TRANSCRIPTION OF THE DICTYOSTELIUM-GLYCOGEN-PHOSPHORYLASE-2 GENE IS INDUCED BY 3 LARGE PROMOTER DOMAINS

The promoter of the Dictyostelium glycogen phosphorylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. T o understand how Dictyostelium achieves specificity during transcripti onal regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure ass ociated with developmental induction of gp2 gene expression. DNase I h ypersensitive analyses indicated the presence of several developmental ly regulated nuclease-sensitive sites located upstream of the start co don: two strong sites at approximately -250 bp and -350 bp and three s ubstantially weaker sites at -290 bp, -445 bp, and -505 bp. In vitro f ootprint analyses using nuclear extracts derived from several stages o f development (corresponding to varying levels of gp2 expression) reve aled three large regions of occupation that were developmentally regul ated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2), and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was c onfirmed by in vivo Footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these fo otprints demonstrated that probes corresponding to domains 1 and 3 bot h interacted with 83 and 77 kDa peptides. The domain 3 probe also inte racted with a 92 kDa peptide, while only a 62 kDa peptide is recognize d by the domain 2 probe. In all cases, peptides capable of binding the se probes were found in nuclear extracts derived from differentiated c ells and not in undifferentiated cell nuclear extract. Using nuclear e xtract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded ban ds for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest hat specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requirin g multiple protein-DNA and/or protein-protein interactions to occur be fore induction can proceed. (C) 1998 Wiley-Liss, Inc.