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

Nat Methods. 2017 Aug;14(8):819-825. doi: 10.1038/nmeth.4343. Epub 2017 Jun 26.

Abstract

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.

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • High-Throughput Nucleotide Sequencing / methods*
  • Protein Interaction Mapping / methods*
  • Proteome / genetics
  • Proteome / metabolism*
  • Sequence Analysis, DNA
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Two-Hybrid System Techniques*

Substances

  • Proteome
  • Transcription Factors