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.