1. Jacques.van.Helden@ulb.ac.be Proteome and interactome Bioinformatics

2. Contents Protein-protein interactions  Two-hybrid assays  Mass spectrometry Cellular localization of proteins  GFP tags Protein-DNA interactions  ChIP-on-chip

3. Jacques.van.Helden@ulb.ac.be Two-hybrid assays Functional genomics

4. Two-hybrid method DNA-binding ORF A DNA-binding Activation ORF B Activation Transcription factor A RNA pol B Hybrid constructions RNA pol A B RNA pol A B Interaction  reporter gene is expressed No interaction  reporter gene is not expressed Bait Prey Bait Prey Bait Prey

5. Two-hybrid Ito et al. (2001) PNAS 98: 4569-4574 Uetz et al. (2000). Nature 403: 623-631

6. Comparison of the results When the second “comprehensive” analysis was published, the overlap between thee results obtained in the two independent studies was surprisingly low. How to interpret this ?  Problem of coverage ? Each study would only represent a fraction of what remains to be discovered.  Problem of noise ? Either or both studies might contain a large number of false positives.  Differences in experimental conditions ? Ito et al. (2001) PNAS 98: 4569- 4574

7. Connectivity in protein interaction networks Jeong et al (2001) calculate connectivity in the protein interaction network revealed by the two-hybrid analysis of Uetz and co- workers. The connectivity follows a power law:  most proteins have a few connections;  a few proteins are highly connected Highly connected proteins correspond to essential proteins. Jeong, H., S.P. Mason, A.L. Barabasi, and Z.N. Oltvai. 2001. Lethality and centrality in protein networks. Nature 411: 41-42.

8. Jacques.van.Helden@ulb.ac.be Mass-spectrometry Functional genomics

9. 1. Construction of a bank of TAG-fused ORFs 2. Expression of the tagged baits in yeast tag Y ORF Y B E A Y C D 4. Affinity purification 3. Cell lysis anti-tag epitope + All cellular proteins,… Other proteins,… Isolation of protein complexes tagged bait Slide from Nicolas Simonis

10. B E A Y C D B E A Y C D 1 dimension SDS-PAGE B C isolation Mass spectrometry B C E E = YLR258w = YER133w = YER054c A Y D = YPR184w = YKL085w = YPR160w Mass spectrometry - Protein identification Slide from Nicolas Simonis

11. Protein complexes High-throughput mass-spectrometric protein complex identification (HMS-PCI) MDS proteomics 493 complexes Tandem Affinity Purification (TAP) CELLZOME: 232 complexes Ho et al. (1999). Nature 415: 180-183 Gavin et al. (1999). Nature 415: 141-147

12. Network of complexes Gavin et al. (1999). Nature 415: 141-147

13. Jacques.van.Helden@ulb.ac.be Assessment of interactome data Functional genomics

14. Assessment of interactome data von Mering et al (2002). Nature.

15. Comparison of large-scale interaction data von Mering et al (2002) compared the results from  Two-hybrid assays  Mass spectrometry (TAP and HMS-PCI)  Co-expression in microarray experiments  Synthetic lethality  Comparative genomics (conservation of operons, phylogenetic profiles, and gene fusion) Among 80,000 interactions, no more than 2,400 are supported by two different methods. Each method is more specifically related to some  functional classes  cellular location Reference: von Mering et al. (2002). Nature 750

16. Comparison of pairs of interacting proteins with functional classes von Mering et al (2002). Nature 750.

17. von Mering et al (2002). Nature. Validation with annotated complexes von Mering et al (2002) collected information on experimentally proven physical protein-protein interactions, and measured the coverage and positive predictive value of each predictive method  Coverage fraction of reference set covered by the data.  Positive predictive value Fraction of data confirmed by reference set. (Note: they call this “accuracy”, but this term is usually not used in this way) Beware: the scale is logarithmic !  This enforces the differences in the lower part of the percentages (0-10), but “compresses” the values between 10 and 50, which gives a false impression of good accuracy.

18. Jacques.van.Helden@ulb.ac.be Cellular localization of proteins Bioinformatics

19. 4156 proteins detected by fluorescence microscopy analysis Nature (2003) 425: 686-691 Slide adapted from Bruno André

20. Global analysis of protein localization This analysis allowed to obtain information for thousands of proteins for which the cellular localization was previously unknown. Slide adapted from Bruno André

21. Localisation and ORF function For historical reasons, the yeast genome is “over-annotated”.  The method used for predicting genes from genome sequences included many false positives, especially among short predicted ORFs. Most of the questionable ORFs were unobserved in the global localization analysis. These mainly correspond to short ORFs. Source: Bruno André

22. Jacques.van.Helden@ulb.ac.be Protein-DNA interactions ChIP-on-chip technology Functional genomics

23. The ChIP-on-chip method Chromatin Immuno-precipitation (ChIP)  Tagging of a transcription factor of interest with a protein fragment recognized by some antibody.  Immobilization of protein-DNA interactions with a fixative agent.  DNA fragmentation by ultrasonication.  Precipitation of the DNA-protein complexes.  Un-binding of the DNA-protein bounds. Measurement of DNA enrichment.  Two extracts are co-hybridized on a microarray (chip),where each spot contains one DNA fragment where a factor is likely to bind (e.g. an intergenic region, or a smaller fragment).. For the yeast S.cerevisiae, chips have been designed with all the intergenic regions (6000 regions, avg. 500bp/region) Recent technology allows to spot 3e+5 300bp DNA fragments on a single slide.  The first extract (labelled in red) is enriched in DNA fragments bound to the tagged transcription factor.  The second extract (labelled in green) has not been enriched.  The log-ratio between red and green channels indicate the enrichment for each intergenic region.

24. Lee et al (2002) In 2002, Lee et al publish a systematic characterization of the binding regions of 106 yeast transcription factors. Lee et al. 2002. Science 298: 799-804.