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锌指蛋白HZF1基因功能和机制研究
【作者】 邓敏捷；
【导师】 张俊武；
【作者基本信息】 中国协和医科大学 ， 生化与分子生物学， 2008， 博士
【摘要】 人体内血细胞的生产是由骨髓中的造血干细胞（Hematopoietic Stem Cells,HSCs）负责的。造血干细胞相继定向到不同链系祖细胞,再进一步成为更为限制的链系前体细胞并进而继续分化发育成不同类型血细胞（红细胞、白细胞、巨核细胞和淋巴细胞等）。这些过程受到转录因子和造血生长因子的调控。这些转录因子有一部分是锌指蛋白。它们在造血发育的各个过程中扮演着重要角色,构成了造血过程中细胞分化的内在基因表达程序的重要部分。2000年,本实验室从人骨髓cDNA文库中筛选到一个新的编码锌指蛋白的cDNA序列（GenBank注册号:AF244088）,命名为HZF1。HZF1编码670个氨基酸残基,其中包含连续的15个C2H2型和2个C2R2型锌指模体。HZF1 mRNA在人各个器官中均有表达,在各种造血细胞系中也都有表达。反义RNA和RNA干扰实验结果都表明,HZF1内源表达被阻遏时,hemin诱导的K562红系分化被抑制,PMA诱导的巨核系分化明显减弱。这表明锌指蛋白HZF1在K562细胞红系分化和巨核系分化过程中具有重要作用。本论文在实验室已有的研究基础上,利用酵母双杂交系统、信号转导激酶检测和基因芯片技术对HZF1基因的功能和机制做了进一步研究。为了寻找在HZF1行使功能过程中与其相互作用的蛋白质,我们利用酵母双杂交系统,以HZF1基因的片段（H2）为诱饵蛋白,从人骨髓cDNA文库中筛选与HZF1相互作用的蛋白。从1478个克隆中初步鉴定出了288个阳性克隆,进行表型检测排除了59个假阳性;对剩下的229个阳性克隆进行分类后,进行序列测定,再利用NCBI网站上的在线软件BLAST对这些cDNA序列进行分析,排除非编码序列,排除编码框不正确的序列,对重复的序列进行归类,最后找到了30个编码正确的cDNA序列,这些序列编码的蛋白可能与HZF1发生相互作用。这些蛋白包括细胞周期相关蛋白、真核转录延伸因子、核糖体蛋白、细胞分化相关蛋白、免疫相关蛋白和肌肉分化相关蛋白等,还包括两个假想蛋白。随后,我们进行二次转化实验,排除了2个自激活的假阳性克隆。经过进一步的分析和比较,我们选择8-23、15-83和11-20这三个阳性克隆作为深入研究的对象。8-23是INCA1（inhibitor of CDK interacting with cyclin A1）基因一个剪接体的编码区的一部分,15-83是FHL1（four and a half LIM domains 1）基因编码区的一部分,11-20是ZNF7（zinc finger protein 7）基因编码区的一部分。我们利用PCR方法克隆得到了FHL1的全长cDNA序列。随后,用免疫共沉淀的方法进一步确认相互作用的存在。首先构建HZF1基因片段H2与FLAG标签的融合表达质粒以及FHL1、INCA1片段和ZNF7片段与myc标签的融合表达质粒,进而将H2-FLAG融合表达质粒分别与FHL1-myc、INCA1-myc和ZNF7-myc融合表达质粒共转染HEK293T细胞,48小时后收集细胞,用小鼠源anti-FLAG单克隆抗体进行免疫共沉淀实验,最后利用小鼠源单克隆抗体anti-FLAG和anti-myc进行Western Blot检测。Western Blot实验结果表明,HZF1可分别与FHL1和INCA1发生相互作用,但是不能和ZNF7发生相互作用。用Real-time PCR检测了INCA1在hemin诱导K562细胞向红系分化过程中的表达变化情况。用hemin诱导K562细胞向红系分化,在0、12、24和48h收集细胞,提取RNA,反转录后,进行Real-time PCR检测。结果表明,INCA1在K562红系诱导分化过程中表达水平上升,24小时达到最高,与HZF1的变化趋势一致。这表明HZF1和INCA1可能在红系分化过程中协同发挥作用。为了分析K562细胞诱导分化过程中HZF1表达阻遏前后ERK信号通路的变化,我们检测了细胞中ERK和MEK磷酸化水平的变化。用HZF1内源表达被RNAi抑制的稳定转染株K562/pAVu6HZFli和对照细胞K562/pAVu6进行实验,我们发现在未使用诱导剂诱导的情况下K562/pAVu6HZFli细胞中ERK和MEK的磷酸化水平要高于对照细胞K562/pAVu6。在诱导过程中,K562/pAVu6HZFli细胞中ERK和MEK的磷酸化水平也比对照细胞K562/pAVu6要高,但变化趋势与对照细胞基本一致。这说明HZF1可能通过调节ERK信号通路参与红系和巨核系分化的调节。为了获得HZF1功能机制的更多线索,我们利用基因芯片检测了RNA干扰抑制HZF1表达前后K562细胞的基因表达差异。使用Affymetrix的在线分析中心(TheNetAffyTM Analysis Center,www,affymetrix.com)对结果进行了分析,并对表达升高或降低的基因根据功能进行了分类,发现与信号转导、发育、分化、转录调节和细胞周期相关的基因占了很大比例。我们的实验结果确认了HZF1与INCA1（cyclinA1-CDK复合物的一个抑制因子）的相互作用。同时,我们也发现HZF1被抑制后,K562细胞分化受到抑制,ERK和MEK的磷酸化水平升高。结合利用基因芯片分析HZF1表达阻遏后基因表达变化的结果,我们推测HZF1可能通过影响ERK信号途径和细胞周期调节促进K562细胞分化。
【Abstract】 Human mature blood cells derive from Hematopoietic Stem Cells（HSCs）. Commitment of stem cells to specific hematopoietic lineages and further differentiation and development mature of the committed cells are controlled at least partially through the combinatorial action of lineage-restricted and more widely expressed transcription factors. Some transcription factors containing C2H2 zinc finger motif have been found to play an important part in differentiation and development of blood cells.In 2000, we cloned a novel zinc finger cDNA HZF1 （GenBank accession No. AF244088） by screening a human bone marrow cDNA library. The HZF1 encodes 670 amino acid residues including 15 typical C2H2 and 2 C2RH zinc finger motifs. The HZF1 mRNA was expressed extensively and highly in brain, heart, skeletal muscle and fetal liver. The HZF1 mRNA was detected in various hematopoietic cell lines. The repression of intrinsic expression of HZF1 was also revealed in K562/pAVu6HZF1i transfectant cells and the hemin-induced erythroid differentiation of K562 cells was significantly blocked and the PMA-induced megakaryocytic differentiation was obviously reduced. All these results proved that the zinc finger protein HZF1 play an important role in the erythroid and megakaryocytic differentiation of K562 cells. Based on these results, my study focused on the research of the functions of HZF1 by yeast two hybrid, monitoring the ERK signaling pathway and the analysis of gene chip.To finding the proteins interacting with HZF1, yeast two hybrid was used by screening a human bone marrow cDNA library and using the fragment of HZF1 as the bait protein. 