Acta Biochim Biophys Sin 2004,36(10)::Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human e-Globin Gene

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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(10): 673-680 CN 31-1940/Q

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human e-Globin Gene

Chun-Hui HOU, Jian HUANG, and Ruo-Lan QIAN*

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

Abstract The developmental control of the human e-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 e-NRAII) was analyzed and one putative NF-kB 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-kB 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 e-NRAII was inserted upstream of the SV40 promoter or e-globin gene proximal promoter (-177 bp to +1 bp), suggesting that e-NRAII might not be a classic silencer. Mutation in the putative NF-kB site or in the GATA site (at -2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or e-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 e-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-kB 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 e-globin gene proximal promoter. Those results suggested that e-NRAII might function differently on the SV40 promoter and e-globin gene proximal promoter.

Key words e-globin gene; regulatory element; NF-kB; GATA

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Received: July 5, 2004 Accepted: September 1, 2004

The work was supported by a grant from the National Natural Science Foundation of China (No. 39870378)

*Corresponding author: Tel, 86-21-54921354; Fax, 86-21-54921011; E-mail, [email protected]