Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping Vanessa Vermeirssen, Bart Deplancke, M Inmaculada Barrasa, John S Reece-Hoyes, H Efsun Arda, Christian A Grove, Natalia J Martinez, Reynaldo Sequerra, Lynn Doucette-Stamm, Michael R Brent & Albertha J M Walhout Supplementary Text and Figures: Supplementary Figure 1 Examples of updated TF gene models. Supplementary Figure 2 Example of a Y1H matrix experiment using Pfat-5 as a promoter bait and the TF array as prey. Supplementary Figure 3 TF dilutions. Supplementary Figure 4 Y2H data with DB-NHR-49 as a protein bait. Supplementary Table 2 All Y1H and Y2H interactions. Supplementary Table 3 Results for Y1H Pfat-5 pooling experiment shown in Figure 4. Supplementary Methods Note: Supplementary Table 1 is available on the Nature Methods website (Excel file).
A Supplementary Figure 1 B C D E Examples of updated TF gene models. (a) Example of gene model change in which the old model extended into an intron at the 5 end. (b) Example of gene model change in which the old model extended outside the 5 end. (c) Example of gene model change in which one gene model is split into two genes, both of which encode predicted TFs. (d) Example in which the old gene model did not include the DNA binding domain. (e) Example in which the DNA binding domain of the old gene model was incomplete. Green bars indicate the location of the DNA binding domain, red dots indicate the gene model used here.
Supplementary Figure 2 1 1 1 5 5 10 11 3 2 3 14 12 14 13 15 2 2 6 6 16 4 5 3 3 6 7 7 7 17 18 4 4 8 8 19 20 16 9 8 25 22 24 21 22 23 19 Example of a Y1H matrix experiment using Pfat-5 as a promoter bait and the TF array as prey. Only HIS3 reporter assays are shown. The mating plates (1 to 8) are shown alongside the corresponding transformation plate (1 to 8 ). Positive spots are numbered (see Supplementary Table 1 for locations of all TFs in the array and Supplementary Table 2 for a list of all interactions). Yeast colonies that are not numbered are not considered to indicate an interaction. Some interactions give a stronger phenotype (e.g. spot 14) than others (e.g. spot 13). Generally, we observed more interactions by transforming the array than by mating. However, interactions were occasionally only observed by mating because the transformation did not work well (e.g. spots 12 and 25).
Pvha-15 1 3 9 27 54 1 3 9 27 54 ZTF-3 CEH-30 C25G4.4 ZC204.2 DSC-1 NHR-3 NHR-34 NHR-49 Pdaf-3 DAF-16A SOX-2 T27B1.2 Y55F3BR.5 EKL-2 1 3 9 27 54 Supplementary Figure 3 1 3 9 27 54 TF dilutions. Two promoter baits, Pvha-15 and Pdaf-3, were used with a total of 14 TFs, one of which was found with both baits (Supplementary Table 2). Empty ppc86 plasmid was used to dilute the TF-encoding plasmid DNA (3, 9, 27 and 54- fold as indicated). Left panels LacZ reporter; right panels HIS3 reporter. NHR-67 EKL-2
Supplementary Figure 4 * A C B D % high-confidence interactions recovered G cdna TF Mini Lib Combi Matrix Mating Matrix Trafo STS G E F Y2H data with DB-NHR-49 as a protein bait. (a,b) matrix mating; (c,d) matrix transformation; (e,f) STS pooling. One plate is shown for each as an example. Left HIS3, right LacZ reporter gene expression. (g) Assay comparison. cdna cdna library; TF mini TF mini-library; Lib combi combined libraries; STS STS smart pooling, Trafo transformation.
Supplementary Table 2: All Y1H and Y2H interactions High-confidence interactions were used in Figure 5. TFs, "M" indicated multiplexed TFs TF cdna screens TF mini-library screens mating transformation pooling uni/multiplex highconfidence? Y1H with Pvha-15 ATHP-1 Y Y Uni Yes C25G4.4 7 Y Multi Yes CEH-20 Y Y Uni Yes CEH-30 Y Y Multi Yes CEH-31 Y Y Y Uni Yes CEH-8 1 Y Y Uni Yes CEH-9 Y Y Y Uni Yes DIE-1 4 1 Y Y Y Uni Yes DMD-4 Y Y Uni Yes DMD-6 1 Y Y Uni Yes DSC-1 1 Y Y Multi Yes EGL-44 4 Y Y Y Uni Yes EKL-2 2 Y Multi Yes F52E4.8 Y Y Uni Yes MAB-5 1 Y Uni Yes MIG-5 Y Y Uni Yes MLS-2 Y Y Uni Yes NHR-1 1 4 Y Y Y Uni Yes NHR-10 1 Y Y Y Uni Yes NHR-111 19 Y Y Y Uni Yes NHR-3 Y Y Y Multi Yes NHR-34 1 Y Y Multi Yes NHR-45 Y Y Uni Yes NHR-49 2 1 Y Y Multi Yes NHR-67 2 Y Y Multi Yes NHR-88 Y Y Y Uni Yes ODR-7 Y Y Uni Yes PQM-1 3 Y Y Uni Yes SEM-4 Y Y Uni Yes VAB-3 Y Y Uni Yes Y47D3B.9 Y Y Uni Yes ZC204.2 Y Y Multi Yes ZTF-1 5 2 Y Y Y Uni Yes ZTF-3 Y Y Multi Yes ZTF-8 Y Y Y Uni Yes C07E3.6 Y Multi No C26C6.1 Y Multi No CEH-17 Y Uni No DMD-5 1 Uni No FKH-4 Y Multi No M163.2 Y Uni No NHR-14 2 Multi No NHR-147 Y Multi No NHR-186 Y Multi No NHR-43 Y Multi No NHR-66 1 Multi No NHR-96 Y Multi No R04A9.5 Y Multi No R05D3.3 Y Multi No T09F3.1 Y Multi No TAB-1 1 Uni No TBX-40 Y Multi No UNC-42 Y Uni No ZTF-6 3 Multi No
Y1H with Pdaf-3 ATHP-1 2 Y Y Uni Yes CEH-17 77 Y Y Y Uni Yes DAF-16A Y Y Multi Yes DIE-1 12 Y Y Y Uni Yes DMD-5 Y Y Uni Yes EKL-2 Y Y Multi Yes MIG-5 1 Y Y Uni Yes MLS-2 Y Y Uni Yes NHR-111 Y Y Uni Yes NHR-45 1 Y Y Uni Yes ODR-7 6 Y Y Y Uni Yes PHP-3 Y Y Uni Yes SOX-2 2 Y Y Y Multi Yes T27B1.2 Y Y Multi Yes UNC-42 Y Y Uni Yes Y47D3B.9 Y Y Uni Yes Y55F3BR.5 Y Y Multi Yes ZTF-1 4 Y Y Y Uni Yes ZTF-8 Y Y Y Uni Yes ALR-1 Y Uni No CEH-20 Y Uni No CEH-30 Y Multi No CEH-6 Y Multi No CEH-8 Y Uni No DMD-4 Y Uni No GAK-1 1 Multi No IRX-1 Y Multi No M163.2 Y Uni No NHR-34 Y Multi No NHR-43 Y Multi No NHR-66 Y Multi No NHR-92 Y Multi No PAL-1 14 Multi No PSA-1 Y Multi No SOX-1 Y Multi No ZTF-7 1 Multi No
Y1H with Pgpa-10 CEH-20 2 Y Y Y Uni Yes DAF-3 Y Y Y Multi Yes DMD-4 Y Y Y Uni Yes EGL-44 11 Y Y Uni Yes IRX-1 Y Y Multi Yes NHR-111 2 Y Y Y Uni Yes NHR-45 Y Y Uni Yes ODR-7 1 Y Y Y Uni Yes PIE-1 11 Y Y Multi Yes ZTF-1 1 11 Y Y Y Uni Yes ZTF-8 Y Y Y Multi Yes AST-1 Y Multi No ATHP-1 Y Uni No CEH-31 Y Uni No CEH-38 2 Multi No CEH-45 Y Multi No CEH-6 Y Multi No CEH-9 Y Uni No DAF-16 Y Multi No HLH-1 Y Multi No M163.2 Y Uni No NHR-43 Y Multi No TAB-1 Y Uni No Y47D3B.9 Y Uni No ZTF-2 Y Multi No
