Functional analysis of multiple transcription factor sites in a regulatory element of human epsilon-globin gene

The developmental control of the human epsilon-globin gene expression is mediated by transcriptional regulatory elements in the 5' flanking DNA of this gene. A previously identified negative regulatory element (-3028 to -2902 bp, termed epsilon-NRAII) was analyzed and one putative NF-kappaB site and two GATA sites locate at -3004 bp, -2975 bp and -2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-kappaB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when epsilon-NRAII was inserted upstream of the SV40 promoter or epsilon-globin gene proximal promoter (-177 bp to +1 bp), suggesting that epsilon-NRAII might not be a classic silencer. Mutation in the putative NF-kappaB site or in the GATA site (at -2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or epsilon-globin gene proximal promoter. However, the mutation of GATA site at -2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by epsilon-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at -2948 bp on SV40 promoter was not affected by the mutation of the putative NF-kappaB site, whereas it could be abolished by the mutation of GATA site at -2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by epsilon-globin gene proximal promoter. Those results suggested that epsilon-NRAII might function differently on the SV40 promoter and epsilon-globin gene proximal promoter.