Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.Author summary: When and where genes are expressed is controlled by regulatory DNA regions known as enhancers. Genes often have multiple enhancers that control expression in different cell types or embryonic regions, but there are also genes that have multiple enhancers that control near-identical expression patterns. These ‘shadow’ enhancers are common features of many animal genomes, but it is unclear to what extent they are truly identical in function. Here we studied a pair of shadow enhancers for the notochord-specific gene Brachyury in the simple model chordate Ciona that are both directly activated by the same signaling pathway. We titrated the activity of this pathway with graded doses of a pharmacological inhibitor and measured the effects in quantitative enhancer assays. We found that the two enhancers had significant differences in sensitivity and cooperativity to the same shared regulator and are thus not identical in function when assessed quantitatively. We also identified subtle differences in sensitivity to this upstream signal between different notochord precursor cells.