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.

Highlights

  • Multi-species conserved non-coding elements occur in the vertebrate genome and are clustered often within large gene deserts in the vicinity of developmentally regulated genes (Boffelli et al, 2004; Woolfe et al, 2005)

  • The point mutations re-direct spatial activity Because the gain-of-function hemimelic extra toes (Hx) mutation lies within the 3′ half of the ZRS, we examined the ability of this mutation to impart autonomous ectopic activity

  • We showed that the conserved DNA sequence that constitutes the ZRS highlights essential interacting regulatory elements

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Summary

Introduction

Multi-species conserved non-coding elements occur in the vertebrate genome and are clustered often within large gene deserts in the vicinity of developmentally regulated genes (Boffelli et al, 2004; Woolfe et al, 2005). A highly conserved 780-bp element called the ZRS is an example of this class of cis-regulator. ZRS sits in an intron of the ubiquitously expressed Lmbr gene and from here it operates over a distance of 1 Mb of DNA to control precisely the spatiotemporal expression of the Shh gene in both the fore and hind limbs (Lettice et al, 2002, 2003; Sagai et al, 2005). SHH is a morphogen that is produced in a single, restricted domain lying at the posterior margin of the developing limb bud called the zone of polarising activity (ZPA).

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