Leaner Virna D, Masemola Agatha, Parker M Iqbal
Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, South Africa.
IUBMB Life. 2005 Apr-May;57(4-5):363-70. doi: 10.1080/15216540500092039.
Transcriptional regulation of the human alpha 2(I) procollagen proximal promoter involves the interaction of trans-acting factors at the inverted CCAAT box (G/CBE) located at position -80 and an adjacent GGAGGCCC-box at -70. Both these elements have previously been shown to be essential for activity of the human promoter. This study investigated nucleotide differences at three sites (-74, -72 and -71) between the human and mouse promoters that were sufficient to abolish trans-acting factor binding with the mouse sequence (GGAGACGT). Two distinct DNA-protein interactions were detected on the human -107/+54 promoter fragment while a single interaction was observed at the equivalent mouse promoter. One of these factors is the CCAAT-binding factor (CBF) and it's binding was observed on both the human and mouse promoters. Although the GGAGGCCC DNA-binding element was not detected on the mouse promoter, GGAGGCC-binding proteins were present in mouse nuclear extracts as observed by their interaction with the human promoter. Functional analysis of the human and mouse -343/+54 and -107/+54 promoter regions revealed significant differences between species; the human constructs having higher activity than the mouse. The differences in promoter activity between species may in part be a result of the nucleotide differences in the GGAGGCCC-box. Mutations in this region of the human -107/+54 promoter prevented DNA-protein interaction and lowered promoter activity. These results support the hypothesis that the GGAGGCCC-box in the human alpha 2(1) procollagen promoter has a regulatory function and that there exists a species-specific difference in transcription factor binding and regulation of the gene.
人类α2（I）前胶原近端启动子的转录调控涉及反式作用因子与位于 -80 位的反向 CCAAT 盒（G/CBE）以及 -70 位相邻的 GGAGGCCC 盒之间的相互作用。先前已表明这两个元件对于人类启动子的活性至关重要。本研究调查了人类和小鼠启动子在三个位点（-74、-72 和 -71）的核苷酸差异，这些差异足以消除反式作用因子与小鼠序列（GGAGACGT）的结合。在人类 -107/+54 启动子片段上检测到两种不同的 DNA - 蛋白质相互作用，而在等效的小鼠启动子上仅观察到一种相互作用。其中一个因子是 CCAAT 结合因子（CBF），在人类和小鼠启动子上均观察到其结合。尽管在小鼠启动子上未检测到 GGAGGCCC DNA 结合元件，但通过它们与人类启动子的相互作用观察到，GGAGGCC 结合蛋白存在于小鼠核提取物中。对人类和小鼠 -343/+54 以及 -107/+54 启动子区域的功能分析揭示了物种之间的显著差异；人类构建体的活性高于小鼠。物种之间启动子活性的差异可能部分是由于 GGAGGCCC 盒中的核苷酸差异所致。人类 -107/+54 启动子该区域的突变阻止了 DNA - 蛋白质相互作用并降低了启动子活性。这些结果支持了这样的假设，即人类α2（I）前胶原启动子中的 GGAGGCCC 盒具有调节功能，并且在转录因子结合和基因调控方面存在物种特异性差异。
