Oshima R G, Trevor K, Shevinsky L H, Ryder O A, Ceceña G
La Jolla Cancer Research Foundation, California 92037.
Genes Dev. 1988 May;2(5):505-16. doi: 10.1101/gad.2.5.505.
The Endo B type-I keratin intermediate filament protein is first expressed at the 4- to 8-cell stage of mouse development. In the adult, its expression is restricted to a variety of simple epithelial cell types. To investigate the mechanisms responsible for the restricted expression of Endo B, the gene coding for Endo B has been identified from among the five different Endo B genes found in the mouse genome by Southern hybridization analysis and cloning all or part of four of the genes. Nuclear run-on experiments demonstrate that Endo B expression is regulated at the level of transcription. The 5' end of the active gene, designated Endo beta 1, was found to be highly methylated and in a relatively nuclease-resistant chromatin conformation in fibroblasts and myoblasts that do not express Endo B, but undermethylated and relatively sensitive to nuclease digestion in endodermal cells or F9 embryonal carcinoma cells. The inactive state of the Endo B beta 1 gene in fibroblast appears to be very stable, because somatic cell hybrids formed by the fusion of HeLa cells, which express the homologous human protein, keratin 18, and mouse fibroblasts, continue to express keratin 18 but do not activate Endo B expression. Similarly, the fusion of mouse endodermal cells and fibroblasts results in hybrids that do not extinguish Endo B expression. These results suggest that Endo B transcription is limited by two different mechanisms. In somatic cells such as fibroblasts or myoblasts, expression may be restricted by methylation and a stable, nonpermissive transcriptional state. However, in embryonal carcinoma cells, the Endo B beta 1 gene is undermethylated and in a relatively nuclease-sensitive conformation, but it is restricted by an additional, negative regulatory mechanism.
内源性B型I角蛋白中间丝蛋白在小鼠发育的4至8细胞阶段首次表达。在成体中，其表达局限于多种简单上皮细胞类型。为了研究内源性B表达受限的机制，通过Southern杂交分析并克隆小鼠基因组中发现的五个不同内源性B基因中的四个基因的全部或部分，已鉴定出编码内源性B的基因。核转录实验表明内源性B的表达在转录水平受到调控。在不表达内源性B的成纤维细胞和平滑肌细胞中，活性基因（命名为内源性β1）的5'端高度甲基化，处于相对抗核酸酶的染色质构象，但在内胚层细胞或F9胚胎癌细胞中甲基化程度较低，对核酸酶消化相对敏感。成纤维细胞中内源性Bβ1基因的非活性状态似乎非常稳定，因为由表达同源人类蛋白角蛋白18的HeLa细胞与小鼠成纤维细胞融合形成的体细胞杂种继续表达角蛋白18，但不激活内源性B的表达。同样，小鼠内胚层细胞与成纤维细胞的融合产生的杂种不会熄灭内源性B的表达。这些结果表明内源性B的转录受两种不同机制的限制。在成纤维细胞或平滑肌细胞等体细胞中，表达可能受甲基化和稳定的、非允许性转录状态的限制。然而，在胚胎癌细胞中，内源性Bβ1基因甲基化程度较低，处于相对核酸酶敏感的构象，但它受另一种负调控机制的限制。
