The Mechanism Of Beta-globin Gene Silencing In Embryonic

点击翻译按钮获取中文摘要

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. We have initiated experiments to establish a transgenic mouse model for specific repression of adult beta-globin expression by the BP1 protein.To alter the developmental specificity of the human beta-globin gene, we introduced a mutated BP1 binding site into the distal promoter of beta-globin gene sequence of 35 kb cosmid constuct containing the micro-LCR (locus control region) and all other essential elements of the human beta-globin gene cluster. This construct and appropriate controls have been mico-injected into mouse eggs. The subsequent transgeneics should permit the in vivo analysis of the role of BP1 silencing motif during the embryonic-fetal developmental stages.