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.     

Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1beta mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1beta-deficient subline Hepa1C4, as well as antibodies raised against HIF-1alpha and ARNT/HIF-1beta confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5' HBS was more efficient in binding HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.     

Restoration of the CCAAT box or insertion of the CACCC motif activates [corrected] delta-globin gene expression

Hemoglobin A2 (HbA2), which contains delta-globin as its non-alpha-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the delta-globin gene in adult erythroid cells, we first compared promoter sequences and found that the delta-globin gene differs from the beta-globin gene in the absence of an erythroid Krüppel-like factor (EKLF) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human delta-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5' flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the delta promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type delta promoter. A set of substitutions that create an EKLF binding site centered at -85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase delta-globin gene expression, with potential future clinical benefit.