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

在K562细胞中研究了控制人类-珠蛋白基因转录发育特异性的分子机制，K562细胞是一种表达少量-珠蛋白的人红细胞白血病系。蛋白质结合分析显示，5'区包含三个由反式作用因子结合的元件，β蛋白1 （BP1）和β蛋白2 （BP2）。将含有氯霉素乙酰转移酶（chloramphenicol acetyltransferase, pCAT）的β -珠蛋白载体中的每个元素进行体外诱变，然后瞬间转染到K562细胞中，使CAT活性水平比wt β -CAT高5.5倍，这与它们的沉默作用一致。然而，这三个元素的诱变导致活性显著低于wt β - cat。BP1和BP2基序与高迁移率基团蛋白（HMG1+2）有重叠的结合位点，环状排列试验显示dna弯曲因子可以从外部弯曲β -珠蛋白远端启动子。理论上，所有β蛋白结合位点的突变都可能影响HMG1+2的结合，足以阻碍dna -蛋白和/或蛋白-蛋白相互作用，从而促进低水平的组成基因表达。在BP1和BP2基序之间放置两个螺旋DNA也使表达增加了3倍，这表明最佳沉默所需的空间限制。然而，插入HMG1+2 dna弯曲基序（也相当于两圈）通过重新建立BP1-BP2邻近性来促进β -沉默。因此，一般dna弯曲和特定转录因子的组合似乎参与了胚胎/胎儿红期的β -珠蛋白沉默。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。制备含有突变BP1沉默基序的uLCRbeta基因座的cosmid将允许在胚胎/胎儿发育阶段对转基因成人β -珠蛋白基因表达进行体内分析。我们使用了两种方法i)。PCR位点定向诱变和ii)。重组-限制性内切酶裂解（RARE）方法，用于创建突变的BP1结合位点与β -珠蛋白粒。后一种技术在K562染色体DNA的背景下也可能被证明是有用的。

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.