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.

Figure 1

CrY2H-seq strain and plasmid design. (a) CrY2H-seq uses yeast strains CRY8930 and Y8800. (b) CrY2H-seq bait and prey plasmids pDBlox and pADlox contain mutant lox sites (lox66 and lox71, respectively) flanking the 3’ end of ORF inserts. Upon Cre/lox-recombination of plasmids, a fused ORF product can be recovered by PCR amplification using activation (AD) and DNA binding (DB) domain specific primers, indicated by the grey arrows. (c) Representative PCR amplicon from AD and DB primers showing fused ORFs. Mutant lox sites are underlined.

Figure 2

The CrY2H-seq screening pipeline. On day 1, archival stocks of bait and prey libraries are combined in one massively-multiplexed mate culture that undergoes diploid selection overnight. On day 2, the diploid culture is plated on media to select for cells with protein interaction-mediated Gal4 reconstitution and subsequent transcriptional activation of the HIS3 and CRE reporter genes. HIS3 expression allows cells to survive on selection media and CRE expression permits unidirectional plasmid linkage, where ORF combinations corresponding to protein-protein interactions become fixed together inside cells. After 3 days of selection, surviving cells are harvested en masse, plasmids are purified in a single prep, and Cre-recombined ORF junctions are amplified in multi-template PCR reactions. From these amplicons, an Illumina sequencing library is prepared and sequenced. A bioinformatics pipeline is used to identify fragments derived from Cre recombination PCR products (see Supplementary Fig. 5 and Online Methods for more details, including media composition).

Figure 3

Coverage of AtTFIN-1. (a) Summary of TF ORFeome screening. (b) Cumulative coverage of unique interacting pairs detected in paired-end sequencing of all ten CrY2H-seq screens after self-activator removal. (c) Sampling sensitivity shown by the average number of new interactions detected after each CrY2H-seq screen considering all possible (10!) orderings of screens. Error bars, standard deviation.

Figure 4

Quality of AtTFIN-1. (a) Fraction of AtTFIN-1 protein-protein interactions (PPIs) that were positive in 1×1 matrix style Y2H retest screen (retest rate) as a function of the number of CrY2H-seq screens that interactions were observed in. Bin sizes, 1–3: 65, 4–6: 342, and 7–10: 249. (b) Fraction of AtTFIN-1 PPIs that were positive in wNAPPA. Error bars, standard error of proportion. P values, one-sided Fisher’s exact test (*** = 3.57e-08, and * = 0.002395). (c) Fraction of 1,368 BioGRID, 1,198 STRING, 1,355 AraNet, 182 Arabidopsis Interactome-1 (AI-1), 501 Arabidopsis Interactome literature-curated interactions (LCI), and 8,577 random interactions in AtTFIN-1. Error bars, standard error of proportion. Literature and database interactions are detected significantly more often than random interactions (P values, one-sided Fisher’s exact test, * = 2.2e-16). (d) Precision-recall curve calculated using the union of known interactions as true positives and a random interaction dataset as false positives plotted as a function of the number of CrY2H-seq screens that interactions were observed in. Interactions observed in two or more replicate experiments are classified as high-confidence interactions as indicated by the pale blue box.

Figure 5

Biological functions underlying TF family interactions in AtTFIN-1. (a) Discrete empirical P values of family interactions observed more frequently in AtTFIN-1 than expected by random chance. Families are hierarchically clustered by common family interactions. Color key: ND = not detected, NS = not significant, * p<0.05, ** p< 0.01, *** p<0.001. Examples of known intra-family and inter-family dimers are highlighted in green and purple, respectively. See Supplementary Fig. 13 for a matrix showing all TF family interactions observed. (b) An ABI3-VP1/B3 transcription factor preferentially interacts with many members of TRIHELIX and GeBP families, a module potentially involved in gynoecium development. (c) GRAS family members preferentially interact with G2-like family members providing a potential molecular link between phosphate sensing and the regulation of root development. (d) Preferential interaction between BBX domain-containing “Orphans” proteins and C2C2-CO-like family members suggest a potential means by which stimulus signals are integrated with circadian rhythms.

Figure 6

An expanded ARF-AUX-IAA transcription factor network. Distinct interactions among AUX-IAA and ARF proteins suggest certain family members have specific functions. IAA17 shows preferential enrichment for TCP family members. IAA2, 10, 17, and 18 commonly interact with MBD proteins. IAA11 shows distinct interactions with hormone and water stress related factors, ERF70 and DRIP2. ARF18 specifically interacts with VAL1 and VAL2 abscisic acid response factors. IAA10 interacts with LOL2 and GEBP defense response-related factors.