Oshima R G, Abrams L, Kulesh D
Cancer Research Center, La Jolla Cancer Research Foundation, California 92037.
Genes Dev. 1990 May;4(5):835-48. doi: 10.1101/gad.4.5.835.
The mouse forms of human keratins 18 and 8 (K18 and K8) are the first members of the large intermediate filament gene family to be expressed during embryogenesis. To identify potential regulatory elements of the human K18 gene, various recombinant constructions were expressed in cultured cells. An enhancer element was found in the first intron that functions on both the K18 and thymidine kinase promoters in differentiated cells. In F9 embryonal carcinoma cells, the level of expression was low in the presence or absence of the first intron. Cotransfection of F9 cells with K18 constructs that include the first intron and increasing amounts of an expression vector of c-jun results in a modest increase in the reporter gene expression. Cotransfection of the same construct with increasing amount of the mouse c-fos gene results in activation of the reporter gene by as much as 15-fold, with a near linear response to the amount of c-fos gene added. Site-specific mutagenesis of a putative AP-1 site within the intron abolishes trans-activation by c-fos in F9 cells. Furthermore, induction of c-fos in a derivative of F9 cells results in increased expression of the endogenous mouse form of K18. Cotransfection with c-jun or c-fos expression vectors had little effect on the expression of the K18 reporter construct in a parietal endodermal cell line already expressing the endogenous mouse gene. These results identify an enhancer within the first intron of K18 that may interact directly with c-jun and c-fos via a conserved AP-1-binding site. K18 expression in undifferentiated F9 cells may be limited by the low levels of c-jun and c-fos.
人角蛋白18和8（K18和K8）的小鼠形式是在胚胎发生过程中表达的大型中间丝基因家族的首批成员。为了鉴定人K18基因的潜在调控元件，在培养细胞中表达了各种重组构建体。在第一个内含子中发现了一个增强子元件，它在分化细胞中对K18和胸苷激酶启动子均起作用。在F9胚胎癌细胞中，无论有无第一个内含子，表达水平都很低。用包含第一个内含子的K18构建体与逐渐增加量的c-jun表达载体共转染F9细胞，导致报告基因表达适度增加。用逐渐增加量的小鼠c-fos基因与相同构建体共转染，导致报告基因激活高达15倍，对添加的c-fos基因量呈近似线性反应。内含子内推定的AP-1位点的位点特异性诱变消除了F9细胞中c-fos的反式激活。此外，在F9细胞衍生物中诱导c-fos导致内源性小鼠形式的K18表达增加。在已经表达内源性小鼠基因的壁内胚层细胞系中，与c-jun或c-fos表达载体共转染对K18报告构建体的表达几乎没有影响。这些结果鉴定了K18第一个内含子内的一个增强子，它可能通过一个保守的AP-1结合位点直接与c-jun和c-fos相互作用。未分化的F9细胞中K18的表达可能受c-jun和c-fos低水平的限制。
