Automated Yeast Two-hybrid Screening for Nuclear Receptor-interacting Proteins

Abstract

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors. High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors. Nuclear receptors are a family of transcription factors involved in the control of many physiological processes including development, sexual differentiation, inflammation, and metabolism (1Giguere V. Orphan nuclear receptors: from gene to function..Endocr. Rev. 1999; 20: 689-725Google Scholar, 2Schwabe J.W. Teichmann S.A. Nuclear receptors: the evolution of diversity.Sci. STKE. 2004; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;stke.2172004pe4Google Scholar). They can bind to DNA directly or via interaction with other proteins. Nuclear receptor activity is regulated by the binding of small molecule ligands to the receptor and/or by posttranslational modifications. Activation of nuclear receptors involves a change in conformation that affects the interaction of the receptor with other proteins, which in turn brings about the effect of the receptor on gene expression (3Baek S.H. Rosenfeld M.G. Nuclear receptor coregulators: their modification codes and regulatory mechanism by translocation..Biochem. Biophys. Res. Commun. 2004; 319: 707-714Google Scholar, 4McKenna N.J. O’Malley B.W. Combinatorial control of gene expression by nuclear receptors and coregulators..Cell. 2002; 108: 465-474Google Scholar). Knowledge about the ligand-dependent binding of nuclear receptors to their cofactors is central to the understanding of their physiological function and their use as targets for drug discovery (5Smith C.L. O’Malley B.W. Coregulator function: a key to understanding tissue specificity of selective receptor modulators..Endocr. Rev. 2004; 25: 45-71Google Scholar). However, the available knowledge is highly biased toward a few intensively studied receptors, and little is known for the potential interaction patterns of the rest of the family (6Albert S. Gaudan S. Knigge H. Raetsch A. Delgado A. Huhse B. Kirsch H. Albers