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
X-box binding protein 1 (XBP-1) plays a critical role in unfolded protein responsesignaling pathway (UPR) and regulate a wide range of cellular responses, includingproliferation, differentiation, migration and apoptosis. It has two splicing variants: XBP-1Sand XBP-1U, both have transcriptional activity. However, how XBP-1 regulatesintracellular events remains largely unknown.
XBP-1 has transcriptional activity suggest that it may play its role through regulatechromatin structure. Here, we report that wide type XBP-1S and XBP-1U protein recruitand induce large-scale chromatin unfolding by targeting XBP-1 to an amplified lacoperator-containing chromosome region in mammalian cells. The transactive domain ofXBP-1S and XBP-1U also can induce large scale chromatin unfolding, but mutants ofXBP-1 lack of transactive domain can not induce chromain unfolding. This unfoldingactivity maps to the subdomains within the transactivition domain of XBP-1. So we inferthis domain interact with some other proteins to induce the transcriptional activity ofXBP-1.
Using yeast two-hybrid screen, XBP-1 was isolated from a human mammary library,with breast cancer relating gene COBRA1 as bait. This study suggests that XBP-1 mayinteract with COBRA1. GST pull-down assay showed that wide XBP-1S and XBP-1Uboth bound to COBRA1,The binding domain of COBRA1 with XBP-1S maps to its135-335 amino acids, and with XBP-1U maps to its 135-335 and 285-480 amino acids. Lacoperator-containing luciferase reporter assay was used to determine the effects ofCOBRA1 on the transcription activity of XBP-1S and XBP-1U, the result suggest thatCOBRA1 can increase the transcription activity of XBP-1S and XBP-1U, but not tomutants without DNA binding domain. Co-immunoprecipitation assay also show thatXBP-1S and XBP-1U both bound to COBRA1 in vivo. The binding of XBP-1U toCOBRA1 was stronger than that of XBP-1S to COBRA1. Large-scale chromatin unfoldingassay show that XBP-1S and XBP-1U induce chromatin unfolding map with the regionhaving COBRA1 expression, but not with mutants. These results showed that COBRA1may participate XBP-1-regulating pathway.
Our laboratory has testified that XBP-1 can interact with estrogen receptor α (ERα),and increase ERα transcription activity in ligand-independent manner, but the mechanismremains unknown. On the basis of report that ERα can induce large scale chromatin
unfolding without its ligand—estrogen. We construct lac-operator and ERα fusion plasmidand transfect it with XBP-1, and find that wide type XBP-1 can enhance ERα induce largescale chromatin unfolding activity. When XBP-1 lacks its DNA binding domain, thisactivity disappeared. Based on the principle that ERα can bind with estrogen responseelement (ERE), we detect how XBP-1 influences the transcription activity of ERα, and wefound wide type XBP-1 can enhance ERα transcriptional activity. These data show a newfunction of XBP-1 that it regulates chromatin unfolding may be the essential hypothesis toincrease the transcriptional activity of ERα, it provides a new research method to healbreast cancer.
引文
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