| 1. GATA-1 is an erythroid specific transcription factor essential for the terminal maturation of proerythroblasts, magakaryocytes and mast cells. The proximal promoter of human GATA-1 gene directs high expression of reporter gene in K562 cells. In order to better understand the transcriptional control of this gene, the 5'proximal promoter of this gene was cloned and analyzed. Reporter gene construct containing -860 to -1bp demonstrated high transcriptional activity and reporter gene construct containing -128 to -1bp had almost no activity. While the reporter gene containing -221 to -1bp had similar activity of the -860bp construct, indicating that this region between -221 to -1bp contains a minimal promoter. Deletion of CGCCC box at -195bp or together with the CACCC box at -173bp reduced the reporter gene activity to 60.4% and 13.2% compared to that of the -860bp construct respectively, indicating that the CGCCC box and the CACCC box are critical for the initiation of the GATA-1 gene transcription. Electrophoresis mobility shift and supershift assays showed the specificity of Sp1 binding to the CACCC box. Reporter construct containing –180bp to -1bp with a mutated CACCC box resulted in a 70% deduction in transcription activity compared to that of the wild type construct containing –180bp to -1bp. Co-transfection of Sp1 expression plasmids with the wild type reporter construct containing –180bp to -1bp increased the reporter gene activity in a dose-dependent manner. Therefore, the core sequence of human GATA-1 erythroid promoter may be localized between -221 to -128bp, in which the Sp1 site at -173 is critical for the basal activity via its Sp1 binding ability.2. A previously identified negative regulatory element (-3028 to -2902bp, termedε-NRAII) was analyzed to characterize the functions of multiple transcription factor sites within this sequence. One putative NF-κB site and two GATA sites locate at -3004bp, -2975bp and -2948bp respectively. Electrophoresis mobility shift assay (EMSA) revealed that nuclear proteins isolated from K562 cells could specifically bind to the putative NF-κB site. Data obtained from transient transfection showed that whenε-NRAII was inserted upstream of the SV40 promoter orε-globin gene proximal promoter (-177bp to +1bp), the expression of reporter gene could be upregulated about50% or 100% respectively, suggesting thatε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at -2975bp) slightly reduced the expression of reporter gene driven by SV40 promoter orε-globin gene proximal promoter. However, the mutation of GATA site at -2948bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not byε-globin gene proximal promoter. To test whether those sites act synergistically, we mutated 2 of these three binding sites with different combinations. Data showed that the negative effect of mutation in GATA site at -2948bp 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 -2975bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven byε-globin gene proximal promoter. Those results suggested thatε-NRAII might function differently on the SV40 promoter andε-globin gene proximal promoter. 3. High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocyte. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the humanε-globin gene promoter (ε-promoter, -177 ~ +1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the nakedε-promoter DNA , however, HMG14/17 couldn't. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mono-nucleosome recon...
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