Y1H with Pfat-5 ATHP-1 Y Y Uni Yes CEH-18 Y Y Multi Yes CEH-6 Y Y Multi Yes CEY-2 Y Y Uni Yes DIE-1 1 5 Y Y Y Uni Yes EGL-44 2 Y Y Y Uni Yes EKL-2 Y Y Multi Yes NHR-111 Y Y Uni Yes NHR-45 Y Y Uni Yes PIE-1 Y Y Multi Yes PQM-1 Y Y Uni Yes R08E3.4 Y Y Multi Yes TBX-9 Y Y Multi Yes Y39B6A.12 Y Y Multi Yes ZTF-1 Y Y Uni Yes ZTF-2 1 22 Y Y Y Multi Yes ZTF-8 Y Y Uni Yes C04F5.9 Y Multi No CEH-17 Y Uni No CES-1 Y Multi No F47H4.1 Y Multi No LIN-26 Y Multi No LSY-2 Y Uni No NHR-35 1 Multi No NHR-72 Y Multi No TBX-8 1 Uni No Y53C12C.1 Y Multi No Y6G8.3 Y Multi No
Y2H with DB-NHR-49 CEH-43 Y Y Multi Yes NHR-105 4 7 Y Y Y Multi Yes NHR-121 1 Y Y Multi Yes NHR-20 1 2 Y Y Y Multi Yes NHR-22 8 102 Y Y Y Multi Yes NHR-234 Y Y Y Multi Yes NHR-256 1 1 Y Y Y Multi Yes NHR-270 1 Y Y Y Multi Yes NHR-274 2 Y Multi Yes NHR-66 1 Y Y Y Multi Yes NHR-71 2 Y Y Y Multi Yes NHR-76 Y Y Y Multi Yes NHR-78 1 Y Y Multi Yes NHR-79 55 Y Y Y Multi Yes UNC-30 Y Y Multi Yes NHR-11 Y Multi No NHR-116 Y Multi No NHR-13 10 Multi No NHR-181 Y Multi No NHR-19 Y Multi No NHR-264 Y Multi No NHR-265 Y Multi No NHR-49 Y Multi No NHR-73 10 Multi No NHR-80 1 Multi No NHR-85 Y Multi No
Supplementary Table 3: Pfat-5 pooling results Pool coordinate TF Uniplex or Multiplex Found in screen? * Found in matrix? Comment 1-A01 ZTF-1 Uni No Yes 1-A05 TAB-1 Uni No No His only ** 1-A08 NHR-111 Uni No Yes 1-A11 LSY-2 Uni No No His only *** 1-B03 CEY-2 Uni No Yes His only, very weak 1-B04 ZTF-8 Uni No Yes 1-B05 DIE-1 Uni 6 times Yes 1-C05 NHR-45 Uni No Yes His only *** 1-C08 EGL-44 Uni 2 times Yes 1-C09 PQM-1 Uni No Yes His only *** 1-C11 ATHP-1 Uni No Yes 1-D06 EKL-2 Multi No Yes 1-D11 EKL-2 Multi No Yes 1-D12 PIE-1 Multi No Yes His only *** 1-E01 EKL-2 Multi No Yes 1-E06 PIE-1 Multi No Yes His only *** 1-F06 PIE-1 Multi No Yes His only *** 2-A01 TBX-9 Multi No Yes 2-A05 CEH-18 Multi No Yes His only *** 2-B05 TBX-9 Multi No Yes 2-E03 R08E3.4 Multi No Yes 2-E04 R08E3.4 Multi No Yes 2-E05 Y39B6A.12 Multi No Yes His only *** 2-E10 ZTF-2 Multi 23 times Yes His only *** 2-E11 R08E3.4 Multi No Yes 2-E12 ZTF-2 Multi 23 times Yes His only *** 2-G02 ZTF-2 Multi 23 times Yes His only *** 2-G07 Y39B6A.12 Multi No Yes His only *** 2-H02 Y39B6A.12 Multi No Yes His only *** 2-H09 CEH-6 Multi No Yes His only *** 2-H12 CEH-6 Multi No Yes His only *** * Deplancke et al., Cell 2006 ** Sticky on his, not considered real positive, see Vermeirssen et al, 2007 *** Not sticky on his, considered real positive because Pfat-5 is somewhat self-active on LacZ
Vermeirssen et al, Supplementary Methods TF predictions. Previously, we predicted that 934 of the ~20,000 C. elegans genes encode regulatory TFs. We found that one gene was recently annotated as a pseudogene (Y60A9.2) and that six genes were missed in previous predictions (T21B4.17, Y55F3AM.7, mig-5, fozi-1, Y55F3BR.5 and ztf-9). We added these genes, removed the pseudogene and also included a newly annotated TF-encoding gene (nhr- 286, Supplementary Table 1). For updated gene models we used WormBase