XBP-1通过调控染色质大规模伸展增强ERα转录活性

发布时间：2018-05-07 14:06

本文选题：XBP-1 + 染色质伸展　；参考：《安徽农业大学》2005年硕士论文

【摘要】：人 X 盒结合蛋白(XBP-1)是非折叠蛋白应答途径(unfolded protein response,UPR)信号途径的重要调节因子,具有转录激活活性,并有两种剪切形式:XBP-1S和 XBP-1U。XBP-1 广泛参与细胞分化、增殖、凋亡等多种信号途径,但对其如何发挥功能还知之甚少。 XBP-1 的转录调节作用提示其功能的发挥可能是通过调节染色质结构来实现的。本文中,利用大规模染色质结构检测技术,在哺乳动物细胞中观察到 XBP-1S 和XBP-1U 表达的蛋白募集,复制染色体上一段含有 lac 操纵子的区域,野生型 XBP-1S和 XBP-1U 以及它们的转录激活结构域都增强了染色质伸展活性,而没有转录激活结构域的突变体则完全消除了这种染色质伸展活性。XBP-1S 和 XBP-1U 具有染色质伸展活性的结构域与其转录激活结构域相吻合。由于蛋白质必须通过与其他蛋白相互作用来发挥功能,因此推断这一区域通过与其它蛋白质的相互作用来发挥 XBP-1 的转录调节功能。实验室已用酵母双杂交实验结果表明,以乳腺癌相关基因 COBRA1 为诱饵,从乳腺文库中分离到 XBP-1 基因,初步显示两者之间存在相互作用关系。GST 沉降实验表明,COBRA1 与野生型 XBP-1S 和 XBP-1U 都能结合,且与 XBP-1S 的结合结构域定位于 COBRA1 的 135-335 个氨基酸;与 XBP-1U 的结合结构域定位于 COBRA1的 135-335 和 285-480 氨基酸两个结构域。在转染含 lac 操纵子应答元件的 lac 荧光素酶报告基因实验中,COBRA1 能增强野生型 XBP-1S 和 XBP-1U 的转录活性,但对于XBP-1S 和 XBP-1U 的 DNA 结合结构域缺失突变体却没有作用,对野生型 XBP-1 的转录活性的刺激倍数低于对其只包含 DNA 结合结构域的突变体的刺激倍数。免疫共沉淀实验结果表明,COBRA1 与 XBP-1U 的结合能力大于 COBRA1 与 XBP-1S 的结合能力。染色质伸展实验表明,野生型 XBP-1S 和 XBP-1U 促进染色质伸展的区域与COBRA1 蛋白表达区域有共定位现象,但缺失突变体均无此现象。这些结果提示COBRA1 有可能参与 XBP-1 调控的信号通路。实验已经证明 XBP-1 能与雌激素受体 ERα相互作用,并且能以激素不依赖的形式提高 ERα的转录活性,但这种作用机制尚不清楚。根据已有文献报道,ERα能在无配体(雌激素)存在时诱导染色质大规模伸展,实验将 ERα基因与 lac 操纵子基因融合,与 XBP-1 基因共转染特殊哺乳动物细胞,结果发现野生型 XBP-1 在雌激素存在的培养基里促进 ERα诱导染色质伸展活性,但当 XBP-1 缺失了 DNA 结合结构域时,这种促进 ERα诱导染色质伸展活性的能力消失;基于 ERα可与雌激素受体应答元件 ERE(estrogen response element)结合的原理,利用 ERE-luc 荧光素酶报告基因,检测
[Abstract]:Human X-cassette binding protein (XBP-1) is an important regulator of unfolded protein response (UPR) signaling pathway. It has transcriptional activation activity and two shearing forms: XBP-1S and XBP-1U.XBP-1 are involved in many signal pathways, such as cell differentiation, proliferation, apoptosis and so on. But little is known about how to function. The transcriptional regulation of XBP-1 suggests that its function may be mediated by regulation of chromatin structure. In this paper, using the technique of large scale chromatin structure detection, we observed the protein recruitment of XBP-1S and XBP-1U expression in mammalian cells, and duplicated a region of chromosome containing lac operon. Wild type XBP-1S and XBP-1U, as well as their transcriptional activation domains, increased chromatin stretching activity. However, the mutant with no transcriptional activation domain completely eliminated the chromatin extension activity. XBP-1S and XBP-1U showed chromatin extension activity, which coincided with its transcriptional activation domain. Since proteins must interact with other proteins to function, it is inferred that this region plays a role in the transcriptional regulation of XBP-1 by interacting with other proteins. The results of yeast two-hybrid experiments have shown that XBP-1 gene was isolated from mammary gland library using breast cancer related gene COBRA1 as bait. The results showed that COBRA1 could bind to wild-type XBP-1S and XBP-1U, and its binding domain with XBP-1S was 135-335 amino acids of COBRA1. The binding domain of XBP-1U was located in the 135-335 and 285-480 amino acid domains of COBRA1. In the experiment of lac luciferase reporter gene transfection with lac operon response element, CoBRA1 could enhance the transcription activity of wild type XBP-1S and XBP-1U, but had no effect on DNA binding domain deletion mutants of XBP-1S and XBP-1U. The stimuli to the transcriptional activity of wild-type XBP-1 were lower than those to mutants containing only DNA binding domain. The results of immunoprecipitation showed that the binding ability of COBRA1 to XBP-1U was higher than that of COBRA1 and XBP-1S. Chromatin stretching experiments showed that the regions of wild type XBP-1S and XBP-1U promoting chromatin extension were colocated with COBRA1 protein expression regions, but no such phenomenon was found in deletion mutants. These results suggest that COBRA1 may be involved in the signaling pathway regulated by XBP-1. It has been proved that XBP-1 can interact with estrogen receptor ER 伪 and increase the transcriptional activity of ER 伪 in the form of hormone independent, but this mechanism is not clear. It has been reported that ER 伪 can induce the extensive expansion of chromatin in the absence of ligands (estrogen). The ER 伪 gene was fused with lac operon gene and co-transfected with XBP-1 gene into special mammalian cells. The results showed that wild-type XBP-1 promoted ER 伪 -induced chromatin stretching activity in estrogen medium, but when XBP-1 lost the DNA binding domain, the ability to promote ER 伪 -induced chromatin stretching activity disappeared. Based on the principle that ER 伪 can be combined with estrogen receptor response element (ERE(estrogen response element), ERE-luc luciferase reporter gene was used to detect the expression of ER 伪.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【共引文献】