【作者】 张亚妮；

【导师】 陈玉林； Ray Lu；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 Zhangfei(ZF),又称为CREBZF,是目前发现的第二个能够与HSV-I相关联的宿主细胞因子HCF-1(Host Cell Factor-1)相互作用的碱性亮氨酸拉链(basic region leucine zipper ,bZIP)转录因子,Zhangfei通过与ATF4形成异二聚体参与哺乳动物的未折叠蛋白应答反应(unfolded protein response ,UPR)。进一步研究Zhangfei基因的转录、翻译调节机制及其蛋白功能对于深入了解其在UPR途径中的调节机制具有重要的理论意义。因此,本研究在构建了相关载体的基础上,应用实时定量PCR、双荧光酶检测系统、Western blotting和染色体免疫沉淀等技术对Zhangfei的转录和翻译调控机制进行了研究,获得以下结果:1.通过分析Zhangfei基因的序列结构,发现在Zhangfei基因mRNA 3′端存在213bp的选择性剪切位点,通过对该位点的选择性剪切可在C端形成携带IFFFR和不携带IFFFR的两种蛋白亚型;此外,Zhangfei基因还存在两个不同的翻译起始位点ATG,选择性翻译起始在第一个ATG开始可在N端添加82个氨基酸,而在第二个ATG开始则缺少N端的82个氨基酸;因此,Zhangfei可以产生4种蛋白亚型:lZF、lZF-IFFFR、sZF和sZF-IFFFR。此外,还以pGL3-Basic为基本载体,构建了包含不同长度Zhangfei启动子的荧光表达载体。2.用包含不同长度的Zhangfei启动子的荧光表达载体转染MDCK细胞24 h后,检测双荧光酶的活性可知,Zhangfei启动子区的-1767—-1112bp对Zhangfei基因的基本转录激活起着重要的作用,但是该区域不能应答化学药物Tunicamycin (Tm)和Thapsigargin(Tg)引起的应激;pGL3-ZF-A转染MDCK细胞24 h后更换培养液,继续培养24 h后通过检测发现,培养液中谷氨酸、亮氨酸、赖氨酸或蛋氨酸缺失可以诱导Zhangfei启动子的转录,尤其是亮氨酸缺失能够大幅度增强Zhangfei的转录活性;进一步用包含Zhangfei启动子序列缺失的表达载体转染MDCK细胞并测定其荧光酶活性,发现位于Zhangfei启动子区9bp的顺式作用元件5′-ATTCACTCA-3′对于氨基酸缺失引起的转录激活是必不可少的,序列比对表明该序列与目前已知的氨基酸应答元件(AARE)序列存在极高的同源性。3.以pcDNA3.1为基本载体,分别将编码lZF、lZF-IFFFR、sZF和sZF-IFFFR的cDNA序列克隆到pcDNA3.1中,形成能够表达Zhangfei 4种不同亚型蛋白的表达载体,Western杂交表明这4种载体都能表达相对应的亚型蛋白;通过对lZF和lZF-IFFFR的第二个ATG进行序列变异产生lZF-M83I和lZF-IFFFR-M83I,发现lZF和lZF-IFFFR能通过选择性翻译起始分别产生sZF和sZF-IFFFR;用Tm处理Hela细胞0 h、24 h和48 h后,提取总RNA、细胞总蛋白后进行实时定量PCR和Western杂交,表明内质网应激因子Tm不仅可以增强Zhangfei mRNA的表达,而且可大量诱导内源性的sZF-IFFFR蛋白表达;当在亮氨酸缺失的细胞培养液中培养Hela细胞0 h,24 h,48 h和72 h,发现72 h氨基酸缺失能够大量诱导sZF-IFFFR表达。4.通过用包含CHOP启动子的荧光表达载体和能表达4种不同Zhangfei亚型蛋白的表达载体共转染Hela细胞后,收获蛋白并进行双荧光酶活性检测,发现lZF-IFFFR和sZF-IFFFR通过CHOP启动子区的(C/EBP)-ATF元件,显著增强CHOP的转录和翻译;用shRNA技术干扰sZF-IFFFR蛋白的表达后,发现CHOP的转录活性明显降低,表明sZF-IFFFR能够与CHOP启动子区的(C/EBP)-ATF元件相结合促进CHOP的转录激活;染色体免疫沉淀结果也证实sZF-IFFFR能够在体外特异性地与CHOP启动子区的(C/EBP)-ATF元件相结合;通过测定caspase-3的活性,表明lZF-IFFFR和sZF-IFFFR可通过激活CHOP的表达而诱导细胞凋亡。总之,本研究发现了Zhangfei蛋白的4种亚型和位于Zhangfei启动子区的AARE序列,并提出lZF-IFFFR和sZF-IFFFR是在CHOP诱导的细胞凋亡途径中出现的两个新的细胞转录因子。更多还原

【Abstract】 The basic leucine zipper (bZIP) transcription factor Zhangfei (also called CREBZF or ZF) was identified through its interaction with Herpes Simplex Virus-1 (HSV-1) related cellular protein HCF-1. Researchers previously found that Zhangfei forms a heterodimer with ATF4 to play a role in the mammalian unfolded protein response. It is necessary to know about Zhangfei’s regulation mechanism under UPR by focusing on the transcription, translation and protein function of Zhangfei. Here, a series of Zhangfei plasmids were construced, the real-time PCR, dual luciferase assay,western blotting and chromatin immunoprecipitation was used to research about the transcription and translation modulation of Zhangfei were conducted, the followed is the research method and results:1. Here report the identification of 213-base alternate mRNA splicing site at the 3′end of Zhangfei. The protein translated from the spliced mRNA will add an IFFFR tail to the C-terminus of the known Zhangfei isoforms. Also found the presence of a conserved in-frame ATG upstream of the