Activation of the δ-Globin Gene by the β-Globin Gene CACCC Motif

Abstract

ABSTRACT: The promoter region of adult β 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 motif is duplicated in humans and others mammals. The human δ-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the β globin gene. Previous studies have shown that the introduction of the β-globin CACCC or CAAT can activate the δ -globin gene promoter, but the effect of the distal CACCC element has not yet been tested. In the present study, using site-specific mutagenesis, we have introduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type δ-globin gene promoter. The resulting mutants, as well as the wild type (wt) δ- and β-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 can increase the transcription efficiency of the δ-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the two CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is more active in adult erythroid cells (MEL). Nonetheless, duplication of this element does not appear to affect the efficiency of the promoter synergistically. Furthermore, to assess the competitive ability of the δ globin promoter containing the proximal or distal CACCC consensus sequences over the wt β globin gene promoter, we have carried out transient expression experiments using DNA constructs in which the δ and β globin gene promoters are linked in cis and are sharing a single enhancer (competitive transient expression). The results show that both CACCC elements are able to activate the δ globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in increasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the β-thalassemia mutations affecting the proximal CACCC box as compared with those within the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a nuclear protein restricted to erythroid cells which specifically bind the CACCC box sequence and activate the β-globin gene. In the present study we carried out transactivation experiments of the mutagenized δ-globin gene promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the distal CACCC element are transactivated. Our results indicate that the proximal CACCC box and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a β-like globin gene, and show that the insertion of a single CACCC motif in the δ-globin gene promoter is sufficient to increase its activity. Nevertheless only the δ globin gene promoter containing the proximal CACCC element is able to compete with the wt β globin gene promoter in the adult erythroid environment. These findings have potential relevance for the future prospective treatment of inherited hemoglobinopathies based on the conversion of the low functioning δ-globin gene into a high functioning β-like globin gene.