How to tune an enhancer.

Every cell’s genome contains two main classes of functional DNA. The best understood type of DNA sequence, which was also the first to be discovered, is that which encodes RNA and protein products via the near-universal “genetic code” (1). A more mysterious but equally important class of functional DNA is cisregulatory sequence, which does not have a physical product but, instead, encodes the conditions under which a particular RNAwill be produced.Cis-regulatory DNA sequences are the primary (although not the only) determinant of gene expression: Not only the rate of RNA production but also the timing, spatial patterning, and environmental control of every gene’s activity are largely controlled by these DNA sequences, which are usually in the general vicinity of the gene they regulate. These DNA segments (often called enhancers) have no enzymatic activity on their own, but act as scaffolds for large complexes of proteins and RNAs that directly control the activity of a gene’s promoter, sometimes over distances of 1 million base pairs or more (2). Transcription factors (TFs), key protein regulators of gene expression, bind DNA in a sequence-specific manner, which means that the nature of the complex assembled at a given enhancer at a given time depends on its DNA sequence (cis information), in conjunction with the set of TFs present and active in the cell at that time (trans information). The chromatin state of regulatory DNA is also a very important factor, but local chromatin states are also specified, indirectly, by DNA sequence, because most chromatin-modifying enzymes are recruited to the genome by sequence-specific factors (3). In part, the DNA sequence of an enhancer encodes a pattern of gene expression through the combinations of sequence-specific regulators that bind to it (and the non–DNA-binding factors they recruit, in turn). However, cis-regulatory DNA sequences do more than merely determine which combinations of TFs will be recruited to an enhancer. The linear arrangement of binding sites (sometimes called “grammar” or “syntax”) can play an important role in controlling enhancer output, especially by setting thresholds for gene activation (4–7). 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 (8–16). 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. The authors of this study build on the results of their recently published high-throughput screen (14) in which hundreds of thousands of short DNA sequences were tested for enhancer activity in transfected Ciona embryos. The library of tested DNAs were all variants of Otx-a, a 69-bp neural enhancer of the Ciona Otx gene (18), in which the 4-bp cores of five binding sites for GATA and ETS transcription factors were preserved but all other base positions were randomized. One synthetic enhancer variant drove strong expression of a GFP reporter gene in the notochord, a tissue that does not normally express Otx. Sequence analysis suggested two likely explanations for the ectopic expression pattern: a new ETS motif with high predicted binding affinity or a new site predicted to bind the transcription factor ZicL. Both ZicL and an ascidian ETS ortholog are expressed in the notochord. DNA stiffness, TF-induced bending prior chromatin state binding site orientation binding site spacing