version WS145. Yeast strain generation. DNA baits were created and integrated into the genome of the Y1H bait strain YM4271 as described 1. Y1H 001 was generated by modifying Y187 (MAT ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, met -, gal4, gal80, URA3::GAL1 UAS -GAL1 TATA -LacZ) 2. Y187 is of the -mating type and is therefore compatible for mating with the a -type bait strains used in Y1H and Y2H assays. However, Y187 contains a LacZ reporter gene in a URA3 reporter construct (URA3::GAL1 UAS -GAL1 TATA -LacZ), which may interfere with Y1H and Y2H assays. URA3 and LacZ were disrupted via PCR-targeting as described 3 using KanMX and Hygromycin B markers respectively (see Supplementary Table 1 for primer sequences). The DB-NHR-49 protein bait was generated and transformed into the MaV103 yeast strain as described 4. Yeast mating. Mating experiments were done as described 4,5. Briefly, aliquots of the TF yeast array glycerol stocks were spotted onto eight 15 cm Sc-T plates in a 96-well
format and incubated overnight at 30 o C. Similarly, lawns of bait yeast strains were prepared on eight 15 cm Sc-U-H (Y1H) or Sc-L (Y2H) plates. Four baits can be analyzed with one freshly spotted copy of the TF-array. Assay readouts were scored after 10-day incubations at 30 o C. Yeast transformations. Transformations were performed as described 4,6. Assay readouts were scored after 10-day incubations at 30 o C. The smart pooling assays were performed twice. Yeast PCR was performed as described 6. PCR products were sequenced by Agencourt Bioscience Corporation. STS modeling To model STS and PI-deconvolution performance, 1,000 random TF-DNA interactions were generated and deconvoluted per number of TFs bound per promoter (the averages were used in Fig. 2e). To model STS performance in the presence of false positives or false negatives, 10,000 random TF-DNA interactions were generated, the corresponding number of random pools was added (false positive) or deleted (false negative), and deconvolution and assessment of results was performed. References 1. Deplancke, B., Dupuy, D., Vidal, M. & Walhout, A. J. M. A Gateway-compatible yeast one-hybrid system. Genome Res 14, 2093-2101 (2004). 2. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805-16 (1993). 3. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793-1808 (1994). 4. Walhout, A. J. M. & Vidal, M. High-throughput yeast two-hybrid assays for largescale protein interaction mapping. Methods 24, 297-306 (2001).
5. Walhout, A. J. M. et al. Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116-122 (2000). 6. Deplancke, B., Vermeirssen, V., Arda, H. E., Martinez, N. J. & Walhout, A. J. M. Gateway-compatible yeast one-hybrid screens. CSH Protocols doi:10.1101/pdb.prot4590 (2006).