Both Ets-1 and GATA-1 are essential for positive regulation of platelet factor 4 gene expression

In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions -880, -75 and -135, -30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either -75Ets, -135GATA, or -30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel-retardation assays revealed that Ets-1 and GATA-1 proteins from HEL and MEG-01 cells bound to the Ets motifs and GATA motifs, respectively. Co-transfection experiments showed that the overexpression of Ets-1 and/or GATA-1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets-1 and GATA-1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets-1 increased 10-fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets-1 and GATA-1 play key roles in the positive regulation of PF4 gene expression.     

Conserved elements containing NF-E2 and tandem GATA binding sites are required for erythroid-specific chromatin structure reorganization within the human beta-globin locus control region

Proper expression of the genes of the human beta-globin gene locus requires the associated locus control region (LCR). Structurally, the LCR is defined by the presence of four domains of erythroid-specific chromatin structure. These domains, which have been characterized as DNase I hypersensitive sites (HSs), comprise the active elements of the LCR. The major focus of this research is to define the cis -acting elements which are required for the formation of these domains of unique chromatin structure. Our previous investigations on the formation of LCR HS4 demonstrated that NF-E2 and tandem, inverted GATA binding sites are required for the formation of the native HS. Similarly arranged NF-E2 and tandem GATA sites are present within the core regions of the other human LCR HSs and are evolutionarily conserved. Using site-directed mutagenesis of human HSs 2 and 3 we have tested the hypothesis that these NF-E2 and GATA sites are common requirements for the formation of all LCR HSs. We find that mutation of these elements, and particularly the GATA elements, results in a decrease or complete loss of DNase I hypersensitivity. These data imply the presence of common structural elements within the core of each LCR HS which are required for erythroid-specific chromatin structure reorganization.     

Cloning and characterization of the 5'-flanking region of the human cardiotrophin-1 gene

To enable the analysis of the regulation of the human cardiotrophin-1 gene expression, the 5'-flanking region of the human cardiotrophin-1 gene was cloned and sequenced. Data bank search revealed several cis- active DNA elements (SP1, CREB, C/EBP, AP1 and AP-2 like and GATA) in the proximal 1.1 kb region. Six nested 5-'terminal deletion mutants from -1091/+39 to -218/+39 were fused to a luciferase reportergene and proved to be functionally active after transfection into COS-7 cells.