currently known translation initiation site, which would add an additional 82-amino acid to the N-terminal. Therefore, Zhangfei can produce four isoforms, lZF, lZF-IFFFR, sZF and sZF-IFFFR.2. pGL3-Basic was used as a backbone and constructs a series of luciferase expression vector including different length fragement of Zhangfei promoter, MDCK cells were used for transfection.MDCK cells were transfected with a series of different Zhangfei promoter for 24 hours and treated with Tm or Tg for 24 hours, harvested protein and conducted dual luciferase assy, the results showed that -1767—-1112bp is essential for the basic transcription of Zhangfei promoter, however, these sequence can not response the stress induced by Tm or Tg. It was well known that nutrients, particularly amino acids, were involved in the control of gene expression. Here examined the molecular mechanisms involved in the regulation of Zhangfei expression upon amino acid deprivation using a transient expression assay, the results showed that transcriptional activation of the Zhangfei gene is regulated by amino acid deprivation, especially regulated by leucine deprivation. Furthermore, a cis-positive element, 5′-ATTCACTCA-3′,is essential for this response on the Zhangfei promoter. This Zhangfei specific sequence is the first described that can regulate a basal promoter in response to amino acid deprivation and therefore can be called an amino acid response element (AARE).3. Western blotting result showed that the constructing vector of pcFLAG-lZF, pcFLAG-lZF-IFFFR, pcFLAG-sZF and pcFLAG-sZF-IFFFR can express four isoforms of Zhangfei accordingly. Mutagenesis studies proved that lZF (SMILE-L) and lZF-IFFFR isoforms can produce the short sZF (SMILE-S) and sZF-IFFFR respectively by the alternate usage of translation initiation site. Also it was found that transcription levels of CREBZF were induced by ER stress. Interestingly, the induction of sZF-IFFFR isoform was identified by tunicamycin treatment and leucine deprivation, both of which are able to induce ER stress.4. CHOP, a gene involved in cell death, was identified as a direct target gene of lZF-IFFFR and sZF-IFFFR. sZF-IFFFR appeared to be a more potent activator of CHOP than lZF-IFFFR. Overexpression of both lZF-IFFFR and sZF-IFFFR activated transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, also called amino acid response element (AARE). shRNA knockdown of overexpressed sZF-IFFFR inhibited transcriptional activity of sZF-IFFFR on the CHOP promoter. Chromatin immunoprecipitation assays showed that sZF-IFFFR physically associated with the CHOP promoter. Overexpression of both lZF-IFFFR and sZF-IFFFR induced apoptosis by measuring caspase-3 activity, likely through activation of expression of CHOP, in both ER stress- and non-ER stress-induced apoptotic conditions.Overall, we report the identification of four CREBZF isoforms. We propose that two Zhangfei isoforms, lZF-IFFFR and sZF-IFFFR, are novel transcription factors that play roles in the CHOP-mediated apoptosis signaling pathway.更多还原