A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene
A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene
复制标题
DOI：
10.1523/jneurosci.22-07-02579.2002
复制
发表时间：
2002-04-01
影响因子：
5.3
通讯作者：
Kim, KS
中科院分区：
文献类型：
--
作者：
Kim, CH;Hwang, DY;Kim, KS
去甲肾上腺素转运体(Net)的表达仅限于去甲肾上腺素能(NA)神经元，它介导去甲肾上腺素重新摄取到突触前神经末梢。我们以前已经证明，9.0kb的上游序列和位于5‘非翻译区的第一个内含子对于高水平和NA细胞特异性转录是至关重要的。在这里，使用瞬时转染分析，我们发现4.0kb的5‘上游序列包含了足够的遗传信息来驱动报告基因以安娜细胞类型特异性的方式表达。有三个功能结构域似乎对人类网络(Hnet)基因转录的调控具有潜在的重要作用：上游增强子区位于-4.0~-3.1 kb之间，近端功能区位于-133~-75 bp之间，以及位于这两个结构域之间的中间沉默区。对近端启动子区域的DNase I足迹分析表明，-128到-80bp的亚区以细胞特异性的方式受到保护。我们提供了多种蛋白质因子与近端启动子结构域相互作用的证据，以关键地调节hNET基因的转录活性。在这个近端亚区的中间有一个同源结构域(HD)结合的核心基序，它以安娜特有的方式与HD因子相互作用，包括Phox2a和HoxA5。共转染分析表明，HoxA5和Phox2a可能反式激活hnet基因启动子。结合以前的研究表明，Phox2a/2b直接激活了多巴胺β-羟基酶的转录，目前的结果支持这样一个模型，即Phox2蛋白可能通过激活NA生物合成和再摄取基因来协调调节NA神经元的表型规格。
Expression of the norepinephrine transporter (NET), which mediates the reuptake of norepinephrine into presynaptic nerve terminals, is restricted to noradrenergic (NA) neurons. We have demonstrated previously that the 9.0 kb upstream sequences and the first intron residing in the 5' untranslated area are critical for high-level and NA cell-specific transcription. Here, using transient transfection assays, we show that 4.0 kb of the 5' upstream sequences contains sufficient genetic information to drive reporter gene expression in an NA cell type-specific manner. Three functional domains appear to be potentially important for the regulation of human NET (hNET) gene transcription: an upstream enhancer region at -4.0 to -3.1 kb, a proximal domain at -133 to -75 bp, and a middle silencer region between these two domains. DNase I footprinting analysis of the proximal promoter region shows that a subdomain at -128 to -80 bp is protected in a cell-specific manner. We provide evidence that multiple protein factors interact with the proximal promoter domain to critically regulate the transcriptional activity of the hNET gene. In the middle of this proximal subdomain resides a homeodomain (HD)-binding core motif, which interacts with HD factors, including Phox2a and HoxA5, in an NA-specific manner. Cotransfection analyses suggest that HoxA5 and Phox2a may transactivate the hNET gene promoter. Together with previous studies indicating direct activation of dopamine beta-hydroxylase transcription by Phox2a/2b, the present results support a model whereby Phox2 proteins may coordinately regulate the phenotypic specification of NA neurons by activating both NA biosynthetic and reuptake genes.