Two types of cis-trans compensation in the evolution of transcriptional regulation K. Ryo Takahasia,b,1, Takashi Matsuoc,2, and Toshiyuki Takano-Shimizu-Kounoa,d,e,1,3 aDepartment of Population Genetics, National Institute of Genetics, Misima 411-8540, Japan; bLab 31, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan; cDepartment of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan; dDepartment of Genetics, Graduate University for Advanced Studies (SOKENDAI), Misima 411-8540, Japan; and eDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan Edited by Gregory Gibson, Georgia Tech University, Atlanta, GA, and accepted by the Editorial Board August 3, 2011 (received for review April 18, 2011) Because distant species often share similar macromolecules, regu- regulatory changes to gene expression divergence would be latory mutations are often considered responsible for much of spuriously exaggerated. their biological differences. Recently, a large portion of regulatory Here, by specifically discriminating two distinct types of cis-trans changes has been attributed to cis-regulatory mutations. Here, we compensation, we show, based on stochastic simulations, that examined an alternative possibility that the putative contribution compensatory regulatory evolution under stabilizing selection of cis-regulatory changes was, in fact, caused by compensatory ac- could reduce heterospecific binding and therefore explain the tion of cis- and trans-regulatory elements. First, we show by sto- hypothetical increase in the relative contribution of cis-regulatory chastic simulations that compensatory cis-trans evolution changes without invoking preferential fixation of cis- over trans- maintains the binding affinity of a transcription factor at a constant regulatory mutations (5). We also show by allele-specific expres- level, thereby spuriously exaggerating the contribution of cis-reg- sion assays that the relative expression of heterospecific alleles ulatory mutations to gene expression divergence. This exaggera- from closely related Drosophila species often varies depending on tion was not observed when changes in the binding affinity were the genetic background. Combining the simulation and experi- compensated by variable transcription factor concentration. Sec- ential observations, we hypothesize that cis-trans hybrid incom- ond, using reciprocal introgressions of Drosophila, we show that patibilities are evolving much faster than previously thought. relative expression of heterozygous alleles from two distinct spe- cies often varied significantly between different species back- Stochastic Simulations cis trans grounds, indicating the possible action of - compensation. One-Activator, One-Target System. In the simplest case with a pair Taken together, we propose that cis-trans hybrid incompatibilities of a transcriptional activator and its target gene, our simulations are accumulating much faster than generally considered. keep track of evolutionary changes in the concentration of the trans-acting activator ([T]) and its dissociation constant to the compensatory evolution | trans-regulatory mutation | epistasis | cis-trans cis-regulatory element of the target gene (K)(Simulation Meth- interaction | reproductive isolation ods). These two properties jointly regulate the expression level of the target gene; more precisely, it is expressed as a monotonically first step to understanding the evolution of gene expression increasing function of the quotient [T]/K. The gene expression Ais to decompose expression variation into cis- and trans- level, in turn, affects the fitness of an individual. The dissociation regulatory components (1–4). This decomposition can be done constant K is also described as a function of two variables (a and by combining an allele-specific expression assay using F1 hybrids c), each variable characterizing the binding properties of the with expression assays in pure species backgrounds (1, 4). From activator and the cis-regulatory element, respectively. Defined in these assays, two kinds of expression differences are determined this way, two distinct types of compensatory cis-trans changes are for a gene: (i) difference between two parental strains and (ii) expected under stabilizing selection: (i) K-[T] compensation, difference between two alleles in an F1 hybrid. When the differ- which refers to correlated evolution of K and [T] in such a way ence between parental strains is entirely due to trans-regulatory that an increase (or decrease) in the transcriptional activator changes, we would not expect any difference between the two abundance cancels out the decreased (increased) binding affinity alleles in the hybrid condition. However, when the difference