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 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.
Ristaldi
M S
MS
Istituto di Ricerche sulle Talassemie e Anemie Mediterranee-CNR-Cagliari, Italy.
Casula
S
S
Porcu
S
S
Marongiu
M F
MF
Pirastu
M
M
Cao
A
A
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D012333:RNA, Messenger; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins