【作者】 杜梅君；

【导师】 梁植权； 刘德培； 吴雪松；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 基因的成簇分布是真核基因在细胞核内分布的一种普遍形式。许多成簇分布的基因以转录后共调控的方式进行调控。基因簇是真核基因成簇分布的方式之一。基因簇的表达通常受共同的调控元件调控。如位点控制区(LCR)、激素应答元件(HRE)等。真核基因的表达是一个高度复杂的过程,是遗传调控和表观遗传调控综合作用的结果。真核基因的表达调控可以分为三个层次:DNA水平、染色质水平和核水平。DNA水平主要通过DNA与蛋白质以及蛋白质与蛋白质相互作用实现。DNA主要指结构基因以及调节基因转录的顺式作用元件,蛋白质主要指RNA聚合酶以及转录调节蛋白(反式作用因子)。其中远距离调控元件是重要的顺式作用元件,在高等真核生物中广泛存在,对于生物体的分化和发育具有重要的作用。β-珠蛋白基因簇是研究远距离调控元件对于基因簇调控作用机制的良好模型,小鼠β-珠蛋白基因簇包括四个结构基因(Ey,βH1,β-major,和β-minor),和上游的由一系列核酸酶Ⅰ高敏位点组成的远距离调控元件LCR。LCR对β-珠蛋白基因簇调控的作用机制长久以来一直是研究珠蛋白基因表达调控的热点。最近,新出现的两项技术RNA-TRAP(Tagging and Recovery of AssociatedProteins)和3C(Chromosome Conformation Capture,3C)对β-珠蛋白基因簇染色质空间构象的研究结果第一次直接证实了β-珠蛋白基因簇远距离增强元件LCR的成环(looping)作用机制。在此基础上Grosveld等提出了活性染色质中心(Active chromatin hub-ACH)的模型。LCR通过成环机制调控下游基因表达的作用方式被证实以后,成环过程的作用机制立即引起了研究者的极大兴趣。研究集中在顺式作用元件和反式作用因子在成环过程中的作用。GATA-1和EKLF是其中两个重要的参与珠蛋白基因激活的因子。EKLF(erythroidKruppel-like factor)结合在CACCC保守序列上,主要调控成年期珠蛋白基因的表达(Marini,Asunis et al.2004)。GATA-1广泛分布在整个珠蛋白基因簇LCR区的高敏位点和下游基因的启动子上,在红系分化和珠蛋白基因的表达调控中起重要作用(Im,Grass et al.2005)。在转录因子EKLF敲除的小鼠中,发现ACH结构的形成依赖于转录因子EKLF的结合。同时,在GATA-1缺失的细胞系GIE中对LCR与珠蛋白结构基因空间距离关系的研究也证实了GATA-1参与成环过程。NF-E2是另一个在红系分化和珠蛋白基因表达调控中起作用的关键因子。NF-E2由大亚基p45和小亚基p18组成,大亚基p45含有反式激活结构域,小亚基p18含有DNA结合结构域。在红系分化过程中,小亚基p18与大亚基p45结合形成NF-E2异源二聚体激活珠蛋白基因的表达。鼠NF-E2异源二聚体的结合位点主要在HS2的AP1/NF-E2序列上。在红系分化过程中,除了LCR区的高敏位点,在β-珠蛋白基因簇的β-maj,β-min的启动子区也有NF-E2的结合。同时,β-maj珠蛋白基因启动子区组蛋白的修饰及RNA聚合酶Ⅱ(RNA PolⅡ)从LCR向β-maj珠蛋白基因启动子的转移也与NF-E2在该区域的结合有密切关系。因此,阐明NF-E2在珠蛋白基因表达调控中的作用机理及其在Looping和ACH形成中的作用是一个亟待研究的重要课题。本研究的目的在于探讨NF-E2在珠蛋白基因表达调控中的作用机制。首先利用逆转录病毒介导的RNA干扰技术在鼠红白血病细胞MEL的亚型DS19细胞系中沉默NF-E2p18小亚基,建立一个NF-E2二聚体功能下降的细胞系。然后运用该细胞克降,研究NF-E2在成环过程中的作用,探讨该因子与RNA PolⅡ在珠蛋白基因簇上结合的关系。研究发现:当NF-E2p18亚基被沉默后,在β-珠蛋白基因簇的LCR高敏位点HS2,HS3及β-maj的启动子区NF-E2 p18,p45两个亚基的结合均明显下降,表明NF-E2异源二聚体的DNA结合活力下降,该下降直接引起β-珠蛋白基因表达的明显下调。此外,CHIP分析发现:RNA聚合酶Ⅱ在HS2,HS3及β-maj启动子区的结合下降,这表明RNA PolⅡ在β-珠蛋白基因簇上的结合是NF E2依赖的。运用3C技术,以HS2为引领引物,与含有Ey,βh1,β-maj,β-min基因片段的引物相匹配,对NF-E2在成环过程中的作用机制进行了探讨。结果表明:NF-E2p18的沉默引起β-maj,β-min珠蛋白基因表达下降的同时,表现为HS2与β-maj,β-min之间交联频率的下降,即:空间接近性下降。而HS2与不表达的基因Ey,βH1之间的交联频率无明显变化。Tolhuis等人认为:珠蛋白基因簇在不同的发育阶段,由不同的基因以成环的方式进入到ACH中开始表达,成年期主要为β-maj和β-min珠蛋白基因参与到ACH中,3C结果证实这两个位点与高敏位点HS2间的交联频率提高,而干扰NF-E2p18亚基后,这两个位点与HS2之间的交联频率下降,可见NF-E2可调节LCR中高敏位点与各个基因之间的接近性,上述结果提示:NF-E2是参与成环过程的转录因子之一。综合NF-E2在细胞核内不同区域的分布对B珠蛋白基因表达激活的影响,NF-E2在β珠蛋白基因簇LCR及结构基因启动子区结合状况及机制的研究,以及我们关于NF-E2参与成环过程的实验结果,我们提出:NF-E2参与珠蛋白基因表达调控的可能机理为:(1).在细胞分化以前,NF-E2p18小亚基与NF-E2p45大亚基分别位于不同的细胞核区域,在一定信号的刺激下,NF-E2 p18小亚基从异染色质区转移到常染色质区,与NF-E2p45大亚基结合,形成有激活功能的NF-E2异源二聚体。(2).NF-E2异源二聚体通过NF-E2/AP1位点结合在LCR区的高敏位点HS2上,同时通过蛋白质与蛋白质相互作用结合到LCR区的其它高敏位点及下游结构基因的启动子上。然后,NF-E2和其它蛋白质因子一起招募RNAPolⅡ,染色质重塑复合物及其它转录起始复合物,分别在LCR与下游基因的启动子上形成不同的蛋白质复合物。(3).转录因子及其它蛋白质复合物的结合引起β珠蛋白基因簇局部区域染色质结构的变化,从而导致成环结构的形成。在成环结构形成的过程中,RNA PolⅡ从LCR被转移到表达基因的启动子区,激活珠蛋白基因的表达。更多还原

