An 80-bp cis-acting regulatory region controls cAMP and development regulation of a prestalk gene in Dictyostelium

We have analyzed an 80-bp region containing the cis-acting sequences necessary for regulation of the Dictyostelium discoideum prestalk gene pst-cathepsin at the appropriate stage during multicellular development and in response to cAMP in single-cell culture. The region lies between approximately -280 and -200 bp upstream from the Cap site and contains two intertwined G/C-rich sequences, including a palindromic sequence and a direct repeat of the 3' portion of the palindrome. In a previous set of experiments, we showed that the direct repeat, or G-box, is important in the regulation of pst-cath expression. In this paper, we use a series of nested internal deletions to define other regions within the promoter required for cAMP and developmental expression, to further examine the importance of the two G-boxes, and to examine the functional relationship of the G-boxes with respect to the other regulatory sequences. Analysis of the expression of these constructs in transformed cells showed that both the 5' portion of the palindrome and the two G-boxes are required for promoter function and are capable of developmentally regulating pst-cath expression. An A/T-rich sequence located 5' to the G/C-rich sequence is also essential for maximal expression, whereas insertion of a linker 5' to this region suggests the presence of a negative element functional during multicellular development. The spacing between the G-box sequences proved to be important for the full induction of gene expression. Constructs containing the G-boxes at wild-type spacing or closer show wild-type or near wild-type levels of expression, whereas expansion of the region between the G-boxes leads to a substantial drop in the level of gene expression in response to cAMP. Insertion of an oligonucleotide containing one of the G-boxes and surrounding sequences can partially complement deletions in which this region has been removed. Analysis of the expression of the cassette constructs, in some cases, revealed significant differences in the quantitative level of expression under the two developmental conditions. This suggests that there are either qualitative or quantitative differences in the factors controlling the expression of this gene under these two conditions.