The Conserved Sonic Hedgehog Limb Enhancer Consists of Discrete Functional Elements that Regulate Precise Spatial Expression

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe basis of embryonic development lies in the spatiotemporal control of gene expression, which is mediated by remote cis-regulatory elements. These cis-acting elements, or enhancers, are fundamental to evolution and disease. Despite these important roles, major unanswered questions remain about the information encoded by the enhancer sequence and the importance of the overall structural architecture to enhancer activity. One class of enhancers that operates during embryogenesis are those that are highly conserved, acting at long distances from their target genes (). Here we focus on a highly conserved element called the zone of polarizing activity regulatory sequence (ZRS) that is responsible for the spatiotemporal expression of Shh during limb bud development (, ) and is essential for specifying digit identity and number. This enhancer is ∼770 bp in length and shows a high degree of similarity in vertebrates across a lengthy evolutionary timescale, including sharks and rays (), and, in accord, the mouse shows >70% similarity with the coelacanth (lobe-finned fish) sequence (). Hence, the ZRS has remained highly invariant against a backdrop of major evolutionary changes to the anatomy of the appendicular skeleton, which includes the transition of fish fins to tetrapod limbs (). The structural organization of this class of deeply conserved vertebrate enhancers is under strong selective constraints, and, even in light of binding site redundancies exhibited by transcription factors, few sequence changes are present.The ZRS is located 800–1,000 kb away from the Shh promoter in the mouse and human and is necessary and sufficient for accurately activating and maintaining Shh expression in the limb (, ). An enhancer evolves not simply as a regulator that switches gene expression on or off but must also solve the challenges of regulating expression from a distance () while controlling gene activity accurately in space and time and at the appropriate levels. Based on the evolutionary stasis of the ZRS, it is reasonable to expect that the sequence was finely honed during evolution so that that there is little tolerance for sequence change. Indeed, point mutations in and duplications of the ZRS result in a spectrum of appendicular skeletal defects (). Point mutations in well over 20 different positions scattered across the ZRS cause autosomal dominant limb defects, called “ZRS-associated syndromes” (). Some of the conditions associated with ZRS mutations include preaxial polydactyly type 2, triphalangeal thumb polysyndactyly, syndactyly type 4, and Werner mesomelic syndrome (WMS).To investigate the structural composition of this highly conserved vertebrate enhancer, we used genome editing technology () to target deletions in three regions within the ZRS. Because ZRS activity is limb-specific, the phenotypes were expected to be overt, accessible, and nonlethal. The regions that were targeted contain the 5-bp site responsible for WMS (), the single mutation responsible for hemimelic extra toes (Hx) () in the mouse, and a previously identified site for binding the HAND2 transcription factor (). This approach generated an overlapping series of mutations and deletions that scan across 250 bp of the endogenous ZRS. Here we show that the ZRS encodes multiple, diverse functions that contribute to the enhancer activity. Spatial restriction of expression is, in part, controlled by a small repressor domain that confines Shh expression to the posterior limb bud margin. In contrast, large overlapping domains regulate expression levels contingent on the number of HOXD binding sites. In addition, in response to insertion mutations, cryptic, unique phenotypes were generated that revealed the functional plasticity potentially encoded in an enhancer. Mutational analysis, however, also showed that, even though the enhancer is highly conserved, it could still tolerate quite substantial losses of sequence information without causing an abnormal phenotype. We propose a collective model for enhancer composition in which discrete activities and redundant sequences in the ZRS accrue to provide a robust regulatory response during development.