A gateway-compatible yeast one-hybrid system
A gateway-compatible yeast one-hybrid system
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DOI：
10.1101/gr.2445504
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发表时间：
2004-10-01
期刊：
影响因子：
7
通讯作者：
Walhout, AJM
中科院分区：
文献类型：
--
作者：
Deplancke, B;Dupuy, D;Walhout, AJM
自从微阵列出现以来，已经产生了大量的基因表达数据。然而，这些微阵列数据未能揭示差异基因表达的转录调控机制，因为负责的转录因子（TF）的身份往往不能直接从这样的数据集推断。调节性TF通过与通常位于基因启动子中的顺式调节元件结合来激活或抑制其靶基因的转录。为了理解差异基因表达的机制，有必要确定调节性转录因子与其靶基因之间的物理相互作用。我们开发了3个网关兼容的酵母单杂交（Y1 H）系统，该系统能够使用两个小的（即，感兴趣的DNA元件）和大的（即，基因启动子）DNA片段。我们使用了四个良好的特点秀丽隐杆线虫启动子作为DNA诱饵来测试这个Y1 H系统的功能。我们可以检测到类似于40%以前报道的TF-启动子相互作用。通过使用两个互补文库进行筛选，我们发现了每个启动子的新的潜在相互作用的TF。我们概括了几个Y1 H为基础的蛋白质-DNA相互作用，在哺乳动物细胞中使用liciferase报告分析。总之，网关兼容的Y1 H系统将允许高通量鉴定蛋白质-DNA相互作用，并可能是一个有价值的工具，破译转录调控网络。
Since the advent of microarrays, vast amounts of gene expression data have been generated. However, these, microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets. Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter. To understand the mechanisms underlying differential gene expressior, it is necessary to identify physical interactions between regulatory TFs and their target genes. We developed 3 Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments. We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system. We could detect similar to40% of previously reported TF-promoter interactions. By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter. We recapitulated several of the Y1H-based protein-DNA interactions using liciferase reporter assays in mammalian cells. Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.