Development of five digits is controlled by a bipartite long-range cis-regulator

Abstract

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.

HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme

“…These results establish the Ets1 and Ets2 genes as bona fide HAND2 target genes in early limb buds. As the ETS binding sites in the ZRS are essential for Shh expression in the posterior limb bud (Lettice et al, 2014; Lettice et al, 2012), HAND2-mediated up-regulation of the Ets transcription factors likely helps to reinforce Shh expression.…”

“…Localized Shh expression depends on the interaction of different transcriptional regulators with the ZRS. In particular, the interaction with HOX, PBX, ETS and HAND2 transcriptional complexes has been implicated in activation of Shh in the limb bud, while TWIST1, ETV and GATA factors prevent anterior ectopic expression (Capellini et al, 2006; Galli et al, 2010; Kmita et al, 2005; Kozhemyakina et al, 2014; Lettice et al, 2014; Lettice et al, 2012; Mao et al, 2009; Zhang et al, 2010; Zhang et al, 2009). How the ZRS integrates these various inputs over time is unknown, but the resulting posterior restriction of SHH signaling is essential for proliferative expansion and anterior-posterior (AP) patterning of the distal limb bud mesenchyme that will form the skeletal elements of the zeugopod and autopod (Ahn and Joyner, 2004; Chiang et al, 2001; Harfe et al, 2004; Zhu et al, 2008).…”

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

“…3C analysis revealed that Shh activation by the ZRS correlates with physical contacts between them (54), suggesting a looping of the chromatin path between the enhancer and Shh. A study that combined genetic manipulation of the enhancer locus, transgenics, and 3D-FISH showed that while the 5= end of the ϳ800-bp ZRS is sufficient to drive an adjacent reporter gene, the 3= end of the enhancer is required for long-range regulation and full expression of Shh in the developing forelimbs and hind limbs and, consequently, complete digit sets (199). The Shh locus was further observed by 3D-FISH to loop out of its chromosome territory when the gene is active (54).…”

Gene regulation at a distance: From remote enhancers to 3D regulatory ensembles

“…Interestingly, the Shh remote limb enhancer comprises a region required for long-but not short-range activation of gene expression [134], suggesting that different factors bound to an enhancer may be involved in its activity and its ability to maintain interactions.…”