Antoniv T T, De Val S, Wells D, Denton C P, Rabe C, de Crombrugghe B, Ramirez F, Bou-Gharios G
Brookdale Center in the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine-New York University, New York, New York 10029, USA.
J Biol Chem. 2001 Jun 15;276(24):21754-64. doi: 10.1074/jbc.M101397200. Epub 2001 Mar 28.
We have examined the chromatin structure around and upstream of the transcriptional start site of the human alpha2(I) collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped approximately 20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretches nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. Characterization of the human element documented functional differences with the mouse counterpart. Enhancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3' of HS3 augmented position-independent expression of the transgene. Hence, subtle differences may characterize the regulation of mammalian alpha2(I) collagen genes by evolutionarily conserved sequences.
我们已经检测了人类α2（I）型胶原蛋白（COL1A2）基因转录起始位点周围及上游的染色质结构。仅在成纤维细胞中检测到四个强DNase I超敏位点（HS2 - 5），在I型胶原蛋白阴性细胞中鉴定出一个较弱的超敏位点（HS1）。在第1内含子中发现了另一个可能与COL1A2沉默有关的超敏位点（HS（In））。HS1和HS2定位于保守的启动子序列内，且位置与小鼠基因相当。HS3、HS4和HS5同样定位于COL1A2上游约20千碱基处，与小鼠远上游增强子位置大致相同，且位于一个高度同源的基因组片段内。DNase I足迹分析确定了远上游区域（FU1 - 12）以及与小鼠序列几乎相同的片段中有12个核酸酶保护区域。发现包含HS3 - 5的区域能使原本活性极低的COL1A2启动子在转基因小鼠中实现高且组织特异性的表达。对人类元件的表征揭示了其与小鼠对应元件的功能差异。没有包含FU1 - 7和HS5的片段时，增强子活性大幅降低，而HS3下游3'端的AluI重复序列的加入增强了转基因的位置非依赖性表达。因此，细微差异可能是进化上保守序列对哺乳动物α2（I）型胶原蛋白基因调控的特征。
