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.
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China.
Chun-Hui
CH
Hou
Jian
J
Huang
Ruo-Lan
RL
Qian
D001665:Binding Sites; D002478:Cells, Cultured; D005809:Genes, Regulator / Q000235:genetics*; D005914:Globins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
D016328:NF-kappa B; D014157:Transcription Factors; D005914:Globins