Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP

Abstract

A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely.

Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica

“…E. histolytica has significant core promoter sequence divergence even compared with other protozoa. The core promoter of the slime mold Dictyostelium contains a consensus TATA element, lacks an Inr element, and has stretches of poly(dT) at or immediately upstream from the start of transcription initiation (16,17). In contrast, the core promoter of Acanthamoeba contains a consensus TATA element and a conventional higher eukaryotic Inr element (18).…”

Transcription of theDictyostelium glycogen phosphorylase-2 gene is induced by three large promoter domains

“…This region of the promoter contains two direct repeats of a C-rich element surrounding a stretch of ten adenines and two direct repeats of a TG-containing element. Deletion analysis [Sucic et al, 1993] and site directed mutagenesis [McCaffery et al, manuscript in preparation] have shown both elements to be transcriptionally relevant. The footprint in this region extends from Ϫ327 to Ϫ393 bp and encompasses the C-rich elements and the tract of adenines.…”

“…Indeed, with an increased concentration of cAMP in the induction experiment described above (1 mM compared to 1 µM during development [Devreotes, 1982] In summary, these experiments demonstrate that a change in chromatin structure accompanies the transcriptional activation of the gp2 gene. The strong hypersensitive site at Ϫ250 bp maps close to the transcription start site of the gene [Sucic et al, 1993], while the Ϫ350 bp site corresponds to a C-rich region in the promoter. The weak site at Ϫ290 bp also maps close to the transcription start site, while the weak upstream site at Ϫ445 bp maps near a TG-rich element that has been implicated in the transcriptional regulation of gp2 by deletion analysis [Rutherford et al, 1997;Sucic et al, 1993].…”

“…The strong hypersensitive site at Ϫ250 bp maps close to the transcription start site of the gene [Sucic et al, 1993], while the Ϫ350 bp site corresponds to a C-rich region in the promoter. The weak site at Ϫ290 bp also maps close to the transcription start site, while the weak upstream site at Ϫ445 bp maps near a TG-rich element that has been implicated in the transcriptional regulation of gp2 by deletion analysis [Rutherford et al, 1997;Sucic et al, 1993]. The weak site at Ϫ505 bp lies close to a repeat sequence in the promoter consisting of TAAT repeated five times and separated by five bases after each repeat.…”

“…4). This region of the promoter contains the transcription start site [Sucic et al, 1993]. The 67 bp footprint encompasses the region from Ϫ227 to Ϫ294 bp and emerges during aggregation when the gene is first expressed ( Fig.…”