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

The molecular mechanisms which govern the developmental specificity of huma n beta-globin gene transcription have been studied in K562 cells, a human e yrthro-leukemia line that expresses minimal beta-globin, Protein-binding an alysis reveals that the 5' region contains three elements bound by trans-ac ting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2), In vitro mutag enesis of each individual element in a beta-globin vector containing chlora mphenicol acetyltransferase (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 thr ee elements, however, resulted in activity significantly lower than wt beta CAT, BP1 and BP2 motifs have overlapping binding sites for high mobility g roup proteins (HMG1+2), DNA-bending factors, shown here to extrinsically be nd the beta-globin promoter. Theoretically, mutations in all beta-protein b inding sites could affect the binding of HMG1+2 sufficiently to impede DNA- protein and/or protein-protein interactions needed to facilitate constituti ve gene expression. Placing two turns of DNA between BP1 and BP2 motifs als o increased expression 3-fold, indicative of spatial constraints required f or optimal silencing. However, insertion of the HMG1+2 DNA-bending motif (a lso equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specif ic transcriptional factors appear to be involved in beta-globin silencing i n the embryonic/fetal erythroid stage.