Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers

Abstract

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.enhancer | gene regulation | transcription | enhancer grammar | regulatory principles P revious studies have highlighted the importance of sequence constraints within developmental enhancers for tissue-specific patterns of gene expression in both Drosophila and Ciona embryos (1-6). For example, the 69-bp orthodenticle homeobox (Otx)-a enhancer mediates restricted expression in the Ciona neural plate in response to pleiotropic fibroblast growth factor (FGF) signaling (7-9). Specificity depends on a series of low-affinity binding sites for the transcription factors ETS (FGF signaling) and GATA (ectoderm determinant) (2). Modification of these sites to improve their binding affinities resulted in augmented levels of gene expression in the neural plate, as well as ectopic expression in additional tissues that respond to FGF signaling (2).These observations prompted the suggestion that the evolution of developmental enhancers depends on the selection of submaximal binding sites. This "suboptimization" might also apply to the organization of enhancers, as changing the spacing of neighboring GATA and ETS binding sites resulted in a significant increase in enhancer activity (2). Modified Otx-a enhancers containing both optimal binding sites and optimal spacing of neighboring sites mediated intense expression in a variety of tissues responding to FGF, including the neural plate, notochord, an...

Mutational analysis of a Drosophila neuroblast enhancer governing nubbin expression during CNS development

“…A second study has shown that an enhancer of the Drosophila Suppressor of Hairless gene is composed of overlapping elements termed submodules that can function independently to activate enhancer activity (Liu & Posakony, 2014). In addition, a recent study of a notochord enhancer structure in Ciona points to the importance of TF binding site affinity and arrangement in conferring tissue specificity on enhancer function (Farley, Olson, Zhang, Rokhsar, & Levine, 2016; reviewed by Barolo, 2016; Crocker, Noon, & Stern, 2016). The presence of repeat sequence motifs in these enhancers, and their conserved positioning, points to the necessity of considering binding site position and TF avidity (Levo & Segal, 2014; Sayal, Dresch, Pushel, Taylor, & Arnosti, 2016).…”

How to tune an enhancer

“…In addition, the affinity with which a TF binds to its DNA site, which is controlled, in part, by the particular sequence of that binding motif, is increasingly recognized as a crucial factor in both the quantitative and qualitative control of gene expression (8)(9)(10)(11)(12)(13)(14)(15)(16). In PNAS, Farley et al (17) investigate how the binding affinity and the spatial arrangement of TF binding sites within enhancers interact to encode precise patterns of gene expression in developing embryos.…”

“…Within the wild-type Otx-a enhancer, certain ETS and GATA sites have relatively poor predicted binding affinity but optimal pairwise spacing, whereas other sites have higher affinity but suboptimal spacing (14). The discovery of a synthetic Otx-a variant with ectopic notochord activity, and comparisons against other synthetic modules with slightly different compositions, suggest that similar trade-offs can modulate gene expression in the notochord (17). This observation led Farley et al (17) to ask an intriguing question: Can we use what we have learned about constraints on TF binding site identity, affinity, and arrangement to find new notochord enhancers in the Ciona genome, using sequence data alone?…”

“…The discovery of a synthetic Otx-a variant with ectopic notochord activity, and comparisons against other synthetic modules with slightly different compositions, suggest that similar trade-offs can modulate gene expression in the notochord (17). This observation led Farley et al (17) to ask an intriguing question: Can we use what we have learned about constraints on TF binding site identity, affinity, and arrangement to find new notochord enhancers in the Ciona genome, using sequence data alone? By first screening for genomic regions containing combinations of ETS and ZicL sites, and then focusing on those clusters with strong binding sites or optimal syntax, Farley et al (17) identify two new enhancers of the notochord-expressed genes Mnx and Brachyury.…”

“…This observation led Farley et al (17) to ask an intriguing question: Can we use what we have learned about constraints on TF binding site identity, affinity, and arrangement to find new notochord enhancers in the Ciona genome, using sequence data alone? By first screening for genomic regions containing combinations of ETS and ZicL sites, and then focusing on those clusters with strong binding sites or optimal syntax, Farley et al (17) identify two new enhancers of the notochord-expressed genes Mnx and Brachyury. Importantly, these tissue-restricted enhancers have either high-affinity ETS/ZicL sites or optimal spacing, but not both: Optimizing both affinity and syntax causes ectopic expression (17).…”