Function of a C-rich region in the transcriptional regulation of the glycogen phosphorylase-2 gene in Dictyostelium discoideum

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Virginia Tech


The cellular slime mold Dictyostelium discoideum is a simple eukaryote that has been used as a model organism for the investigation of eukaryotic cell signaling, regulation of gene expression, and development. Transcription of the gp-2 gene is induced during development and is regulated by extracellular morphogens such as cyclic AMP (cAMP; Sucic et al., 1993) and differentiation induction factor (DIF; Yin et al., 1994a). These morphogens are known to be involved in regulating cellular differentiation and pattern formation in the multicellular development of Dictyostelium. This makes the gp-2 gene a good candidate for the investigation of the mechanisms of regulation of gene expression during cellular differentiation. The gp-2 gene has been cloned and previous analyses of the promoter with 5' deletions have revealed the presence of several regulatory regions which contain repeated sequence elements; TA-boxes, the TAG-boxes, and C-boxes (Sucic et al., 1993; Rutherford et al., submitted). The C-rich regulatory region contains two C-box repeats and is the most proximal of the regulatory regions. The aims of this investigation are the precise definition of regulatory elements within the C-rich regulatory region and identification of the role played by these sequences in the transcriptional regulation of the gp-2 gene. The effects of disrupting regulatory sequences within the C-rich region with upstream regulatory elements intact were investigated using internal deletions and site-directed mutations. Using the luciferase reporter gene system it was shown that site-directed mutation of the downstream C-box (CB-2) and adjacent bases results in a 50-fold decrease in the developmentally induced luciferase levels compared to a wild-type promoter construct (OS). An internal deletion of this mutant construct that deletes the upstream Cbox (CB-1) and intervening sequences has no further effect on levels of expression induced during development. These data suggest that CB-2 and adjacent sequences are the regulatory sequences within the C-rich region that are involved in the transcriptional regulation of gp-2. The mutant promoter constructs regulate gene expression during development with the same temporal profile as the wild-type construct indicating that these sequences are not involved with the timing of induction of expression of the gp-2 gene. The induction of the gp-2 promoter constructs by extracellular cAMP was analyzed. The wild-type construct was induced approximately 10-20-fold by extracellular cAMP. However, the site-directed mutated promoter construct was not induced by cAMP under the same conditions. This suggests that CB-2 and adjacent sequences are involved with the induction of gp-2 expression by extracellular cAMP. Previously, 5' deletion analyses have shown that upstream regulatory elements are involved in cAMP-responsive expression of the gp-2 gene (Sucic et al., 1993). The data presented here indicate that the regulation of expression of the gp-2 gene by extracellular cAMP also requires sequences in and around CB-2.