应用体内足迹法研究小鼠β-珠蛋白基因簇重要调控元件上的DNA-蛋白质间的相互作用
摘要
β-珠蛋白基因簇是研究真核基因表达调控的良好模型，在个体发生中β-珠蛋白基因簇表现为从5′到3′依次表达，具有高度的红系组织特异性和发育阶段特异性，虽然α和β两个珠蛋白基因簇不在同一条染色体上，但两类珠蛋白基因表达的终产物始终维持平衡。
基因表达是由众多顺式作用元件和反式作用因子相互作用调控的。β-珠蛋白基因的正确表达主要依赖于两种类型的调控元件：位于基因簇5′端的位点控制区(Locus Control Region，LCR)以及各珠蛋白基因的启动子等近端调控序列。LCR由4个DNase 历高敏位点(5'HS1-5'HS4)构成，每个HS的活性又都集中在核心序列上。研究发现，LCR的HSs以及单个基因的启动子上，都含有红系特异和公共表达的反式作用因子的结合位点，LCR及其HSs的形成、启动子上转录装置的建立、LCR与下游基因调控元件间的相互联系(如：Looping)等，都需要这些顺式作用元件(DNA)与相关反式作用因子(蛋白质)间的相互作用来介导。因此，研究DNA-蛋白质之间的相互作用对于阐明LCR的作用机制、珠蛋白基因表达调控的开关机制具有重要意义。
体内足迹法(In Vivo Footprinting)是一种研究DNA-蛋白质相互作用的方法，它能真实反映体内蛋白．DNA相互作用的情况，而且可以检测体内的DNA构象的变化，连接介导的聚合酶链反应(Ligation-Mediated polymerase chain Reaction，LM-PCR)的发明，使得体内足迹研究的敏感性和特异性大大提高，有力地促进了体内足迹技术在真核基因表达调控研究方面的应用。
本文应用连接介导的聚合酶链反应及体内足迹法研究了诱导前后的MEL细胞、处于不同发育时期的红系组织、药物处理后的成年造血组织中β珠蛋白基因启动子及LCR-HS2上的DNA-蛋白质之间的相互作用。
(1)标准条件下培养MEL细胞，经2％DMSO诱导分化三天，收集状态良好的细胞，悬浮于无血清DMEM培养基中，调整细胞浓度为5×10~7／ml。
(2)正常健康昆明小鼠交配，待胚胎发育至14天时，处死怀孕母鼠，取胎鼠肝脏，用另一组成年小鼠制备骨髓；然后应用40％-70％Percol1梯
β-globin gene cluster has been one of the favorite model systems for analyzing the control of gene expression,particularly developmental regulation. During erythroid development, the β-like globin genes are expressed sequentially from 5' to 3' according to the genome orientation in a erythroid-specific and developmental stage-specific manner. Although clustered in distinct chromosomal loci, the expression of α-like and β-like globin chains is balanced.
Gene expression is regulated by the complex interaction of many cis-acting elements and trans-acting factors. The correct expression of β-globin genes mainly depends on two kinds of regulatory elements: the locus control region located 5' of this cluster and promoter regions of individual genes. The β-globin gene LCR consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4),every HS activity is defined to its core sequences. It is known now that: the HS core sequences of the LCR and the promoter of individual globin genes have many binding sites for erythroid -specific as well as ubiquitous proteins. The involvement of specific DNA-protein interaction in the formation of hypersensitive sites, in the assembly of basal transcription apparatus and in the contact between LCR and downstream gene promoter (eg. looping) is suggested by a large body of evidence. Therefore, studying DNA-protein interaction is important and significant to elucidate the mechanism of LCR action and globin gene switching.
In vivo footprinting is a method of studying DNA-protein interaction. It can reflect the authentic status of DNA-protein in vivo, and can also detect the change of DNA conformation. The introduction of Ligation-Mediated PCR(LM-PCR) greatly improves the sensitivity and specificity of in vivo footprinting study, and has facilitated the executation of in vivo footprinting in the regulation of eucaryotic gene expression.
In this article, we have used in vivo footprinting and LM-PCR to study DNA - Protein interaction at HS2 of LCR and β-globin gene promoter of MEL cells with or without induction, of mouse bone marrow-derived and fetal liver-derived erythroid cells, and of bone marrow-derived erythroid cells of mice treated with myleran or hydroxyurea.
