Modulation of gene expression by DNA-protein and protein-protein interactions in the promoter region of the zein multigene family.

A common cis-acting element in the promoter region of many genes expressed during endosperm development of cereal seeds, the prolamine-box or P-box, is only 20bp upstream of the alpha-class 22-kDa zein gene-specific cis element, the O2-box, which is recognized by the b-ZIP transcription factor, Opaque-2 (O2). The proximity of these two boxes has prompted a study of how two DNA-binding proteins of a different hierarchy might be involved in the activation and modulation of the 22-kDa zein-encoding genes. This was accomplished by utilizing a highly purified P-box-binding-factor-1 (PBF-1) and a bacterially expressed truncated form of the O2 protein. After adding the recombinant O2 to the purified fraction of PBF-1, binding studies were performed with a series of DNA probes combining the P- and O2-boxes from zein promoters. These studies have revealed an interesting inhibitory effect of PBF-1 over O2 function dependent on their ratio, consistent with its in-vivo properties and the developmental expression profiles of zein genes. We also could show that the P-box is specifically recognized by topoisomerase II and single-strand DNA-binding proteins, indicating a possible additional linkage between P-box and the scaffold-attachment-region (SAR).