Identification of novel Pax-2 binding sites by chromatin precipitation
Identification of novel Pax-2 binding sites by chromatin precipitation
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DOI：
10.1074/jbc.271.14.7978
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发表时间：
1996-04-05
影响因子：
4.8
通讯作者：
Dressler, GR
中科院分区：
文献类型：
--
作者：
Phelps, DE;Dressler, GR
Pax基因编码一个发育转录因子家族，其通过配对结构域与特定的DNA序列结合，并且是多种组织的形态发生所必需的。小鼠Pax-2基因通过选择性剪接编码两种核蛋白Pax-2A和Pax-2B，其在特定神经细胞类型的分化和早期肾脏形成期间瞬时表达。为了在体内鉴定潜在的Pax-2靶序列，用Pax-2特异性抗体免疫沉淀胚胎神经管的染色质并克隆。两个独特的免疫沉淀克隆含有三个特定的Pax-2结合位点的功能结合试验，使用Pax-2蛋白在大肠杆菌和真核细胞中产生的确定。在体外DNA结合试验中，使用Pax-5和Pax-8 DNA识别序列以及三个免疫纯化的Pax-2结合位点，证明了两种形式的Pax-2蛋白以相似的特异性结合DNA，并且这种结合由配对结构域介导。本报告中鉴定的结合位点在它们之间以及与先前定义的Pax-5和Pax-2的共有序列之间具有显著的同源性。基因组克隆现在可以用作序列标签来鉴定潜在的靶基因座。
The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues, The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.