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 motif is duplicated in humans and other 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-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
delta-globin gene promoter. The resulting mutants, as well as the wild type (wt) delta- and
beta-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
delta-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
delta globin promoter containing the proximal or distal CACCC consensus sequences over the wt
beta globin gene promoter, we have carried out transient expression experiments using
DNA constructs in which the delta and
beta 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
delta 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
beta-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
beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized
delta-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 beta-like
globin gene, and show that the insertion of a single CACCC motif in the
delta-globin gene promoter is sufficient to increase its activity. Nevertheless only the
delta 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 have potential relevance for the future prospective treatment of inherited
hemoglobinopathies based on the conversion of the low functioning
delta-globin gene into a high functioning beta-like
globin gene.