Deplancke Bart, Vermeirssen Vanessa, Arda H Efsun, Martinez Natalia J, Walhout Albertha J M
Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
CSH Protoc. 2006 Oct 1;2006(5):pdb.prot4590. doi: 10.1101/pdb.prot4590.
INTRODUCTIONProtein-DNA interactions (PDIs) between transcription factors (TFs) and their target genes form the backbone of transcription regulatory networks. Such PDIs can be identified with either a TF or a gene as a starting point. The Gateway-compatible yeast one-hybrid (Y1H) system provides a high-throughput, gene-centered method for the identification of interactions between a "DNA bait" (e.g., cis-regulatory DNA elements or gene promoters) and "protein preys" (e.g., TFs). The Y1H system is a genetic system to detect PDIs based on selection of reporter gene expression in yeast. DNA baits are fused by Gateway cloning to two reporter genes, HIS3 and lacZ, and the resulting DNA bait::reporter constructs are subsequently integrated into the genome of the host yeast strain. After integration, baits are examined for self-activation (i.e., their ability to drive reporter gene expression in the absence of an exogenous prey protein). Subsequently, each DNA bait is screened for interacting proteins by transforming a library of preys into the corresponding Y1H DNA bait yeast strain. Preys are hybrid proteins composed of a protein from the organism of interest and a heterologous transcription activation domain. When a prey protein binds to the DNA bait, the heterologous activation domain activates reporter gene expression. Thus, physical interactions between both repressors and activators and their DNA targets can be identified.
引言
转录因子（TFs）与其靶基因之间的蛋白质 - DNA 相互作用（PDIs）构成了转录调控网络的核心。此类蛋白质 - DNA 相互作用可以以转录因子或基因作为起点来进行识别。Gateway 兼容的酵母单杂交（Y1H）系统提供了一种以基因为中心的高通量方法，用于鉴定“DNA 诱饵”（例如顺式调控 DNA 元件或基因启动子）与“蛋白质猎物”（例如转录因子）之间的相互作用。Y1H 系统是一种基于在酵母中选择报告基因表达来检测蛋白质 - DNA 相互作用的遗传系统。DNA 诱饵通过 Gateway 克隆与两个报告基因 HIS3 和 lacZ 融合，然后将所得的 DNA 诱饵::报告基因构建体整合到宿主酵母菌株的基因组中。整合后，检测诱饵的自激活情况（即它们在没有外源猎物蛋白时驱动报告基因表达的能力）。随后，通过将猎物文库转化到相应的 Y1H DNA 诱饵酵母菌株中，对每个 DNA 诱饵进行相互作用蛋白的筛选。猎物是由感兴趣生物体的一种蛋白质和一个异源转录激活域组成的杂交蛋白。当猎物蛋白与 DNA 诱饵结合时，异源激活域会激活报告基因的表达。因此，阻遏物和激活物与其 DNA 靶标之间的物理相互作用都可以被识别出来。
