title: 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.
authors.affiliation: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; ekfarley@princeton.edu msl2@princeton.edu.
authors.forename: Emma K
authors.identifier: 0000-0002-9241-247X
authors.initials: EK
authors.lastname: Farley
authors.affiliation: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544;
authors.forename: Katrina M
authors.initials: KM
authors.lastname: Olson
authors.affiliation: Department of Medicine, University of California, San Diego, CA 92093-0688;
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development, Center for Integrative Genomics, University of California, Berkeley, CA 94720-3200; Innovative Genomics Initiative, University of California, Berkeley, CA 94720.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Rokhsar
authors.affiliation: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; ekfarley@princeton.edu msl2@princeton.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Levine
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002938:Ciona intestinalis / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D009672:Notochord / Q000378:metabolism; D009928:Organ Specificity; D014157:Transcription Factors / Q000235:genetics
chemical_list: D014157:Transcription Factors
keywords: enhancer; enhancer grammar; gene regulation; regulatory principles; transcription