The role of trans-acting factors and DNA-bending in the silencing of human β-globin gene expression

Abstract

The molecular mechanisms which govern the developmental specificity of human β-globin gene transcription have been studied in K562 cells, a human eyrthroleukemia line that expresses minimal β-globin. Protein-binding analysis reveals that 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 individual element in a beta-globin vector containing chloramphenicol acetyl-transferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5.5-fold higher than wild-type (wt) βCAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt βCAT. BP1 and BP2 motifs have overlapping binding sites for high mobility group proteins (HMG1+2), DNA-bending factors, shown here to extrinsically bend the β-globin 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 constitutive gene expression. Placing two 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 β-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in β-globin silencing in the embryonic/fetal erythroid stage.