Bonovich M T, List H J, Zhang S, Danielsen M, Riegel A T
Department of Biochemistry and Molecular Biology, Georgetown University, Washington, DC 20007, USA.
Exp Cell Res. 1998 Mar 15;239(2):454-62. doi: 10.1006/excr.1997.3920.
It has previously been determined that the mouse mammary tumor virus (MMTV) promoter when integrated in the genome assumes a defined chromatin structure which is disrupted upon addition of glucocorticoids. In contrast, a transiently introduced MMTV promoter has a random nucleoprotein structure. To reveal glucocorticoid receptor (GR) domains necessary for transcriptional activation of the MMTV promoter we compared the effects of mutations of the GR on transcriptional activation of the stably integrated versus transiently introduced MMTV promoter. For this purpose we generated a GR-negative cell line which has an MMTV promoter/reporter construct integrated in the genome and studied the transcriptional activation of this construct by different GR mutants introduced into the cells. Transcriptional activation of the integrated and transiently introduced promoter was achieved by the wild-type GR or a chimeric receptor in which the MR hormone-binding domain (HBD) replaced the GR HBD. In contrast, we found that deletion of the HBD of the GR or replacement of this region with the equivalent domain of the estrogen receptor produced receptors that were unable to activate the MMTV promoter integrated in the genome although these receptors efficiently activated the transiently introduced MMTV promoter. The HBD was not the sole determinant of MMTV transcriptional activation when integrated in the genome. Chimeric receptors which harbored the MR amino terminal domain or the wild-type MR were also unable to activate the integrated MMTV promoter. Taken together, these data indicate a rigid requirement for sequences in both the GR amino and the carboxy terminal domains for transcriptional activation of a hormone response element in the defined chromatin context of the MMTV promoter.
先前已经确定，整合到基因组中的小鼠乳腺肿瘤病毒（MMTV）启动子呈现出一种特定的染色质结构，在添加糖皮质激素后这种结构会被破坏。相比之下，瞬时导入的MMTV启动子具有随机的核蛋白结构。为了揭示MMTV启动子转录激活所需的糖皮质激素受体（GR）结构域，我们比较了GR突变对稳定整合型与瞬时导入型MMTV启动子转录激活的影响。为此，我们构建了一个GR阴性细胞系，其基因组中整合有MMTV启动子/报告基因构建体，并研究了导入细胞的不同GR突变体对该构建体的转录激活作用。野生型GR或一种嵌合受体（其中MR激素结合结构域（HBD）取代了GR HBD）可实现整合型和瞬时导入型启动子的转录激活。相比之下，我们发现，GR的HBD缺失或用雌激素受体的等效结构域替换该区域后产生的受体无法激活基因组中整合的MMTV启动子，尽管这些受体能有效激活瞬时导入的MMTV启动子。当整合到基因组中时，HBD并非MMTV转录激活的唯一决定因素。含有MR氨基末端结构域或野生型MR的嵌合受体也无法激活整合的MMTV启动子。综上所述，这些数据表明，在MMTV启动子特定的染色质环境中，GR的氨基末端和羧基末端结构域中的序列对于激素反应元件的转录激活具有严格要求。
