Abstract
Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'-untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.
Highlights
Interaction between the stage selector element (SSE) in the proximal y.globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the p·globin promoter in the fetal developmental stage
To determine if the core enhancer was sufficient for the competitive advantage of the v-promoter in the fetal environment, the -378 (3-promoter/CAT reporter gene and -260 v-promoter/luciferase reporter gene were linked in competition for hypersensitivity site 2 (HS2) or HS2 M (HS2(3CAT)'LUC, HS2M(3CAT)'LUC) and transfected into K562 cells
The reduction in y-promoter activity observed in construct 5 by comparison with construct 2 is consistent with the loss of the co-enhancer effect
Summary
Interaction between the stage selector element (SSE) in the proximal y.globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the p·globin promoter in the fetal developmental stage. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-l These results indicate that the 5'UTR of the l'-gene contains sequences that may be important for its transcriptional and developmental regulation. The concept of competition between globin gene promoters for a shared enhancer as a mechanism of developmental regulation was originally proposed for the chicken globin locus In this system, silencing of the embryonic e-globin gene in the adult stage of erythropoiesis is mediated by a sequence in the f3-promoter, the stage selector element (SSE) [12]. CP2 has recently been demonstrated to represent the human homologue of chicken NF-E4 [16]
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