Centro de Estudo do Genoma Humano, Department Genetica e Biologia Evolutiva, Instituto de Biociencias/Universidade de São Paulo, Brazil.
Dev Biol. 2010 Jan 15;337(2):496-505. doi: 10.1016/j.ydbio.2009.10.028. Epub 2009 Nov 3.
Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short.
XVIII 型胶原是基底膜的组成部分，在眼睛、血管、肝脏和中枢神经系统中表达明显。COL18A1 基因的纯合突变导致诺布洛克综合征，其特征是眼部缺陷和枕骨脑膨出。然而，关于 XVIII 型胶原在发育中的作用，人们知之甚少，其组织特异性表达模式的调控也一无所知。我们利用斑马鱼转基因技术来鉴定和描述控制人类基因表达的顺式调控序列。候选增强子是根据哺乳动物之间的序列保守性，从与 COL18A1 相关的非编码序列中选择的。尽管这些序列与同源的斑马鱼序列没有明显的保守性，但其中的四个区域在斑马鱼胚胎中仍作为组织特异性转录增强子发挥作用，并共同再现了 col18a1 表达的主要方面。对阳性增强子序列的额外事后计算分析揭示了哺乳动物和硬骨鱼序列之间的排列，我们假设这些排列预测了相应的斑马鱼增强子；对于其中一个，我们证明了与同源人类增强子序列的功能重叠。我们的研究结果为 COL18A1 的生物学功能和调控提供了重要的见解，并指出了其他可能与涉及 COL18A1 的复杂疾病有关的序列。更广泛地说，我们表明，将功能数据与针对系统发育保守性的靶向分析相结合，可以揭示大量情况下保守的顺式调控元件，而仅通过计算比对则无法实现。