(1) MEL cells are grown in DMEM medium,induction with 2%DMSO for three days.collect cells and resuspend in DMEM medium without serum,adjust cell concentration to 5×10~7.
(2) Healthy kunming mice mate to produce embryo. sacifice prognant mice at
基因表达是由众多顺式作用元件和反式作用因子相互作用调控的。β-珠蛋白基因的正确表达主要依赖于两种类型的调控元件：位于基因簇5′端的位点控制区(Locus Control Region，LCR)以及各珠蛋白基因的启动子等近端调控序列。LCR由4个DNase 历高敏位点(5'HS1-5'HS4)构成，每个HS的活性又都集中在核心序列上。研究发现，LCR的HSs以及单个基因的启动子上，都含有红系特异和公共表达的反式作用因子的结合位点，LCR及其HSs的形成、启动子上转录装置的建立、LCR与下游基因调控元件间的相互联系(如：Looping)等，都需要这些顺式作用元件(DNA)与相关反式作用因子(蛋白质)间的相互作用来介导。因此，研究DNA-蛋白质之间的相互作用对于阐明LCR的作用机制、珠蛋白基因表达调控的开关机制具有重要意义。
体内足迹法(In Vivo Footprinting)是一种研究DNA-蛋白质相互作用的方法，它能真实反映体内蛋白．DNA相互作用的情况，而且可以检测体内的DNA构象的变化，连接介导的聚合酶链反应(Ligation-Mediated polymerase chain Reaction，LM-PCR)的发明，使得体内足迹研究的敏感性和特异性大大提高，有力地促进了体内足迹技术在真核基因表达调控研究方面的应用。
本文应用连接介导的聚合酶链反应及体内足迹法研究了诱导前后的MEL细胞、处于不同发育时期的红系组织、药物处理后的成年造血组织中β珠蛋白基因启动子及LCR-HS2上的DNA-蛋白质之间的相互作用。
(1)标准条件下培养MEL细胞，经2％DMSO诱导分化三天，收集状态良好的细胞，悬浮于无血清DMEM培养基中，调整细胞浓度为5×10~7／ml。
(2)正常健康昆明小鼠交配，待胚胎发育至14天时，处死怀孕母鼠，取胎鼠肝脏，用另一组成年小鼠制备骨髓；然后应用40％-70％Percol1梯
β-globin gene cluster has been one of the favorite model systems for analyzing the control of gene expression,particularly developmental regulation. During erythroid development, the β-like globin genes are expressed sequentially from 5' to 3' according to the genome orientation in a erythroid-specific and developmental stage-specific manner. Although clustered in distinct chromosomal loci, the expression of α-like and β-like globin chains is balanced.
Gene expression is regulated by the complex interaction of many cis-acting elements and trans-acting factors. The correct expression of β-globin genes mainly depends on two kinds of regulatory elements: the locus control region located 5' of this cluster and promoter regions of individual genes. The β-globin gene LCR consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4),every HS activity is defined to its core sequences. It is known now that: the HS core sequences of the LCR and the promoter of individual globin genes have many binding sites for erythroid -specific as well as ubiquitous proteins. The involvement of specific DNA-protein interaction in the formation of hypersensitive sites, in the assembly of basal transcription apparatus and in the contact between LCR and downstream gene promoter (eg. looping) is suggested by a large body of evidence. Therefore, studying DNA-protein interaction is important and significant to elucidate the mechanism of LCR action and globin gene switching.
In vivo footprinting is a method of studying DNA-protein interaction. It can reflect the authentic status of DNA-protein in vivo, and can also detect the change of DNA conformation. The introduction of Ligation-Mediated PCR(LM-PCR) greatly improves the sensitivity and specificity of in vivo footprinting study, and has facilitated the executation of in vivo footprinting in the regulation of eucaryotic gene expression.
In this article, we have used in vivo footprinting and LM-PCR to study DNA - Protein interaction at HS2 of LCR and β-globin gene promoter of MEL cells with or without induction, of mouse bone marrow-derived and fetal liver-derived erythroid cells, and of bone marrow-derived erythroid cells of mice treated with myleran or hydroxyurea.
(1) MEL cells are grown in DMEM medium,induction with 2%DMSO for three days.collect cells and resuspend in DMEM medium without serum,adjust cell concentration to 5×10~7.
(2) Healthy kunming mice mate to produce embryo. sacifice prognant mice at
引文
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