【Abstract】 Eukaryotic gene expression is a complex process that relies on the collective action of genetic and epigenetic regulations.It can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels,i.e.the sequence level,the chromatin level and the nuclear level.At the sequence level,gene expresson is controlled by the interplay between DNA and DNA or DNAs and proteins.DNA mainly refers to the structural genes and cis-regulatory elements.Protens include RNA polymeraseⅡ,trans-acting factors and other co-regulatory proteins.Distal regulatory elements are the most important regulatory elements existing in gene cluster and widely dispersed in genome of higher eukaryotes.They occur so frequently and play critical roles in the process of cellular diffierentication and organic,development.They can exert their effects from several hundred bps to several hundred kbs far away from the promoters of their target genes.β-globin gene cluster is an exellent model for the study of distal enhance actions.The murineβ-globin locus contains fourβ-like globin genes(εy,βh1,β-maj,andβ-min) and an upstream LCR consisting of six DNaseⅠhypersensitive sites(HSs).How such distal LCR work is one of the basic and pivotal problems in the regulation of globin gene expression.Three models have been proposed to explain the action of LCR.They are:Looping modcl,Linking model and Tracking model. Recently,a notable advance has been achieved in the studies of distal enhance action. By using two novel techniques,RNA-TRAP(Tagging and Recovery of Associated Proteins) and 3C(Chromatin Conformation Capture),researchers demonstrated for thc first time that LCR spatial locates close to the active globin gene promoter.It is the first time that people get direct evidence supporting looping model.On this base, Grosvel et al put forward novel concept,the active chromatin hub(ACH),to explain the action of distal enhance action in nuclei.The ACH is local nucleic compartment formed by distal enhance elements and promoters which concentrates the transcription factors and isolates the gene from surrounding enviroment.The question is what factors involved in the looping and ACH formation ?GATA-1 and EKLF are two important factors which are involved in globin gene activation.EKLF mainly binds to conserved CACCC sequences,and regulates adult globin gene expression.GATA-1 distributed widely on the wholeβ-globin gene cluster,including LCR and gene promoter.In EKLF knocked-out mouse,the structure of ACH dispeared.While in G1E cell in which GATA-1 is absent, proximity between the LCR and theβ-major promoter reduced,indicating that GATA-1 and EKLF are two factors which are involved in the looping or ACH formation.NF-E2 is another important factor which played important roles in erythroid differentiation and globin gene expression.NF-E2 composed of a tissue-specific subunit,NF-E2 p45 and a smaller subunit NF-E2 p18 that is widely expressed. During erythroid differentiation,p18 formed heterodimer with p45 to activate globin genes expression.NF-E2 binds two’tandom AP-1/NF-E2 sites in HS2 which form the core of its enhancer activity.During globin gene activation,both hypersensitive sites of LCR and the promoters ofβ-major andβ-minor globin genes were occupied by NF-E2.In addition,the histone modification pattern and the transfer of RNA PolⅡfrom LCR toβ-major promoter have a close relationship with the transcription factor NF-E2.So it is interesting and