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

Abstract

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.

Quantitative Dissection of the Proximal Ciona brachyury Enhancer

“…We conclude that the loss of reporter expression in the ZIC TFBS mutant construct partially involves an increase in transgene mosaicism, but it also involves a graded decrease in reporter expression at the level of individual expressing cells. This is similar to what we previously observed quantifying the response of proximal and distal Bra enhancer reporter constructs to graded MAPK inhibition with intermediate doses of the MEK inhibitor U0126 (Harder et al, 2021).…”

“…Several previous studies have mapped out cis-regulatory modules controlling brachyury expression in ascidian models (Corbo et al, 1998;Fujiwara et al, 1998;Takahashi et al, 1999;Yagi et al, 2004;Matsumoto et al, 2007;Farley et al, 2016;Harder et al, 2021;Reeves et al, 2021). Various transcription factor binding motifs of interest have been mutated, but these efforts have been split between Halocynthia and Ciona and also between the proximal bra enhancer and the more distal "shadow" bra enhancer in Ciona.…”

“…30 micrograms of Bra>Histone H2B:HA plasmid were included in each electroporation as an internal control for electroporation efficiency. This construct is from Harder et al (2021) and contains 2.2 kB of bra upstream sequence spanning both the proximal and distal enhancers. Most individual experiments involved 4 or 5 different electroporations, one of which was always the 377-bp wildtype bra enhancer>Venus plasmid.…”

“…That is our goal here, using the proximal enhancer for the notochord transcription factor Brachyury (Bra) as a test case. Brachyury induction in both the primary A-line and secondary B-line notochord lineages has been extensively investigated and numerous upstream regulators are known (Miya and Nishida, 2003;Yagi et al, 2004;Hudson and Yasuo, 2006;Imai et al, 2006;Matsumoto et al, 2007;Farley et al, 2016;Veeman, 2018;Harder et al, 2021;Reeves et al, 2021). Several of these interactions are likely direct based on mutating predicted TFBSs in reporter assays (Corbo et al, 1998;Fujiwara et al, 1998;Takahashi et al, 1999;Yagi et al, 2004;Matsumoto et al, 2007;Farley et al, 2016;Reeves et al, 2021), but there has been no systematic attempt to predict and disrupt sites for all of these putative upstream regulators in the same quantitative framework.…”

Origin of the Chordate Notochord

“…While some genes including brachyury and tropomyosin-like are expressed uniformly along the notochord, differential expressions were found in TGF-beta, ERM, Bcam, TGF-β, etc. [69,70]. In addition, ascidian notochord is deprived of hox expression, which marks the notochord of other chordate groups and the proposed annelid axochord, likely due to clade-specific loss of expression during evolution after the split between Urochordata and Vertebrata [21].…”