MRC Human Genetics Unit at the MRC IGMM at the University of Edinburgh, Edinburgh EH4 2XU, United Kingdom.
Institute of Molecular and Cell Biology, Agency for Science Technology and Research (A⁎STAR), Biopolis, Singapore 138673, Singapore.
Dev Biol. 2014 Mar 15;387(2):214-28. doi: 10.1016/j.ydbio.2014.01.007. Epub 2014 Jan 17.
Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.
细胞类型和发育过程之间的生物学差异的特征在于基因表达谱的差异。基因远端增强子是指定组织特异性表达模式驱动胚胎发育和细胞命运决定的调控网络的关键组成部分，其序列的变异是遗传疾病和疾病易感性的主要原因。尽管在发现推定顺式调控序列的方法上取得了进展，但在哺乳动物模型系统中对其时空增强子活性进行特征描述仍然是一个主要的瓶颈。我们采用了一种策略，该策略结合了有颌类动物序列保守性以及转基因小鼠和斑马鱼报告基因检测，来调查发育控制基因 PAX6 的基因组位点是否存在新的顺式调控元件。人类和软骨板鳃鱼（Callorhinchus milii）之间的序列比较揭示了几个古老的有颌类动物保守非编码元件（agCNEs）广泛分布在 PAX6 基因座中，将已知的 PAX6 顺式调控景观的范围扩展到包含完整的上游 PAX6-RCN1 基因间区。我们的数据表明，即使在斑马鱼中不保守，古老的保守调控序列也可以在转基因斑马鱼中进行有效的测试。该策略还允许对以前在转基因小鼠中评估的复合调控区域进行有效的剖析。由一组 agCNEs 驱动的表达模式的显著重叠表明 PAX6 位于多个组织特异性调控群岛的景观中。
