Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers

Abstract

The clustering of transcription factor binding sites in developmental enhancers and the apparent preferential conservation of clustered sites have been widely interpreted as proof that spatially constrained physical interactions between transcription factors are required for regulatory function. However, we show here that selection on the composition of enhancers alone, and not their internal structure, leads to the accumulation of clustered sites with evolutionary dynamics that suggest they are preferentially conserved. We simulated the evolution of idealized enhancers from Drosophila melanogaster constrained to contain only a minimum number of binding sites for one or more factors. Under this constraint, mutations that destroy an existing binding site are tolerated only if a compensating site has emerged elsewhere in the enhancer. Overlapping sites, such as those frequently observed for the activator Bicoid and repressor Krüppel, had significantly longer evolutionary half-lives than isolated sites for the same factors. This leads to a substantially higher density of overlapping sites than expected by chance and the appearance that such sites are preferentially conserved. Because D. melanogaster (like many other species) has a bias for deletions over insertions, sites tended to become closer together over time, leading to an overall clustering of sites in the absence of any selection for clustered sites. Since this effect is strongest for the oldest sites, clustered sites also incorrectly appear to be preferentially conserved. Following speciation, sites tend to be closer together in all descendent species than in their common ancestors, violating the common assumption that shared features of species' genomes reflect their ancestral state. Finally, we show that selection on binding site composition alone recapitulates the observed number of overlapping and closely neighboring sites in real D. melanogaster enhancers. Thus, this study calls into question the common practice of inferring “cis-regulatory grammars” from the organization and evolutionary dynamics of developmental enhancers.

Neutral forces acting on intragenomic variability shape the Escherichia coli regulatory network topology

“…Within a promoter, complex binding-site patterns like clustering, which were once thought to be adaptive, may in fact emerge from neutral evolution (23). At the subgraph level, commonly accepted motifs are poorly conserved across homologous genes (24); thus, convergent evolution of subgraphs (24, 25) is more simply explained as the result of neutral forces acting similarly in divergent species rather than as an adaptive response to environmental changes.…”

De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

“…In this context, we found that loss of a TATA box at enhancer iHMRs predicts loss of hypomethylation in chimp, suggesting a role for TBP and the transcriptional machinery, in addition to the function of other specific TFs, in the establishment of these iHMRs. These comparisons may thus serve to distinguish those enhancer iHMRs that fit an ''enhanceosome'' model, whereby exclusion of a single binding event can wipe out enhancer function in one species (possibly by loss of hypomethylation), or a ''billboard'' model in which bindingsite rearrangements within the enhancer are tolerated as long as the sum of TF interaction is constant (Arnosti and Kulkarni 2005;Lusk and Eisen 2010).…”

Changes in Selective Effects Over Time Facilitate Turnover of Enhancer Sequences

“…In one study to focus on the effects of purifying selection and regulatory function, Lusk and Eisen (2010) simulated enhancer evolution using binding site composition as the measure of fitness. They considered the case in which having too few TF binding sites is lethal and all alleles above the threshold number of binding sites are functionally equivalent (Lusk and Eisen 2010).…”

“…They considered the case in which having too few TF binding sites is lethal and all alleles above the threshold number of binding sites are functionally equivalent (Lusk and Eisen 2010). The authors required a binary classification of sequence into what is or is not a binding site.…”

“…Accordingly, my model allows the nature of turnover to be an emergent property not tied to a priori architectural requirements; for example, I do not necessarily view turnover as a two-step process, in which a new binding site arises, obviating an existing one. Such twostep models are forced to assume functional equivalency of alleles with redundant binding sites (Lusk and Eisen 2010) or to assume an arbitrary selection coefficient for the intermediate form (Durrett and Schmidt 2008). Binding-site-oriented models may portray the turnover process unrealistically because, in a population setting, even a fewfold variation in a selection coefficient can be the difference between having an appreciable or vanishingly small fixation probability.…”