 HbSBP。 HbSBP 可以作为 TGF-β3 基因转录调节因子发挥作用，并且可以以细胞类型特异性的方式表达。
Transforming growth factor-β3 (TGF-β3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-β3 5′-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5′-deletion encompassing DNA sequences from −2297 to −1003bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the −742 to 104bp regions, suggesting both positive and negative transcriptional regulation in the TGF-β3 promoter. The fragment containing 1.8kb had the highest luciferase activity. Characterization of this 1.8kb 5′-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-β3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-β3 gene transcriptional regulator and may be expressed in a cell type-specific manner.