We have been investigating the molecular mechanisms which govern the tissue and developmental stage-specific expression of the adult beta-like globin genes. Our efforts have focused on establishing the combination of trans-acting factors and DNA cis-elements that determines the low level of expression of the adult beta-like globin genes in immature erythroid tissues. It is our hope that elucidating the mechanisms of globin gene regulation will facilitate the manipulation of hemoglobin synthesis in individuals affected by underexpressed or structurally abnormal globin genes. Our current studies of hemoglobin ontogeny have employed the K562 human erythroleukemia cell line as a tissue culture model of globin gene expression. Previous studies of this cell line revealed an embryonic- fetal phenotype, which is unique for low level expression of the beta globin gene in the absence of beta globin transcription. This phenotype contrasts markedly with normal erythrocytes from all stages of development, where expression of beta globin far exceeds delta, ranging from a ratio of 75:1 in fetal tissues to 40:1 in normal adults. Using transient transfection assays, we have been exploring the possibilities that both the reversal of normal relateive levels of synthesis of delta and beta globin, and the overall low level of expression of the two adult genes in K562 cells are due to the differential binding of trans- acting proteins to upstream regulatory regions. DNA fragments from the 5'flanking regions of the delta and beta globin genes were cloned into expression vectors containing the CAT reporter gene (pSV CAT), then transiently transfected into the K562 cells. We have clearly demonstrated the existence of a negative transcription element in the -500 to -250bp region of the beta globin gene, consistent with previous studies by Berg (N.A.R., 1989) which identified two negative regulatory elements (BP-1 and BP-2) 5' to the beta globin gene. We also identified an important positive transcription element in the comparable region flanking the delta globin gene. Structural analysis suggests that a potential regulatory element with extensive homology to the beta globin BP-2 motif exists in the delta globin 5' flanking region. We suggest that the negative regulatory elements upstream from the beta globin gene play an important role in expression of the adult beta-like globin genes in immature erythroid tissue.
NIH ExPorter
NIH ExPorter