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.
In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients
.
We noted that enhancers of several Hh-responsive
Drosophila
genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of
dpp
,
wingless
and
stripe
, by transplanting optimal sites from the
patched
gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.
The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure—one of the oldest cis-regulatory features discovered in animal genomes—explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways.DOI:
http://dx.doi.org/10.7554/eLife.13550.001
The Hedgehog (Hh) signaling pathway is an important regulator of embryogenesis and tissue homeostasis during adulthood. This pathway is conserved from flies to humans, where it controls the transcription of key target genes. Cubitus interruptus (Ci), a member of the Gli family of transcription factors (TFs), regulates the transcription of Hh target genes by binding to regulatory sequences known as enhancers. In the Drosophila wing, preliminary data show that enhancers that contain consensus Ci binding sites, GACCACCCA, are repressed in cells that lack Ci. We hypothesized that an unidentified transcriptional repressor binds preferentially to consensus Ci binding sites to refine the expression patterns of some Hh target genes. In this project, we identified in silico nine related TFs that have similar Ci binding motifs, and tested whether they selectively recognize Ci binding sites in vitro. We transcribed and translated the DNA binding domains of Button head, Drosophila Specificity Protein 1, Lameduck (Lmd), Sugarbabe, Odd paired (Opa), Klumpfuss (Klu), Stripe, Scratch, and Longitudinals Lacking. Using Electrophoretic Mobility Shift Assays, we tested if these proteins differentially recognized consensus Ci binding sites versus non‐consensus binding sites, which are sequences that deviate from the consensus by one or more base pairs. We found that Opa, Klu, and Lmd recognized consensus Ci sites, but not non‐consensus sites. In addition, we performed in vivo genetic experiments to knockdown these proteins in the wing and the embryo. Knocking down Klu in the wing, resulted in a posterior cross vein phenotype, and knocking down Opa resulted in lethality. Knocking down Klu and Opa in embryos caused derepression of the Hedgehog target gene patched. These results posit a potential role for these TFs in refining the expression of Hh target genes.
How do gene regulatory networks evolve? A new study in yeasts shows that cis- and trans-regulatory changes resulted in a hybrid state of coexisting ancestral and derived regulatory circuits. This hybrid state then diversified into a variety of modern networks.
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