meaningful to elucidate the mechanism about how NF-E2 involved in globin gene activation.In this reaserch,we tried to study the mechanism about how NF-E2 regulating globin gene expression.We first knocked down NF-E2p18 subunit by retrovirus vector-directed RNAi in MEL DS19 cell line in order to establish NF-E2 activity reduced cell line.Then we examined the function of NF-E2 in NF-E2p18 silenced cell pools.At last,we explored the’mechanism of NF-E2 in LCR-directed looping formation and discussed the relationship between NF-E2 and how RNA PolⅡinitiating globin gene transcription.The results showed that:In NF-E2p18 knocked-down cell line,the occupancy of NF-E2 p18 and p45 on both HSs of LCR and globin gene promoter declined, accompanied by low expression level of a andβglobin genes.In additon,the binding activity of RNA PolⅡat both HSs of LCR and globin gene promoters also reduced,indicating that the binding of RNA PolⅡatβglobin gene promoter relied on NF-E2 binding.To assay the effect of NF-E2 on LCR proximity with each active globin gene,a primer within a BglⅡfragment containing HS2 was used in pair-wise combination with a primer within a BglⅡfragment in the vicinity of eachβ-globin gene,such as:β—maj,β—min,Ey andβh1.The results showed that the relative proximity of eachβ-like globin gene with(the LCR) HS2 decreased in a distance-dependent manner in DS19 cell line before differentiation.After DMSO induction,the ligation frequency between HS2 andβ—maj,β—min increased, HS2-β-major ligation product increased 2.2-fold.A moderate increase in proximity was observed between HS2 and 13-minor,which likely reflects the LCR-dependent activity of this gene.Proximity of the LCR with Ey andβh1 embryonic globin genes was not significantly increased,consistent with low-level transcription of these genes in DS19sip18 cells.However,when NF-E2p18 knocked down,HS2-βmajor ligation product decreased 0.43 fold in accordance with low NF-E2 binding frequency and low expression of globin genes.The results of 3C analysis indicate that NF-E2 is necessary to the transition of globin locus into a looped conformation as determined by 3C analysis. Combined the research results on transcription factor NF-E2 in other labs and the research work about this factor in our lab,we think that the mechanism of NF-E2 involvement in activiatingβ-globin gene expression includes three steps:1. Before erythroid terminal differentiation,LCR was occupied by NF-E2p18 and BachI,globin gene expression was inhibited.During the process of globin gene activation,p18 heterodimerized with p45 forming NF-E2 complex.2.NF-E2 occupied NF-E2/AP-1 binding site on LCR,recruited other protein activators,RNA PolⅡetc,forming LCR preinitiation complex.At the same time,NF-E2 was also recruited toβ-maj promoter through protein-protein interaction,forming another protein complex.3.The binding of protein complex on LCR and promoter lead to chromatin structure alteration,forming loop structure,further interaction of these complexes promoted the transfer of RNA PolⅡfrom LCR toβ-globin gene promoter,leading to transcription initiation.更多还原