MRC-Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK.
Development. 2014 Apr;141(8):1715-25. doi: 10.1242/dev.095430.
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.
内含子 DNA 中的保守性通常突出了远距离控制基因表达的调控元件。单个元件中的保守性与调控信息的关系以及内部组成与功能的关系尚不清楚。在这里，我们研究了高度保守的 ZRS（也称为 MFCS1）顺式调控因子的结构特征，该因子负责控制肢芽中 Shh 的时空表达。通过在转基因检测和内源性基因座中系统地剖析 ZRS，我们表明 ZRS 实际上由两个不同的活性域组成：一个域指导时空活性，但主要从短距离起作用，而第二个域则需要促进长距离活性。我们进一步表明，这两个域编码的活性高度整合，第二个域对于促进与基因活性相关的染色体构象变化至关重要。在肢芽发育过程中，ZRS 编码的这些活性在前后肢中被不同地解释；如果没有第二个域，前肢中就没有 Shh 活性，而在后肢中，Shh 的低水平会导致从两个到四个手指的变体数字模式。因此，在胚胎中，第二个域稳定了发育程序，为 SHH 形态发生素活性提供了缓冲，从而确保两组肢都形成五个手指。
