Liu J J, Wilson S E
Department of Ophthalmology, University of Washington School of Medicine, Seattle 98195-6485, USA.
Invest Ophthalmol Vis Sci. 2001 Nov;42(12):2776-83.
Angiogenesis refers to the latter stage of vascular development. It has been reported that angiopoietin-like factor cornea-derived transcript 6 (CDT6) encodes a protein homologous to angiopoietins that could play a critical role in blocking a receptor of angiopoietin (Tie2) and therefore contribute to the avascularity and transparency of the cornea in the developing embryo and the adult. This study was focused on isolation and characterization of the CDT6 promoter.
Rapid amplification of cDNA ends (5'-RACE) was used to isolate the CDT6 promoter from an adaptor-ligated genomic DNA fragment library and to identify the transcription initiation site of the CDT6 gene. The RNase protection assay was performed to confirm the initiation site. The sequence similarity, binding sites for putative transcription factors, and transcriptional activity of human and mouse CDT6 promoters were compared. Corneal and noncorneal cells from humans and other animals were transiently transfected with CDT6 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs to analyze the transcriptional activity of the promoter.
A 2956-bp human CDT6 promoter fragment and a 3142-bp mouse CDT6 promoter fragment were isolated. The major transcription initiation sites of the human and mouse CDT6 genes were located at 224 and 168 bp, respectively, upstream of the translation initiation site. Human and mouse CDT6 promoter sequences were very similar. Both promoters were minus TATA and CAAT boxes close to the transcription initiation site. Transfection into human corneal and noncorneal cells and into nonhuman cells revealed that the human CDT6 promoter probably contains positive and negative cis-regulatory elements that modulate cell, tissue, and species specificity. The human CDT6 promoter contains four interferon (IFN)-stimulated response elements (ISREs). No ISREs could be identified in the mouse promoter. IFN-alpha stimulated transcriptional activity of the human promoter.
The human and mouse CDT6 promoters have similar sequences and share many cis-regulatory elements. IFN-alpha appears to have an important role in regulating transcription of the human, but not the mouse, CDT6 promoter.
血管生成是指血管发育的后期阶段。据报道，角膜衍生转录本6血管生成素样因子（CDT6）编码一种与血管生成素同源的蛋白质，该蛋白质可能在阻断血管生成素受体（Tie2）中起关键作用，从而有助于发育中的胚胎和成年个体角膜的无血管性和透明度。本研究聚焦于CDT6启动子的分离与特性分析。
采用cDNA末端快速扩增（5'-RACE）从连接接头的基因组DNA片段文库中分离CDT6启动子，并鉴定CDT6基因的转录起始位点。进行核糖核酸酶保护试验以确认起始位点。比较了人和小鼠CDT6启动子的序列相似性、假定转录因子的结合位点及转录活性。将人和其他动物的角膜及非角膜细胞用CDT6启动子-氯霉素乙酰转移酶（CAT）报告基因构建体进行瞬时转染，以分析启动子的转录活性。
分离出一个2,956 bp的人CDT6启动子片段和一个3,142 bp的小鼠CDT6启动子片段。人和小鼠CDT6基因的主要转录起始位点分别位于翻译起始位点上游224和168 bp处。人和小鼠CDT6启动子序列非常相似。两个启动子在靠近转录起始位点处均无TATA盒和CAAT盒。转染人角膜和非角膜细胞以及非人类细胞表明，人CDT6启动子可能含有调节细胞、组织和物种特异性的正负顺式调节元件。人CDT6启动子含有四个干扰素（IFN）刺激反应元件（ISRE）。在小鼠启动子中未鉴定到ISRE。IFN-α刺激人启动子的转录活性。
人和小鼠CDT6启动子具有相似的序列并共享许多顺式调节元件。IFN-α似乎在调节人而非小鼠CDT6启动子的转录中起重要作用。
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