Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis
Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis
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DOI：
10.1016/j.ydbio.2009.10.028
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发表时间：
2010-01-15
影响因子：
2.7
通讯作者：
Fisher, Shannon
中科院分区：
文献类型：
--
作者：
Kague, Erika;Bessling, Seneca L.;Fisher, Shannon
XVIII型胶原是基底膜的一种成分，在眼睛、血管、肝脏和中枢神经系统中显著表达。COL 18 A1的纯合突变导致Knobloch综合征，其特征为眼部缺陷和枕部脑膨出。然而，关于XVIII型胶原在发育中的作用的描述相对较少，并且关于其组织特异性表达模式的调节也一无所知。我们已经使用斑马鱼转基因识别和表征控制人类基因表达的顺式调控序列。基于哺乳动物之间的序列保守性，从与COL 18 A1相关的非编码序列中选择候选增强子。虽然这些显示没有明显的保护与orthopathic斑马鱼序列，四个区域仍然作为组织特异性的转录增强子在斑马鱼胚胎，并一起概括了col 18 a1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比对，我们假设预测相应的斑马鱼增强子;对于其中之一，我们证明了与orthopathic人类增强子序列的功能重叠。我们的研究结果为COL 18 A1的生物学功能和调控提供了重要的见解，并指出了可能导致涉及COL 18 A1的复杂疾病的其他序列。更一般地说，我们表明，结合功能数据与有针对性的分析系统发育保守可以揭示保守的顺式调控元件在大量的情况下，计算对齐单独福尔斯短。(C)2009 Elsevier Inc. All rights reserved.
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. (C) 2009 Elsevier Inc. All rights reserved.