ICSB

2016 Poster

presented at:107-P-S

Evangelia Petsalaki DOI: 10.3252/pso.eu.17ICSB.2016

Large-Scale Networks

Nozomu Yachie1,2*, Evangelia Petsalaki1,2,* Joseph C. Mellor1,2, Yves Jacob3,4, Marta Verby1,2, Sedide B. Ozturk1,2, Siyang Li1,2, Atina G. Cote1,2, Roberto Mosca5, Jennifer J. Knapp1,2, Jochen Weile1,2,9, Minjeong Ko1,2, Analyn Yu1,2, Marinella Gebbia1,2, Nidhi Sahni4,6, Song Yi4,6, Tanya Tyagi1,2, Dayag Sheykhkarimli1,2,9, Jonathan F. Roth1,2,9, Cassandra Wong1,2, Louai Musa1,2, Jamie Snider1, Yi-Chun Liu1, Haiyuan Yu7, Pascal Braun4,6,8, Igor Stagljar1,9, Tong Hao4,6, Michael A.

Calderwood4,6, Laurence Pelletier2,9, Patrick Aloy5,10, David E. Hill4, Marc Vidal4,6 & Frederick P. Roth1,2,4,9,11,12

*These authors contributed equally to this work.

1Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. 2Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada. 3Unit of Genetics, Papillomavirus and Human Cancer, Pasteur Institute, Paris, France. 4Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 5Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain. 6Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 7Weill Institute for Cell and Mo-lecular Biology, Cornell University, Ithaca, New York, USA. 8Technische Universität München, Wissenschaftszentrum Weihenstephan, Department of Plant Systems Biology, Freising, Germany. 9Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 10Institució Catalana de Recerca i Estudis Avançats

(ICREA), Barcelona, Spain. 11Department of Computer Science, University of Toronto, Toronto, Ontario, Canada. 12Canadian Institute For Advanced Research (CIFAR)

Barcode-Fusion Genetics Yeast-two hybrid (BFG-Y2H): Towards a multidimensional protein interaction map.

AbstractProtein interaction networks are inherently multidimensional, e.g., they are regulated and dependent on environment and sequence variation. While current technologies for binary protein interactions discovery are highly efficient, the dramatic increase in the complexity of the query space may require new technology.

We developed Barcode-Fusion Genetic Yeast-two-Hybrid (BFG-Y2H) that uses DNA barcodes, Cre-Lox recombination and next generation sequencing, to increase the potential throughput of traditional Y2H, enabling routine parallel screens of millions of protein pairs in pools.

To assess the scalability we applied BFG-Y2H on 4 matrices of increasing size (2.3 x 104 to 5.6 x 106 protein interaction pairs). Using a calibration matrix within these sets we observed that BFG-Y2H performed similarly, regardless of matrix size indicating that it is scalable. Moreover we showed that our method performs comparably to state-of-the-art Y2H (Rolland et al, 2014, Cell) when evaluated against a literature curated protein interaction dataset.

We expect BFG-Y2H to open large-scale Y2H screens to labs that don't have extensive robotic facilities and resources, and to have a range of applications such as conditional interactome screening, Y2H screening of mutant proteins and isoforms, contributing to a better understanding of dynamic, multidimensional protein interactomes.

METHODS

Generation of Barcoded Reagents

FIGURE 1. Traditional yeast-two hybrid (Fields & Song, 1989) is a protein complementation assay which relies on the reconstitution of an

active transcription factor by the interaction of two proteins of interest X and Y fused to its DNA Binding (DB) and Activation Domain (AD)

components, thus activating the transcription of a selectable marker. In BFG-Y2H (Yachie & Petsalaki et al, 2016) the plasmids carrying

the DB-X and AD-Y fusion genes are tagged with a double barcode (UP-TAG and DN-TAG) flanked by site specific recombination sites.

TRADITIONAL YEAST-TWO HYBRID BARCODE FUSION GENETICS-YEAST-TWO HYBRID

BFG-Y2H Overview

FIGURE 3. In BFG-Y2H barcoded DB-X and AD-Y libraries are grown and mated en masse. After diploid cell enrichment and Y2H selection

the cells are treated with doxocyclin to activate the transcription of Cre recombinase, which then specifically recombines the DB-X UPTAG

with the AD-Y UPTAG and the DB-X DNTAG with the AD-Y DNTAG. These plasmids now hold the information of the X-Y pairs and are subse-

quently PCR-amplified at the barcode locus and sequenced using an Illumina platform. The read counts from the selective conditons are

normalized appropriately resulting in an interaction score for each X-Y pair.

FIGURE 2. Two approaches were used for the generation of barcoded DB-X and AD-Y plasmids.

