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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]