The cis-Regulatory Logic of Hedgehog Gradient Responses: Key Roles for Gli Binding Affinity, Competition, and Cooperativity

Abstract

Gradients of diffusible signaling proteins control precise spatial patterns of gene expression in the developing embryo. Here, we use quantitative expression measurements and thermodynamic modeling to uncover the cis-regulatory logic underlying spatially restricted gene expression in a Hedgehog (Hh) gradient in Drosophila. When Hh signaling is low, the Hh effector Gli, known as Cubitus interruptus (Ci) in Drosophila, acts as a transcriptional repressor; when Hh signaling is high, Gli acts as a transcriptional activator. Counterintuitively and in contrast to previous models of Gli-regulated gene expression, we found that low-affinity binding sites for Ci were required for proper spatial expression of the Hh target gene decapentaplegic (dpp) in regions of low Hh signal. Three low-affinity Ci sites enabled expression of dpp in response to low signal; increasing the affinity of these sites restricted dpp expression to regions of maximal signaling. A model incorporating cooperative repression by Ci correctly predicted the in vivo expression of a reporter gene controlled by a single Ci site. Our work clarifies how transcriptional activators and repressors, competing for common binding sites, can transmit positional information to the genome. It also provides an explanation for the widespread presence of conserved, nonconsensus Gli binding sites in Hh target genes.

Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning

“…In contrast to our data here, high-affinity sites governing ptc activity correlated with high expression restricted close to the source of Hh, while broad dpp expression was regulated through low-affinity interactions (Parker et al 2011). In our analysis of GBS affinity, we focused on Gli A -dependent outputs to control for potential differential binding responses between Gli A and Gli R forms encoded by different mammalian Gli genes (Sasaki et al 1997;Ding et al 1998;Lei et al 2006;Vokes et al 2007).…”

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

“…S8A). The enhancer of ptc, unlike that of other Hh pathway targets, responds in a cooperative manner to both the levels and active state of Ci; thus, a change in the levels of Ci that modulates the amplitude of Hh signaling might not necessarily result in an effect on the expression of ptc (Parker et al, 2011). Any gene that modified the levels of Ci, but not necessarily Ptc, when independently knocked down with at least two non-overlapping IRs was classified as a regulator of the Hh pathway ( Fig.…”

A Gli silencer is required for robust repression of gremlin in the vertebrate limb bud

“…Our study was limited to the contribution of a single CRM, and future studies will be required to determine if there are higher-order chromatin interactions among the individual CRMs regulating Grem1, as has been suggested for the Fgf8 and HoxD loci (Marinićet al, 2013;Montavon et al, 2011). In Drosophila, Ci (Gli) repressors have been proposed to work cooperatively by binding to several distinct sites within a CRM regulating dpp (Parker et al, 2011). The presence of an additional Gli CRM in the gremlin locus raises the intriguing possibility that Gli proteins binding to distinct CRMs might nonetheless be able to cooperatively repress Grem1 in the context of a higher order chromatin structure.…”