288 candidated positive clones which was identified from 1478 clones was retested phenotypes, then 59 false positive clones was excluded. 229 candidated positive clones was sorted and sequenced, then blasted on the NCBI website. The non-coding cDNA sequence and the cDNA sequence which could not the correct protein was excluded, finally 30 cDNA sequences which ORF were correct were found. These protein was involved in eukaryotic translation elongation, differatiation, cell cycle and immune etc. Two false positive clones that could autonomously the report gene was excluded by the bait and prey plasmid cotransform experiment. Three positive clones 8-23、15-83 and 11-20 was choosed to proceed the next study. 8-23 is the part of the ORF of inhibitor of CDK interacting with cyclin A1 （INCA1）. 15-83 is the part of the ORF of four and a half LIM domains 1 （FHL1）. 11-20 is the part of the ORF of zinc finger protein 7 （ZNF7）. The full coding sequence of FHL1 was cloned from the cDNA. The interaction of HZF1 and INCA1, as well as the interaction of HZF1 and FHL1, were confirmed by CoIP and Western Blot experiments, but the interaction of HZF1 and ZNF7 wasn’t found.The expression of INCA1 during the the hemin-induced erythroid differentiation of K562 cells was detected by real-time PCR. The result showed INCA1 mRNA expression increased following erythroid differentiation of K562 cells induced by hemin and reached its crest at 24h, which was consistent with the case of HZF1.To study the change of ERK signaling pathway during the hemin-induced erythroid differentiation or the PMA-induced megakaryocytic differentiation of K562 cell line when the intrinsic expression of HZF1 was repressed, phosphorylation of ERK and MEK in K562/pAVu6HZF1i （intrinsic expression of HZF1 repressed by RNAi） and the control K562/pAVu6 were detected by Western Blot experiment. Whether the induce agents were exist or not, the phosphorylation of ERK and MEK were higher than the control cells K562/pAVu6. During the hemin- or PMA-induced differentiation of K562/pAVu6HZF1i, the change of phosphorylation of ERK and MEK as time was going were the same as the K562/pAVu6 cells. These results suggested that the repression of HZF1 probably effected the expression of some genes upstream MEK gene, resulting in the increase of phosphorylation of ERK and MEK.In order to find more clues to study the function of HZF1, the differention of gene expression between K562/pAVu6HZF1i and K562/pAVu6 was detected by using gene chip. Then, the results were analysized using the The NetAffyTM Analysis Center（www.affymetrix.com）. The genes that their expression increased or decreased were found and sortsed according to their functions. A number of genes involved signal transduction, cell cycle, differentiation, development and transcript regulation etc.INCA1, the interaction partner of HZF1, was found and confirmed in our study. INCA1（ inhibitor of CDK interacting with cyclin A1） is a inhibitor of cyclin A1-CDK complex. The results also showed when the differentiation was repressed, the phosphorylation of ERK and MEK increased. Combination with the results of gene chip analysis, these results suggested HZF1 probably promotes the differentiation of K562 cell line by effecting ERK signaling pathway and regulating cell cycle.