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

The molecular mechanisms which govern the develop-mental specificity of human [beta]-globin gene transcription have been studied in K562 cells, a human eyrthroleukemia line that expresses minimal [beta]-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) [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 for high mobility group proteins (HMG1+2), DNA-bending factors, shown here to extrinsically bend the [beta]-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 [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.