【作者】 蒋磊；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 锌指是一种能与DNA结合的结构域，为锌指蛋白中的半胱氨酸和／或组氨酸与二价的锌离子结合形成的一种特定的二级结构。锌指蛋白可以通过与双链DNA、单链DNA及RNA的结合发挥转录调节及RNA剪接等功能。根据与锌离子结合的氨基酸的不同，锌指蛋白可以分为许多亚家族，其中C₂H₂（或Kruppel）型锌指蛋白是其最大的亚家族，它的锌指序列的特征为CX₂CX₃FX₅LX₂HX₃H；两个锌指之间的保守序列为TGEKP（Y／F）X（X代表在保守氨基酸之间的任意氨基酸）。而C₂H₂型锌指蛋白又可以根据其N端含有的结构域分成4大类：FAX（finger-associated boxes）型，FAR（finger-associatedrepeats）型，POZ（pox virus and zinc fingers，also known as Zin）和KRAB（Kruppel-associated box）型。含KRAB结构域的锌指蛋白也称KRAB型锌指蛋白（KRAB-containingzinc finger protein，KZNF），占人类基因组中所有锌指蛋白（799种）的约三分之一（290种），是哺乳动物中最大的转录调控因子家族，其中超过220种在胚胎发育、细胞分化、细胞转化及细胞周期的调控中发挥重要功能，其表达水平随时间和空间的不同而变化。Mipu1是本室从经历短暂缺血-再灌注的大鼠心肌中分离克隆的新基因，Genbank登录号为AY221750。其ORF的全长为1827bp，编码608个氨基酸。其编码肽链的N-端含一KRAB结构域，C-端含14个C₂H₂型锌指，故为一典型的KRAB／C₂H₂型锌指蛋白。本室近年研究发现，Mipu1基因在缺血-再灌注心肌中表达明显升高；Mipu1过表达可减轻去血清对C₂C₁₂细胞的生长抑制作用，在去血清应激状态下具有促细胞生长及修复损伤的功能；Mipu1过表达可明显抑制氧化应激（H₂O₂处理）导致的LDH释放率和细胞凋亡发生率，具有细胞保护功能。本文拟进一步阐明Mipu1的功能及结构-功能关系。为了探讨新基因Mipu1的DNA结合活性，我们首先构建了Mipu1的多个原核和真核表达质粒。利用原核表达质粒pGEX-Mipu1，诱导表达并纯化了Mipu1的融合蛋白GST-Mipu1。将GST-Mipul固定在Glutathione-Sepharose上后，从随机寡核苷酸文库中筛选到了一个Mipu1共同的DNA结合序列5’-TGTCTTATCGAA-3’。经化学合成包含这一序列的探针，末端同位素标记后进行EMSA发现，GST-Mipu1能与探针结合，并呈剂量依赖性，但纯化的GST蛋白不能与探针结合；GST-Mipu1与探针的结合信号能被非标记探针（冷探针）竞争，但不能被含突变核心序列（CTTA）的冷探针竞争。在EMSA的结合缓冲液中加入EDTA，则融合蛋白与探针的结合作用被废除。这些结果说明，5’-TGTCTTATCGAA-3’是Mipu1的特异性DNA结合位点（Mipul DNAbinding site，MDBS），其结合作用具有锌离子依赖性，其中CTTA为结合位点的核心序列。为了进一步揭示Mipul与DNA的结合作用，我们表达与纯化了不包含KRAB结构域及KRAB与锌指之间的连接序列，仅包含14个锌指的融合蛋白GST-ZF。化学合成包含Mipul的特异性结合位点的探针，并用生物素进行末端标记。Target detection asssay显示，探针能与融合蛋白GST-Mipul和GST-ZF结合，但不能与GST结合，并且上述结合作用具有锌离子依赖性。为探讨Mipul中锌指结构域的功能，我们将Mipul的14个锌指分成二大段，表达与纯化了只包含部分锌指的融合蛋白，即GST-ZFl（包含1-8个锌指）和GST-ZF2（包含9-14个锌指）。Target detection asssay显示，只有其中的GST-ZF2能与探针结合。为了进一步探讨Mipul是否具有转录因子功能，将含三个MDBS的寡核苷酸序列插入报告基因载体pGL3-promoter构建了重组报告基因载体pGL3-promoter-MDBS，并将三个MDBS核心序列突变构建了突变的重组报告基因载体pGL3-promoter-MDBS^mut。将pGL3-promoter-MDBS和pGL3-promoter-MDBS^mut分别与Mipul的真核表达质粒pcDNA3.1-Mipul共转染小鼠巨噬细胞株Raw264.7或小鼠肌原细胞株C₂C₁₂，经荧光素酶测定发现，Mipul的过表达能抑制pGL3-promoter-MDBS的荧光素酶活性，并呈剂量依赖性，但不能抑制pGL3-promoter-MDBS_mut的荧光素酶活性，表明Mipul具有转录抑制功能。将Mipul开放阅读框的全长，或KRAB结构域或锌指（ZF）结构域分别与绿色荧光蛋白（GFP）融合，构建了pEGFP-Mipul、pEGFP-KRAB及pEGFP-ZF三个真核表达质粒。将它们导入C₂C₁₂，12小时后将细胞置荧光显微镜下观察。结果显示，Mipul的全长和KRAB定位于核，而空载体和ZF在细胞中呈散在分布。本文进一步对Mipul调控的靶基因进行了初步分析。根据Mipul的DNA结合位点，经生物信息学分析发现，多个促凋亡基因启动子区含有该结合位点的核心序列。将其中Bax基因启动子的全长标上生物素后，Target detection assay显示，生物素标记的Bax的全长启动子能与GST-ZF及GST-ZF2相互作用，表明Bax是Mipul的潜在靶基因之一。综上所述，本研究得到如下结论：①Mipul是一个DNA位点特异性的核酸结合蛋白，其特异性的结合位点为5’-TGTCTTATCGAA-3’，核心序列为CTTA，C-末端的六个锌指为其与DNA结合所足够与必需；②Mipul是一个核蛋白，其核定位信号位于KRAB结构域或KRAB与锌指之间的连接序列中；③Mipul是一个转录抑制因子，可能通过抑制促凋亡基因的表达而抑制细胞凋亡，在细胞凋亡的发生过程中发挥调控作用。更多还原

【Abstract】 Zinc fingers are of importance by binding to specific DNA sequences with characteristics of forming a secondary structure in which cysteines and/or histidines coordinate zinc ions. Zinc finger proteins function by binding to double-stranded DNA （dsDNA）, single-stranded DNA or RNA in transcriptional control and splicing of RNA. According to the amino acid residues that coordinate zinc ion, zinc finger proteins are classified into a lot of sub-families, among which the subfamily of C2H2 （Kruippel） zinc fmger proteins is the largest, with zinc finger CX2CX3FX5LX2HX3H and conserved linker sequence TGEKP（Y/F）Ⅹ（whereⅩdenotes any amino acid）. C2H2 zinc finger proteins can be further divided into 4 groups by their domains at the N-terminus, including FAⅩ（finger-associated boxes）, FAR （finger-associated repeats）, POZ （pox virus and zinc fingers, also known as Zin） and KRAB （Kruppel-associated box）.Zinc finger proteins containing KRAB domain （also known as KRAB-containing zinc finger proteins） account for about one-thirds （209） of total zinc finger proteins （799） arranged in human genome, forming the largest family of transcriptional factors. 220 of KRAB-containing zinc finger proteins express differentially in different tissures and developmental stages, functioning importantly in the control of embryo development, cellular differentiation, cellular transformation and cell cycle.Mipul is a new gene cloned in our lab from transiently ischemic and reperfused rat heart （Genbank accession number AY221750） that was up-regulated after transient myocardial ischemia/reperfusion. It has an open reading frame of 1827 bp, encoding a 608 amino acid polypeptide with an N-terminal KRAB domain and 14 C-terminal C2H2 ZinC fingers. Preliminary study has shown that over-expression of Mipul in C2C12 myogenic cells were more resistant to oxidative injury when compared with the control, as indicated by lower LDH release and apoptosis occurrence in Mipul-overexpressed cells than in the control cells, and that overexpression of Mipul could alleviate cellular growth arrest in the stress induced by a seral withdrawal. The present study is to clarify the function and structure-function relationship of Mipul.In order to investigate the DNA binding activity of Mipul, we first constructed more than ten Mipul bacterial and eukaryotic expression plasmids. Using the bacterial expression plasmid pGEX-Mipul we prepared Mipul fusion protein GST-Mipul. GST-Mipul was immobilized on glutathione-bound Sepharose beads to capture specific oligonucleotide sequences from a random oligonucleotide library, and a consensus sequence （5’-TGTCTTATCGAA-3’） was obtained.To determine if the consensus was specific Mipul DNA binding site, a probe containing the consensus sequence was synthesized and ³²p end- labelled. EMSA showed that GST-Mipul can bind to the probe in a protein concentration dependent and zinc dependent manner while GST can not. Mutation analysis showed that nonlabelled wild type （wt） probe was good competitor, but the nonlabelled mutant probe with putative core base substitution at the consensus sequence could not compete with the wt labeled probe. These results suggest that the consensus sequence 5’-TGTCTTATCGAA-3’ is specific Mipul DNA binding site （MDBS）, in which CTTA is the core sequence.To further study the Mipul-DNA interaction, a truncated Mipul fusion protein GST-ZF containing 14 zinc fingers （ZF） but without the KRAB domain and the linker was expressed and purified. A probe containing MDBS was also synthesized and biotin-labelled. Target detection assay showed that GST-Mipul and GST-ZF can interact with the probe in a zinc dependent manner while GST protein can not, which is consistent with the results above in EMSA, suggesting that the consensus sequence is Mipul DNA binding site, and the zinc finger region of Mipul is necessary and sufficient for its DNA binding activity.To further study the structure-function relationship of Mipul, two more truncated Mipul fusion proteins were prepared: GST-ZF1 which consisted of zinc fingers 1-8, and GST-ZF2 which was composed of 9-14 zinc fingers. Target detection assay also showed that GST-ZF2 can bind to above biotin-labelled probe while GST-ZF1 can not, suggesting that the last 6 zinc fingers of Mipul is required and sufficient for its DNA binding.To clarify the potential function of Mipul as a transcription factor, an oligonucleotide fragment containing 3 MDBSs was chemically synthesized and inserted into pGL3-promoter vector to produce pGL3-promoter-MDBS, and the mutant reporter plasmid pGL3-promoter-MDBS^mut was also constructed with mutant core sequence （TCTTA→AGCGC）. Co-transfection of Raw 264.7 cells with pGL3-promoter-MDBS and pcDNA3.1-Mipul showed that Mipul overexpression reduced the reporter activity of pGL3-promoter-MDBS reproducibly, but Co-transfection of Raw 264.7 cells with pGL3-promoter-MDBS^mut and pcDNA3.1-Mipul showed that Mipul overexpression failed to reduce the reporter activity of pGL3-promoter-MDBS^mut, suggesting that Mipul suppressed promoters containing MDBS.Full-length ORF （Mipul） or the KRAB region plus the linker sequence （KRAB） or 14 zinc fingers （ZF） of Mipulwas fused with EGFP, respectively. C₂C₁₂ cells were transfected with the pEGFP recombinant plasmids overnight, and observed under a fluorescence microscopy. Although the cells transfected with pEGFP empty vector or pEGFP-ZF had a generalized fluorescence distribution in the cytoplasm and nucleus, the cells transfected with either pEGFP-Mipul or pEGFP-KRAB showed distinct nuclear fluorescence localization. Thus, the ZF domain was not required for nuclear localization, whereas the presence of the KRAB region and/or the remaining 57 amino acids of the linker region appeared to be sufficient for directing nuclear localization of Mipul.We further analyzed the possible target genes of Mipul and found that many apoptosis-related genes contain MDBS or its core sequence at their promoters by using bioinformatic techniques. It was found that Bax, an important proapoptotic gene, contains several MDBSs, and biotin-labelled Bax promoter could bind to GST-ZF and GST-ZF2.It was concluded that:①Mipul is a DNA binding protein that binds to the specific sequence 5’-TGTCTTATCGAA-3’, with CTTA as the core sequence;②Mipul is a nuclear protein that is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region; ③Mipul is a transcriptional repressor that may repress apoptosis by repressing the expression of proapoptotic genes which may up-regulate in myocardial ischemia-reperfusion injury.更多还原