Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity

Abstract

Developmental enhancers integrate graded concentrations of input transcription factors (TFs) to create sharp gene expression boundaries. Here we examine the hunchback P2 (HbP2) enhancer which drives a sharp expression pattern in the Drosophila blastoderm embryo in response to the transcriptional activator Bicoid (Bcd). We systematically interrogate cis and transfactors that influence the shape and position of expression driven by HbP2, and find that the prevailing model, based on cooperative binding of Bcd to HbP2 is not adequate. We demonstrate that other proteins, such as pioneer factors, mediator and histone modifiers influence the shape and position of the HbP2 expression pattern. By comparing our results to theory, we assess how higher-order cooperativity and energy expenditure impact boundary location and sharpness. Our results emphasize that the bacterial view of transcription regulation, where pairwise interactions between regulatory proteins dominate, must be re-examined in animals, where multiple molecular mechanisms collaborate to shape the gene regulatory function.