The Mechanism of Beta-Globin Gene Silencing in Embryonic-Fetal Erythroid Cells

胚胎-胎儿红细胞中β-珠蛋白基因沉默的机制

• 批准号: 6432082
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432082
• 关键词: DNA binding protein; DNA footprinting; cell line; developmental genetics; embryo /fetus protein; gel mobility shift assay; gene expression; gene induction /repression; genetic promoter element; genetic regulation; genetic regulatory element; globin; intermolecular interaction; nonhistone nucleoprotein; reporter genes; site directed mutagenesis; transcription factor; transfection

The molecular mechanisms which govern the developmental specificity of human beta-globin gene transcription has been studied in K562 cells, a human eyrthroleukemia line that expresses minimal beta-globin. Protein-binding analysis reveals the 5' region contains three elements bound by trans-acting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2). In vitro mutagenesis of each element in a beta-globin vector containing chloramphenicol acetyltransferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5.5-fold higher than wt beta-CAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt beta-CAT. BP1 and BP2 motifs have overlapping binding sites with high mobility group proteins (HMG1+2), DNA-bending factors shown here by circular permutation assay to extrinsically bend the beta-globin distal promoter. Theoretically, mutations in all beta-protein binding sites could affect the binding of HMG1+2 sufficiently to impede DNA-protein and/or protein-protein interactions needed to facilitate the low level, constitutive gene expression. Placing two helical turns of DNA between BP1 and BP2 motifs also increased expression 3-fold, indicative of spatial constraints required for optimal silencing. However, insertion of the HMG1+2 DNA-bending motif (also equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in beta-globin silencing in the embryonic/fetal erythroid stage. To further define this relationship an in vivo study using transgenic animals is underway. Preparation of a cosmid containing the uLCRbeta locus with a mutated BP1 silencing motif will permit an in vivo analysis of adult beta-globin gene expression in transgenics during the embryonic/fetal developmental stages. We are using two approaches i). PCR site directed mutagenesis and ii). Recombinant-A Restriction endonuclease-cleavage(RARE) method to create of the mutated BP1 binding site with the beta-globin cosmid. The latter technique might also prove useful in testing the hypothesis directly in the context of K562 chromosomal DNA.

控制人类β-珠蛋白基因转录发育特异性的分子机制已经在K562细胞中进行了研究，K562细胞是一种表达最低β-珠蛋白的人红细胞白血病株。蛋白质结合分析表明，5‘端含有三个被反式作用因子结合的元件，即β蛋白1(BP1)和β蛋白2(BP2)。在体外诱变含有氯霉素乙酰转移酶(PCAT)的β-珠蛋白载体中的每个元件，然后瞬时转染K562细胞，CAT活性水平比wtβ-CAT高5.5倍，这与它们的沉默作用一致。然而，所有这三种元素的突变导致活性显著低于wtβ-CAT。BP1和BP2基序与高迁移率基团蛋白(HMG1+2)有重叠的结合部位，DNA弯曲因子在这里通过循环排列实验显示，以外部弯曲β-珠蛋白远端启动子。从理论上讲，所有β蛋白结合位点的突变都可以影响HMG1+2的结合，从而阻止DNA-蛋白质和/或蛋白质-蛋白质之间的相互作用，这是促进低水平、结构性基因表达所必需的。在BP1和BP2基序之间放置两个螺旋DNA也使表达增加3倍，表明最佳沉默所需的空间限制。然而，插入HMG1+2 DNA弯曲基序(也相当于两个旋转)通过重新建立BP1-BP2邻接关系来促进β沉默。因此，一般DNA弯曲和特定转录因子的组合似乎与胚胎/胎儿红系阶段的β-珠蛋白沉默有关。为了进一步确定这种关系，一项使用转基因动物的体内研究正在进行中。含有突变的BP1沉默基序的uLCRbeta基因的粘粒的制备将允许在体内分析在胚胎/胎儿发育阶段转基因的成人β-珠蛋白基因的表达。我们正在使用两种方法：i)。聚合酶链式反应定点突变和II)。重组-一种限制性内切酶裂解(RARE)方法，用于创建突变的BP1与β-珠蛋白粘粒的结合位点。后一种技术也可能被证明在K562染色体DNA的背景下直接检验这一假设是有用的。