We have been investigating the molecular mechanism which govern the tissue and developmental stage-specific expression of the 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. Our current studies of hemoglobin ontogeny have employed the K562 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 delta 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 expression assays, we have been exploring the possibilities that both the reversal of normal relative levels of synthesis of delta and beta globin, and the overall low levels 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 from the 5' flanking regions of the delta and beta globin genes was cloned into the expression vector PSVoCAT, then transfected into K562 cells. Our findings clearly demonstrate the existence of a negative transcription element in the -500 to -250bp region of the beta globin gene, consistent with previous studies by Berg, et al. (N.A.R., 1989), which identified 2 regulatory elements (BP-1 and BP-2)5' to the beta globin gene. We have 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 repression of the adult beta-like globin genes in immature erythroid tissues.
