Systems Biology

1. Systems Biology • The search for the syntax of biological information, that is, the study of the dynamic networks of interacting biological elements. • The aim is to obtain, integrate and analyze complex data from multiple experimental sources using interdisciplinary tools. • Some typical technology platforms are: • Transcriptomics:whole cell or tissue gene expression measurements. • Proteomics:complete identification of proteins and protein expression patterns of a cell or tissue • Metabolomics :identification and measurement of all small-molecules metabolites within a cell or tissue • Glycomics:identification of the entirety of all carbohydrates in a cell or tissue. • Interactomics:encompasses interactions between all molecules within a cell • Fluxomics:which deals with the dynamic changes of molecules within a cell over time

2. Illustrative example of the interaction and contrast between a traditional approach and a Systems Biology approach Systems Biology is characterized by: Integration of the levels of biological organization and complexity addressed and Interaction between experimentation and predictive modeling

3. Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network Trey Ideker, Vesteinn Thorsson, Jeffrey A. Ranish, Rowan Christmas, Jeremy Buhler, Jimmy K. Eng, Roger Bumgarner, David R. Goodlett, Ruedi Aebersold, Leroy Hood

4. The Integrated Approach Microarrays DATA Integration and Assimilation Into BIOLOGICAL MODELS Global Quantification and Measurement Of Protein Two Hybrid System

5. The Strategy If possible, define an initial model of the molecular interactions governing pathway function. Design additional perturbation experiments D) Formulate new hypotheses to explain observations not predicted by the model. A)Define all of the genes in the genome and the subset of genes, proteins, and other small molecules constituting the pathway of interest. C)Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global network of protein-protein, protein-DNA, and other known physical interactions. B)Perturb each pathway component through a series of genetic or environmental manipulations Detect and quantify the corresponding global cellular response

6. A)Define all of the genes in the genome and the subset of genes, proteins, and other small molecules constituting the pathway of interest.

7. A) BASICS of Galactose Utilization(GAL) pathway in Saccharomyces cerevisiae • Controlled by an operon-like system. • It converts Galactose to Glocose-6-phosphate. • It produces the enzymes essential for galactose breakdown to glucose. • In the absence of galactose ENZYMES ARE NOT PRODUCED. • In the presence of galactose STRUCTURAL GENES(GAL 1, GAL 7, GAL 10) ARE ACTIVATED. • The above enzymes are regulated by Gal 4 and Gal 80 regulatory genes.

8. REGULATION of Galactose Utilization GAL) pathway in Saccharomyces cerevisiae Galactose GALACTOSE INDUCTION LOOP Gal3p GAL2 GAL3 Gal 80p GAL80 Gal 4p GAL7 GAL1 GAL10

9. B)Perturb each pathway component through a series of genetic or environmental manipulations

10. B) Application of 20 perturbations to the GAL pathway • Wild type and nine genetically altered strains were examined. These were: • Transport(gal∆2) • Enzymatic(gal∆1, gal∆5, gal∆7, gal∆10) • Regulatory(gal∆3, gal∆4, gal∆6, gal∆80) • The strains were perturbed environmentally by growth in presence (+gal) or absence(-gal) of galactose. • Global changes in mRNA of 6200 nuclear yeast genes were seen. • Identified 997 genes whose mRNA level significantly differed from control. • These were then divided into 16 clusters in which each cluster represented genes with similar expression responses over all perturbations.

11. PERTURBATION MATRIX RESULTS

12. Comparison of Northern vs. microarray analysis Medium-gray representing no change, darker or lighter shades representing increasing or decreasing amounts of expression respectively,

13. Wt+gal Protein extracts Wt-gal Protein extracts Labeled with isotopically heavy and normal ICAT reagents Combined and digested with trypsin Fractionated by Multidimensional chromatography Are the observed changes in mRNA expressionalso reflected at the level of protein abundance? • RESULTS: • As a whole, protein-abundance ratios were moderately correlated with their mRNA counterparts. • 30 proteins showed clear changes in abundance between wt+gal and wt-gal conditions. • mRNA of 15 did not change significantly in response to perturbation. • Many ribosomal-protein genes increased three-to five fold in mRNA but not in protein abundance. Analyzed by MS/MS

15. C)Integrate the observed mRNA and protein responses with the current, pathway-specific model and with the global network of protein-protein, protein-DNA, and other known physical interactions.

16. C) Can we attribute the observed mRNA and protein changes to underlying regulatory interactions in the cell? • They assembled a catalogue of previously observed physical interaction in yeast by COMBINING: • 2709 protein-protein interactions • 317 protein-DNA interactions • Out of the total genes above they observed 348 genes that were affected in mRNA or protein expression by at least one perturbation and also involved in two or more interactions with the affected genes….

17. 348 Genes along with their 362 associated interactions as a PHYSICAL INTERACTION NETWORK

18. POSSIBILITIES • A protein DNA interaction may be responsible for directly transmitting an expression change from a transcription factor to a highly co-related target gene. e.g. Mcm1 Far1and Mig1 Fbp1 • Two genes may be under control of a common transcription factor which is the 3rd gene. C (A,B) e.g. GAL enzymes regulated by Gnc4 Class of gluconeogenic genes controlled by Sip4 (Fbp1, Pck1, Ic11) • Scanning of network for indirect effects, such as a change in one gene transmitted to the other through a protein-protein interaction with a signaling protein. e.g. Gcr2-Gcr1 Tpi1 • Gal4p directly regulates genes in several processes of other networks through novel protein DNA interaction. e.g. Cluster 1,2,3 contained genes with Gal4 binding sites • The above genes were involved in glycogen accumulation, protein metabolism and others with unknown function

19. Tree comparing gene-expression changes resultingfrom different perturbations to the GAL pathway.

20. D) How do the observed responses Of GAL genes compare to their predicted behavior? • In general the results were same as predicted.

21. D) Formulate new hypotheses to explain observations not predicted by the model.

22. New Observations, Hypotheses, and possible tests

23. Contd….

24. Tree comparing gene-expression changes resultingfrom different perturbations to the GAL pathway.

25. Refinements in the GAL Pathway Model of galactose utilization. Yeast metabolize galactose through a series of steps involving the GAL2 transporter and enzymes produced by GAL1, GAL7, GAL10, and GAL5. These genes are transcriptionally regulated by a mechanism consisting primarily of GAL4, GAL80, and GAL3. GAL6 produces another regulatory factor thought to repress the GAL enzymes in a manner similar to GAL80. Dotted interactions denote model refinements supported by this study.

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