3 years ago

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping
Adeline Goubil, Shao-shan C Huang, Anna Bartlett, Joseph Feeney, Andrew MacWilliams, Shelly A Trigg, Zhuzhu Z Zhang, Ronan O'Malley, Joseph R Ecker, Rosa Castanon, Renee M Garza, Mary Galli, Joseph R Nery
Broad-scale protein–protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq′s improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

Publisher URL: http://dx.doi.org/10.1038/nmeth.4343

DOI: 10.1038/nmeth.4343

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