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

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.

Authors:
 [1];  [2];  [2];  [2];  [2];  [2];  [3];  [3];  [3];  [2];  [2];  [3];  [4]
  1. Salk Inst. for Biological Studies, La Jolla, CA (United States). Genomic Analysis and Plant Biology Lab.; Univ. of California, San Diego, CA (United States). Division of Biological Sciences
  2. Salk Inst. for Biological Studies, La Jolla, CA (United States). Genomic Analysis and Plant Biology Lab.
  3. Salk Inst. for Biological Studies, La Jolla, CA (United States). Genomic Analysis and Plant Biology Lab.; National Inst. for Agricultural Research, Paris (France)
  4. Salk Inst. for Biological Studies, La Jolla, CA (United States). Genomic Analysis and Plant Biology Lab., and Howard Hughes Medical Inst.; Univ. of California, San Diego, CA (United States). Division of Biological Sciences
Publication Date:
Research Org.:
Salk Inst. for Biological Studies, La Jolla, CA (United States)
Sponsoring Org.:
USDOE; National Science Foundation (NSF)
OSTI Identifier:
1473848
Grant/Contract Number:  
SC0007078; IOS-1650227; IOS1456950; IOS1546873; DGE-1650112
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Methods
Additional Journal Information:
Journal Volume: 14; Journal Issue: 8; Journal ID: ISSN 1548-7091
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Trigg, Shelly A., Garza, Renee M., MacWilliams, Andrew, Nery, Joseph R., Bartlett, Anna, Castanon, Rosa, Goubil, Adeline, Feeney, Joseph, O'Malley, Ronan, Huang, Shao-shan Carol, Zhang, Zhuzhu Z., Galli, Mary, and Ecker, Joseph R. CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping. United States: N. p., 2017. Web. doi:10.1038/NMETH.4343.
Trigg, Shelly A., Garza, Renee M., MacWilliams, Andrew, Nery, Joseph R., Bartlett, Anna, Castanon, Rosa, Goubil, Adeline, Feeney, Joseph, O'Malley, Ronan, Huang, Shao-shan Carol, Zhang, Zhuzhu Z., Galli, Mary, & Ecker, Joseph R. CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping. United States. doi:10.1038/NMETH.4343.
Trigg, Shelly A., Garza, Renee M., MacWilliams, Andrew, Nery, Joseph R., Bartlett, Anna, Castanon, Rosa, Goubil, Adeline, Feeney, Joseph, O'Malley, Ronan, Huang, Shao-shan Carol, Zhang, Zhuzhu Z., Galli, Mary, and Ecker, Joseph R. Mon . "CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping". United States. doi:10.1038/NMETH.4343. https://www.osti.gov/servlets/purl/1473848.
@article{osti_1473848,
title = {CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping},
author = {Trigg, Shelly A. and Garza, Renee M. and MacWilliams, Andrew and Nery, Joseph R. and Bartlett, Anna and Castanon, Rosa and Goubil, Adeline and Feeney, Joseph and O'Malley, Ronan and Huang, Shao-shan Carol and Zhang, Zhuzhu Z. and Galli, Mary and Ecker, Joseph R.},
abstractNote = {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.},
doi = {10.1038/NMETH.4343},
journal = {Nature Methods},
number = 8,
volume = 14,
place = {United States},
year = {Mon Jun 26 00:00:00 EDT 2017},
month = {Mon Jun 26 00:00:00 EDT 2017}
}

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Works referenced in this record:

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome
journal, April 1995