A. In-yeast assembly (IYA;Ma et al 1987;Gibson et al 2009) B. En masse LR (EMLR)

B. EN MASSE LR (EMLR)A. IN-YEAST ASSEMBLY (IYA)

FIGURE 8. BFG-Y2H’s quantitative IS

score is indicative of number of inter-

face residue contacts and hence of

interaction strength

Results

bfg-y2h Performs similarly to state-of-the-art y2h

Acknowledgements

ReferencesFIELDS, S. & SONG, O. A NOVEL GENETIC SYSTEM TO DETECT PROTEIN-PROTEIN INTERACTIONS. NATURE 340, 245-246 (1989)

YACHIE, N.*, PETSALAKI, E.* ET AL, POOLED COMBINATORIAL PROTEIN INTERACTION SCREEN USING BARCODE FUSION GENETICS, MOL SYS BIOL 12(4):863 (2016)

MA, H., KUNES, S., SCHATZ, P.J. & BOTSTEIN, D. PLASMID CONSTRUCTION BY HOMOLOGOUS RECOMBINATION IN YEAST. GENE 58, 201-216 (1987).

GIBSON, D.G. SYNTHESIS OF DNA FRAGMENTS IN YEAST BY ONE-STEP ASSEMBLY OF OVERLAPPING OLIGONUCLEOTIDES. NUCLEIC ACIDS RES 37, 6984-6990 (2009).

CASSONNET, P. ET AL. BENCHMARKING A LUCIFERASE COMPLEMENTATION ASSAY FOR DETECTING PROTEIN COMPLEXES. NAT METHODS 8, 990-992 (2011).

ROLLAND, T. ET AL. A PROTEOME-SCALE MAP OF THE HUMAN INTERACTOME NETWORK. CELL 159, 1212-1226 (2014)

FU, J. ET AL. THE RNA-BINDING PROTEIN RBPMS1 REPRESSES AP-1 SIGNALING AND REGULATES BREAST CANCER CELL PROLIFERATION AND MIGRATION. BIOCHIM BIOPHYS ACTA 1853, 1-13 (2015).

.

BFG-Y2H is Reproducible and scalable

FIGURE 6. All BFG-Y2H screens, provide

consistent results when tested against

a calibration set, indicating scalability

This work was supported by the Canada Excellence Research Chairs Program , the Krembil Foundation, and US National Institutes of Health (NIH) grants HG001715 (M.Vidal, D.E.H and F.P.R.) and HG004233 (M.Vidal and F.P.R.). N.Y. was

supported by a JSPS fellowship (Research Abroad), Japan Society for the Promotion of Science, a Banting Postdoctoral Fellowship, National Sciences and Engineering Research Council of Canada, and PRESTO research grant by Japan Science

and Technology Agency (JST). L.P. was supported by Canadian Institutes of Health Research (CIHR) grants (MOP-123468 and MOP-130507).

BFG HITS NOVEL BFG HITS KNOWN NOT BFG HITS BUT KNOWN

FIGURE 7. BFG-Y2H networks discovered.

FIGURE 5. Interaction Score correlations

between huCENT BFG-Y2H screens

INTERNAL BARCODES DOUBLE BARCODESSAME CELL SAME SCREEN

DIFFERENT SCREEN

FIGURE 9. huCENT BFG-Y2H hits ranked by IS score. The higher scores show a clear enrich-

ment in known interactions. 49% and 79% tested pairs were confirmed by pairwise Y2H

and GPCA (Cassonnet et al, 2011), an orthogonal assay in human cells, respectively.

FIGURE 12. A. log(GPCA) scores for the huCENT top 100 hits. There is no significant diffe-

rence between the retest positive and the top 55 hits. BFG-Y2H hits that were found to

be autoactivators in the pairwise Y2H testing show a significant increase in GPCA score.

FIGURE 4. 4 spaces of increasing size were screened:

a) huCENT (2.3*10^4 pairs): centrosomal proteins

b) huCCC (1.5*10^5 pairs): cancer & cell cycle pro-

teins

c) huCV (3.5*10^5 pairs): viral host target proteins

d) huCVA (2.3*10^6): huCV plus random proteins.

IYA EMLR

FIGURE 10. We compared the performance of BFG-Y2H

to the state-of-the-art Y2H (Rolland et al, 2014) against

the literature-curated Lit-BM-13 dataset

FIGURE 13. The TGF pathway transcription

factor RBPMS interaction with SMAD3,

found in the huCCC screen, was shown (Fu

et al 2015) to play a role in cell growth and

migration inhibition in breast cancer cell

lines.

Conclusions Future Plans BFG-Y2H is a scalable, low cost, low effort techno-

logy for high throughput protein interaction screen-

ing.

- can search autoactivator space

- provides indication of interaction strength

- eliminates the need for pairwise y2h testing

- is easily scalable at the same effort and minimal

cost increase

A. Conditional Y2H (BFG-C2H)

- in the presence of kinases, Drugs

B. alternative reporters

- gfp reporters

C. Application of BFG to other assays

e.g. MAPPIT, Bi-FC

Figure 11. The huCV and huCVA

spaces were highly reproduc-

ible in their overlapping space.