Identifying cis-acting DNA elements within the control region of glycogen phosphorylase 2 by DNaseI footprinting in Dictyostelium discoideum

Abstract

Glycogen phosphorylase 2 (encoded by gp2) is a key enzyme expressed during the development of Dictyostelium discoideum. The Gp2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. In this report we present the identification of several putative cis-acting elements of gp2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an end-labeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for the protein sources. However, using a more sensitive footprint strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It is concluded that the TAG and C boxes are cis-acting elements involved in the regulation of gp2 expression.