Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene
Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene作者机构：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 SciencesShanghai 200031 China
出 版 物：《Acta Biochimica et Biophysica Sinica》 (生物化学与生物物理学报(英文版))
年 卷 期：2004年第36卷第10期
页 面：673-680页
核心收录：
学科分类：0710[理学-生物学] 07[理学] 071007[理学-遗传学]
主 题：ε-globin gene regulatory element NF-κB GATA
摘 要：The developmental control of the human ε-globin gene expression is mediated by transcrip- tional regulatory elements in the 5’ flanking DNA of this gene. A previously identified negative regulatory element (–3028 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB 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-κB 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 ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (–177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at –2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-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 ε-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-κB 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 ε-globin gene proximal promoter. Those results suggested that ε- NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.
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