Identification of upstream regulatory elements that repress expression of adult beta-like globin genes in a primitive erythroid environment.

Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcription activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage.