Activation of the delta-globin gene by the beta-globin gene CACCC motif

The promoter region of adult beta globin genes in humans and other mammals contains conserved regions of pivotal importance for their regulated tissue specific expression. These include the CACCC and CAAT motifs. The CACCC mo tif is duplicated in humans and others mammals. The human delta-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the beta globin gene. Previous studies have shown that the introduction of the beta-globin CACCC or CAAT can activate the delta-globi n gene promoter, but the effect of the distal CACCC element has not yet bee n tested. In the present study, using site-specific mutagenesis, we have in troduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type delta-globin gene pr omoter. The resulting mutants, as well as the wild type (wt) delta- and bet a-globin gene promoters, have been analyzed in a transient expression assay in Cos7, K562, and MEL cell lines. The results show that the CACCC boxes c an increase the transcription efficiency of the delta-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the t wo CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is mor e active in adult erythroid cells (MEL). Nonetheless, duplication of this e lement does not appear to affect the efficiency of the promoter synergistic ally. Furthermore, to assess the competitive ability of the delta globin pr omoter containing the proximal ol distal CACCC consensus sequences over the wt beta globin gene promoter, we have carried out transient expression exp eriments using DNA constructs in which the delta and beta globin gene promo ters are linked in cis and are sharing a single enhancer (competitive trans ient expression). The results show that both CACCC elements are able to act ivate the delta globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in i ncreasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the beta-thal assemia mutations affecting the proximal CACCC box as compared with those w ithin the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a n uclear protein restricted to erythroid cells which specifically bind the CA CCC box sequence and activate the beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized delta-globin ge ne promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the disral CACCC element are transactivated. Our results indicate that the proximal CACCC bo x and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a beta-like globin gene, and show that the in sertion of a single CACCC motif in the delta-globin gene promoter is suffic ient to increase its activity. Nevertheless only the 6 globin gene promoter containing the proximal CACCC element is able to compete with the wt beta globin gene promoter in the adult erythroid environment. These findings hav e potential relevance for the future prospective treatment of inherited hem oglobinopathies based on the conversion of the low functioning delta-globin gene into a high functioning beta-like globin gene.