A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice
A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice
复制标题
DOI：
10.1083/jcb.134.5.1333
复制
发表时间：
1996-09-01
影响因子：
7.8
通讯作者：
deCrombrugghe, B
中科院分区：
文献类型：
--
作者：
BouGharios, G;Garrett, LA;deCrombrugghe, B
我们已经确定了三个DNase I-超敏感位点之间的15和17 kb的小鼠前α 2（I）胶原基因上游的染色质。这些位点在产生I型胶原的细胞中检测到，但在不表达这些基因的细胞中没有检测到。与先前在携带较短启动子片段的动物中观察到的水平相比，含有小鼠前α 2（I）胶原基因的-17 kb至+54 bp序列的构建体（克隆在大肠杆菌β-半乳糖苷酶或萤火虫荧光素酶报告基因的上游）在转基因小鼠中显示出较强的增强子活性。报告基因在真皮、筋膜和许多内脏器官的纤维层中的表达水平特别高。在一些成骨细胞中也可以检测到高水平的表达，当将5'侧翼序列的各种片段克隆到与lacZ基因连接的350-bp近端pro α 2（I）胶原启动子的上游时，负责增强的顺式作用元件定位在-13.5和-19.5kb之间的区域，包含三个DNA酶I超敏感位点的同一区域。此外，从-13.5到-19.5kb的DNA片段也能够驱动220-bp小鼠pro α 1（I）胶原启动子的细胞特异性表达，该启动子在转基因小鼠中是沉默的。因此，我们的数据表明，远上游增强子元件在调节小鼠pro α 2（I）胶原基因的高水平表达中起作用。
We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2(I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2(I) collagen gene, cloned upstream of either the Escherichia coli beta-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells, When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice, Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.