本文選題：XBP-1 + 染色質(zhì)伸展�。� 參考：《安徽農(nóng)業(yè)大學》2005年碩士論文

【摘要】：人 X 盒結(jié)合蛋白(XBP-1)是非折疊蛋白應(yīng)答途徑(unfolded protein response,UPR)信號途徑的重要調(diào)節(jié)因子,具有轉(zhuǎn)錄激活活性,并有兩種剪切形式:XBP-1S和 XBP-1U。XBP-1 廣泛參與細胞分化、增殖、凋亡等多種信號途徑,但對其如何發(fā)揮功能還知之甚少。 XBP-1 的轉(zhuǎn)錄調(diào)節(jié)作用提示其功能的發(fā)揮可能是通過調(diào)節(jié)染色質(zhì)結(jié)構(gòu)來實現(xiàn)的。本文中,利用大規(guī)模染色質(zhì)結(jié)構(gòu)檢測技術(shù),在哺乳動物細胞中觀察到 XBP-1S 和XBP-1U 表達的蛋白募集,復(fù)制染色體上一段含有 lac 操縱子的區(qū)域,野生型 XBP-1S和 XBP-1U 以及它們的轉(zhuǎn)錄激活結(jié)構(gòu)域都增強了染色質(zhì)伸展活性,而沒有轉(zhuǎn)錄激活結(jié)構(gòu)域的突變體則完全消除了這種染色質(zhì)伸展活性。XBP-1S 和 XBP-1U 具有染色質(zhì)伸展活性的結(jié)構(gòu)域與其轉(zhuǎn)錄激活結(jié)構(gòu)域相吻合。由于蛋白質(zhì)必須通過與其他蛋白相互作用來發(fā)揮功能,因此推斷這一區(qū)域通過與其它蛋白質(zhì)的相互作用來發(fā)揮 XBP-1 的轉(zhuǎn)錄調(diào)節(jié)功能。 實驗室已用酵母雙雜交實驗結(jié)果表明,以乳腺癌相關(guān)基因 COBRA1 為誘餌,從乳腺文庫中分離到 XBP-1 基因,初步顯示兩者之間存在相互作用關(guān)系。GST 沉降實驗表明,COBRA1 與野生型 XBP-1S 和 XBP-1U 都能結(jié)合,且與 XBP-1S 的結(jié)合結(jié)構(gòu)域定位于 COBRA1 的 135-335 個氨基酸;與 XBP-1U 的結(jié)合結(jié)構(gòu)域定位于 COBRA1的 135-335 和 285-480 氨基酸兩個結(jié)構(gòu)域。在轉(zhuǎn)染含 lac 操縱子應(yīng)答元件的 lac 熒光素酶報告基因?qū)嶒炛?COBRA1 能增強野生型 XBP-1S 和 XBP-1U 的轉(zhuǎn)錄活性,但對于XBP-1S 和 XBP-1U 的 DNA 結(jié)合結(jié)構(gòu)域缺失突變體卻沒有作用,對野生型 XBP-1 的轉(zhuǎn)錄活性的刺激倍數(shù)低于對其只包含 DNA 結(jié)合結(jié)構(gòu)域的突變體的刺激倍數(shù)。免疫共沉淀實驗結(jié)果表明,COBRA1 與 XBP-1U 的結(jié)合能力大于 COBRA1 與 XBP-1S 的結(jié)合能力。染色質(zhì)伸展實驗表明,野生型 XBP-1S 和 XBP-1U 促進染色質(zhì)伸展的區(qū)域與COBRA1 蛋白表達區(qū)域有共定位現(xiàn)象,但缺失突變體均無此現(xiàn)象。這些結(jié)果提示COBRA1 有可能參與 XBP-1 調(diào)控的信號通路。 實驗已經(jīng)證明 XBP-1 能與雌激素受體 ERα相互作用,并且能以激素不依賴的形式提高 ERα的轉(zhuǎn)錄活性,但這種作用機制尚不清楚。根據(jù)已有文獻報道,ERα能在無配體(雌激素)存在時誘導(dǎo)染色質(zhì)大規(guī)模伸展,實驗將 ERα基因與 lac 操縱子基因融合,與 XBP-1 基因共轉(zhuǎn)染特殊哺乳動物細胞,結(jié)果發(fā)現(xiàn)野生型 XBP-1 在雌激素存在的培養(yǎng)基里促進 ERα誘導(dǎo)染色質(zhì)伸展活性,但當 XBP-1 缺失了 DNA 結(jié)合結(jié)構(gòu)域時,這種促進 ERα誘導(dǎo)染色質(zhì)伸展活性的能力消失;基于 ERα可與雌激素受體應(yīng)答元件 ERE(estrogen response element)結(jié)合的原理,利用 ERE-luc 熒光素酶報告基因,檢測
[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 偽 and increase the transcriptional activity of ER 偽 in the form of hormone independent, but this mechanism is not clear. It has been reported that ER 偽 can induce the extensive expansion of chromatin in the absence of ligands (estrogen). The ER 偽 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 偽 -induced chromatin stretching activity in estrogen medium, but when XBP-1 lost the DNA binding domain, the ability to promote ER 偽 -induced chromatin stretching activity disappeared. Based on the principle that ER 偽 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 偽.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

【共引文獻】

相關(guān)期刊論文 前5條

1 劉耀華;楊光;張旭;陳曉豐;于洪偉;鄭天虎;汪立剛;趙世光;;X盒結(jié)合蛋白1調(diào)控膠質(zhì)瘤細胞氧化應(yīng)激的研究[J];中國神經(jīng)腫瘤雜志;2010年01期

2 呂曉濤;張繼新;李保玉;;B淋巴細胞分化相關(guān)的基因調(diào)控[J];臨床與實驗病理學雜志;2009年03期

3 ;Effects of transfected adenovirus-mediated transcription factor X-box binding protein 1 on hippocampal-derived neural stem cell proliferation and apoptosis under hypoxia[J];Neural Regeneration Research;2010年13期

4 范麗霞;高安慧;周宇波;李佳;;靶向IRE1/XBP1信號通路的細胞水平高通量篩選模型建立[J];中國生物工程雜志;2012年01期

5 劉雨飛,丁麗華,郝春芳,方言,趙福弟,黃翠芬,楊曉,葉棋濃;轉(zhuǎn)錄因子XBP1的融合表達、純化及多克隆抗體的制備[J];中國生物化學與分子生物學報;2004年06期

相關(guān)碩士學位論文 前3條

1 范麗霞;靶向內(nèi)質(zhì)網(wǎng)應(yīng)激的小分子調(diào)節(jié)劑的發(fā)現(xiàn)與生物活性研究[D];華東師范大學;2011年

2 李婧;pET32a-XBP1u蛋白的原核表達、純化及多克隆抗體的制備[D];重慶醫(yī)科大學;2009年

3 林建煒;轉(zhuǎn)錄因子XBP1s與細胞增殖及凋亡的實驗研究[D];重慶醫(yī)科大學;2010年

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