as measured by the inverse of the dissociation constant (and vice between parental strains is entirely due to cis-regulatory changes, versa), and (ii) a–c compensation, which refers to coordinated this difference should be reproduced as an allelic difference changes in a and c that maintain the binding affinity at a con- in the hybrid. A recent application of this method identified stant level. a greater contribution of cis-regulatory changes in inter- than Following the previous study (4), we studied the linear re- intraspecific comparisons, suggesting that cis-regulatory muta- gression of the relative allelic expression in F1 hybrids on the tions are fixed more preferentially during evolution than trans- relative expression difference between parental strains. When regulatory mutations (5). both types of compensatory changes were allowed in the simu- In the above decomposition, allelic differences in F1 hybrids are entirely attributed to cis-regulatory variation under the as- sumption that trans-regulatory factors should affect both alleles Author contributions: T.T.-S.-K. designed research; K.R.T., T.M., and T.T.-S.-K. performed research; T.M. and T.T.-S.-K. contributed new reagents/analytic tools; K.R.T. and T.T.-S.-K. equally. However, some types of cis-trans interactions can violate analyzed data; and K.R.T. and T.T.-S.-K. wrote the paper. this assumption by differently affecting the two alleles. For ex- The authors declare no conflict of interest. ample, by adapting to an evolving cis-regulatory element of the fi This article is a PNAS Direct Submission. G.G. is a guest editor invited by the Editorial same species, a transcription factor may bind less ef ciently with Board. the cis-regulatory element from the distinct species. In the ex- 1K.R.T. and T.T.-S.-K. contributed equally to this work. treme case where the binding between species does not occur at 2Present address: Department of Agricultural and Environmental Biology, University of all, an allelic difference in a hybrid would be similar to the pa- Tokyo, Tokyo 113-8657, Japan. rental difference as if there were only cis-regulatory variations. 3To whom correspondence should be addressed. E-mail: [email protected]. Thus, when incompatibilities between cis- and trans-regulatory This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. elements reduce heterospecific binding, the contribution of cis- 1073/pnas.1105814108/-/DCSupplemental. 15276e15281 | PNAS | September 13, 2011 | vol. 108 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1105814108 Downloaded by guest on September 27, 2021 0.06 By contrast, when a was held constant so that a–c compen- sation was prohibited, no increasing tendency of the linear re- Conspecific binding ) 0.05 gression was observed (Fig. 2, gray line), despite the action of K- K [T] compensation. In this case, the expression level as a function 0.04 of [T]/K was even larger in the hybrids (for which K is expected to 0.03 be uncorrelated with [T]) than in the parental strains (for which K is expected to be positively correlated with [T] under K-[T] 0.02 compensation). We also observed many cis × trans genes but an Heterospecific binding Binding affinity (1 / almost complete absence of the cis + trans genes (Fig. S2B). 0.01 One-Activator, Two-Target System. Many transcription factors 0.00 regulate the expression of multiple genes pleiotropically. This 0 10000 20000 30000 40000 50000 pleiotropic effect imposes strong selective constraints on both Time (in units of 1 / U generations) [T] and a, which may retard the accumulation of compensatory cis trans Fig. 1. Temporal changes in the binding affinity of the transcriptional acti- - changes. For example, when a transcriptional activator vator to its cis-regulatory element. Coevolutionary dynamics of the tran- affects multiple targets simultaneously, mutational changes in scriptional activator concentration [T] (as a function of f)(Simulation [T] or a that optimize the expression level of a target gene may Methods) and its dissociation constant K (as a function of a and c) were sim- deviate the expression levels of other targets away from their op- ulated for 10,000 pairs of orthologous genes. Mutation effects and rates per tima. Because of this potentially deleterious side effect, trans- ± ± ± generation are df and vf for f, da and va for a, and dc and vc for c,re- regulatory mutations have been considered to play only a limited spectively. The average binding affinities as quantified by the inverse of the role in the evolution of transcriptional regulation. fi dissociation constant (1/K) are illustrated for conspeci c (black) and hetero- To see how constraints on trans-regulatory changes affect the specific (gray) binding. Under