A DNA transcription code for cell-specific gene activation by Notch signaling

Background: Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or "architecture" within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to "S" DNA sites and proneural bHLH activator proteins bound to nearby "A" DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an "S+A" transcription code) can mediate the Notchproneural transcriptional synergy. Results: By contrast, we show that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ("SPS"), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, "SPS+A", that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. We also present evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. Conclusions: The SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression.