1. 1.Introduction to transcriptional networks 2.Regulation of the expression of the Lac operon 3.Finding Biclusters in Bipartite Graphs Today’s lecture will cover the following three topics Systems Biology

2. Unlike protein-protein interaction networks the transcriptional networks are directed networks By the term transcriptional networks we generally mean gene regulatory networks transcriptional networks

3. transcriptional networks: Basic mechanism of gene regulation

4. transcriptional networks

5. Most genes are regulated at transcription level and it is assumed that 5-10% of protein coding genes encode regulatory proteins. Some regulatory proteins play targeted role i.e. they take part in regulation of a few genes. Some regulatory proteins play more general role in initiating transcription (for example the eukaryotic transcription factors of type II or the RNA polymerase itself that is essential for the transcription of all genes). It is considered that dedicated regulatory proteins are those that affect up to 5% genes of a genome. However the boundary between the generalist and dedicated regulatory proteins is blurred. transcriptional networks

6. Experiments and methods used to determine regulatory relations 1.Complementary DNA microarrays 2.Oligonucleotide chips 3.Reverse transcription polymerase chain reaction 4.Serial analysis of gene expression 5.Chromatin Immunoprecipitation 6.Bioinformatics—e.g. by way of identifying binding sites transcriptional networks

7. Transcriptional Networks: Case study 1 An extended transcriptional regulatory network of Escherichia coli and analysis of its hierarchical structure and network motifs Hong-Wu Ma, Bharani Kumar, Uta Ditges2, Florian Gunzer2, Jan Buer1,2 and An-Ping Zeng* Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649 This work combined data sets from 3 different sources: 1.RegulonDB (version 4.0, http://www.cifn.unam.mx/Computational_Genomics/regulondb/) http://www.cifn.unam.mx/Computational_Genomics/regulondb/ 2.Ecocyc (version 8.0, www.ecocyc.org)www.ecocyc.org 3.Shen-Orr,S.S., Milo,R., Mangan,S. and Alon,U. (2002) Network motifs in the transcriptional regulation network of Escherichia coli. Nature Genet., 31, 64–68.

8. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649 Comparison of the TRN of E.coli from three different data sources (A) Based on number of genes (B) Based on number regulatory interactions

9. A combined network that includes all the 2624 interactions from the three data sets has been produced. In addition, this work extended this network by adding 23 additional genes and around 100 regulatory relationships through literature survey. The final TRN altogether includes 1278 genes and 2724 interactions. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

10. This work discovered a hierarchical structure in the TRN. The hierachical structure was identified according to the following way: (1)genes which do not code for transcription factors (TFs) or code for a TF which only regulates its own expression (auto-regulatory loop) were assigned to layer 1 (the lowest layer); (2) then we removed all the genes in layer 1 and from the remaining network identified TFs which do not regulate other genes and assigned the corresponding genes in layer 2; (3)we repeated step 2 to remove nodes which have been assigned to a layer and identified a new layer until all the genes were assigned to different layers. As a result, a nine layer hierarchical structure was uncovered. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649 From BMC Bioinformatics 2004, 5:199 of the related authors

11. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

12. The hierarchical structure implies absence of cycles in the network i.e. feedback loops (though auto regulatory and inter-regulatory loops exist) As the network is not complete, we cannot say that feedback loop could not be found in future however it seems they would not be too many. A possible biological explanation for the existence of this hierarchical structure is that the interactions in this particular TRN are between proteins and genes without involving metabolites. Only after a regulating gene has been transcribed, translated and eventually further modified by cofactors or other proteins, it can regulate the target gene. A feedback from the regulated gene at transcriptional level may delay the process for the target gene to access a desired expression level in a new environment. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

13. Feedback control may be mainly through other interactions (e.g. metabolite and protein interaction) at post-transcriptional level rather than through transcriptional interactions between proteins and genes. For example, a gene at the bottom layer may code for a metabolic enzyme, the product of which can bind to a regulator which in turn regulates its expression. In this case, the feedback is through metabolite–protein interaction to change the activity of the transcription factor and then to affect the expression of the regulated gene. Therefore, to fully understand the gene expression regulation, an integrated network that includes different interactions is needed. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

14. To calculate network motifs in the E.coli TRN, this work removed all the loops in the network (including the autoregulatory loops and the two- gene regulatory loops). Then they used the program Mfinder developed by Kashtan et al. to generate the motif profiles. The first four types are the so-called coherent FFLs in which the direct effect of the up regulator is consistent with its indirect effect through the mid regulator. In contrast, the last four types of FFLs are incoherent because the direct effect of the up regulator is contradictive with its indirect effect Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

15. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649 (A) Gene gadA is regulated by six FFLs (B)Gene lpd is regulated by five FFLs (C) Gene slp is regulated by 17 regulators

16. Transcriptional Network: Case study 1 Nucleic Acids Research, 2004, Vol. 32, No. 22 6643–6649

17. Topological and causal structure of the yeast transcriptional regulatory network Nabil Guelzim1,2, Samuele Bottani3, Paul Bourgine2 & François Képès1 Transcriptional Network: Case study 2 In this work the yeast transcriptional network was constructed by manual inspection of the websites of MIPS, SwissProt, Yeast Protein Database, S. cerevisiae Promoter Database and the Saccharomyces Genome Database The network consists of 491 genes and 909 regulatory relations nature genetics volume 31 may 2002

18. Transcriptional Network: Case study 2 nature genetics volume 31 may 2002 The network consists of 491 genes and 909 regulatory relations Bold type indicates self-activation, bold italics indicates self-inhibition and borders indicate essential genes. Thick lines represent activation, thin lines represent inhibition and the dashed gray line represents dual regulation.

19. Indegree distribution of this yeast transcriptional network is exponential Typical exponential distribution on normal scale Transcriptional Network: Case study 2 nature genetics volume 31 may 2002

20. Indegree distribution of this yeast transcriptional network is exponential Indegree distribution of the transcriptional network on semi-log scale open squares, full line --for all 402 regulated genes (367 nonregulatory and 35 interregulatory genes), 909 connections, p(k)=157e–0.45k; R=0.99) filled circles, broken line ---for the subset of 35 interregulatory genes, 72 connections; p(k)=15e–0.43k; R=0.94 Transcriptional Network: Case study 2 nature genetics volume 31 may 2002

21. Outdegree distribution of this yeast transcriptional network follows power law Typical power law distribution on normal scale Transcriptional Network: Case study 2 nature genetics volume 31 may 2002

22. Outdegree distribution of this yeast transcriptional network follows power law Outdegree distribution of the transcriptional network on log-log scale Open squares, full line --for all 124 regulating proteins (909 connections; P(k)=23k−0.87; R=0.95) filled circles, broken line – for 37 regulating proteins that control regulatory genes (72 connections; P(k)=19k−1.14; R=0.99) Transcriptional Network: Case study 2 nature genetics volume 31 may 2002

23. an operon is a functioning unit of genomic material containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately. The result of this is that the genes contained in the operon are either expressed together or not at all. Originally operons were thought to exist solely in prokaryotes but since the discovery of the first operons in eukaryotes in the early 1990s, more evidence has arisen to suggest they are more common than previously assumed. The operon

24. The lac operon of e.coli consists of three genes LacZ, LacY and LacA They are the codes of enzymes needed for processing lactose LacI is an adjacent gene which is a regulator ( transcriptional repressor) of the Lac operon Besides the promoter operator region there is a region where a complex called CAP binds which affect the transcription positively LacZ codes for the enzyme B-galactosidase and LacY codes for lactose permease, an enzyme that facilitates the flux of lactose through the cell membrane LacA is not directly involved in processing Lactose Source: Models of cellular regulation by Baltazar D. Aguda and Avner Friedman The Lac operon

25. Source: Models of cellular regulation by Baltazar D. Aguda and Avner Friedman Static model of the regulation of the expression of the Lac operon The LacI tetramer binds at the promoter region and stops the transcription The CAP complex binds the cap region and enhance the binding of RNA polymerase The Lac operon

26. cAMP binds and LacI is suppressed by Allolactose cAMP cannot bind and repressor protein LacI binds cAMP binds and repressor protein LacI binds cAMP cannot bind and LacI is suppressed by Allolactose Summary in Table

27. The technique of finding biclusters can be used to determine co-expressed gene groups 1.Introduction to transcriptional networks 2.Regulation of the expression of the Lac operon 3.Finding Biclusters in Bipartite Graphs

28. Given a nxp data matrix X, where n is the number of objects (e.g. genes) and p is the number of conditions (e.g. array), a bicluster is defined as a submatrix XIJ of X within which a subset of objects I express similar behavior across the subset of conditions J. A nxp data matrix X can be easily converted to a bipartite graph by considering a threshold or so. Finding bicluster (densely connected regions) in a bipartite graph is a similar problem. Definition of a bicluster

29. A Graph G=(V,E) is bipartite if its vertex set V can be partitioned into two subsets V 1, V 2 such that each edge of E has one end vertex in V 1 and another in V 2. V1 V2

30. Biclusters are densely connected regions in a bipartite graph CdAaGgIfKk DcAbGhIgLi DdBaHeIhLj EcBbHfJfLk EdCaHgJgMl FcCbHhKhMm FdDaIeGfNl GdDbKiCcNm Kj

31. Gene expression data can be represented as bipartite graphs gene/cond.cond0cond1cond2cond3cond4 YAL005C2.853.34000 YAL012W0.210.030.18-0.27-0.32 YAL014C-0.03-0.070.280.32-0.27 YAL015C-0.250.580.770.280.32 YAL016W0.110.040.750.820.21 YAL017W0.240.310.950.120.18 YAL021C-0.30.220.02-0.640.06 gene/cond.cond0cond1cond2cond3cond4 YAL005C11000 YAL012W00000 YAL014C00000 YAL015C00000 YAL016W00010 YAL017W00100 YAL021C00000 By transforming highest 5% values to 1 Before transforming, the data can be normalized Biclusters in gene expression data represents transcription modules/co-expressed gene groups

32. Tanay,A. et al. (2002) Discovering statistically significant biclusters in gene expression data. Bioinformatics, 18 (Suppl. 1), S136–S144. Ihmels,J. et al. (2002) Revealing modular organization in the yeast transcriptional network. Nat. Genet., 31, 370–377. Ben-Dor,A., Chor,B., Karp,R. and Yakhini,Z. (2002) Discovering local structure in gene expression data: the order-preserving sub-matrix problem. In Proceedings of the 6th Annual International Conference on Computational Biology, ACM Press, New York, NY, USA, pp. 49–57. Cheng,Y. and Church,G. (2000) Biclustering of expression data. Proc. Int. Conf. Intell. Syst. Mol. Biol. pp. 93–103. Murali,T.M. and Kasif,S. (2003) Extracting conserved gene expression motifs from gene expression data. Pac. Symp. Biocomput., 8, 77–88.

33. We propose a biclustering method incorporating DPClus G/Eabcdefghijklm A1100000000000 B1100000000000 C1111000000000 D1111000000000 E0011000000000 F0011000000000 G0001111000000 H0000111100000 I0000111100000 J0000110000000 K0000000111100 L0000000011100 M0000000000011 N0000000000011 An example bipartite graph and its corresponding matrix (for i  k)

34. BiClus:Biclustering method incorporating DPClus Concerning each row i (i=0 to |G|-1) of M CN, we calculate threshold i =avg i +(max i - avg i )  G margin and set (M SG ) ik =(M SG ) ki =1if (M CN ) ik  threshold i and threshold i is not an indeterminate number (for k=0 to |G|-1). Here, avg i = SUM i /n i where n i is the number of non-zero entries in row i of M CN and max i is the maximum value of the entries in row i of M CN G margin is a user defined value  1. ABCDEFGHIJKLMN A02220000000000 B20220000000000 C22042210000000 D22402210000000 E00220210000000 F00222010000000 G00111103320000 H00000030421000 I00000034021000 J00000022200000 K00000001100300 L00000000003000 M00000000000002 N00000000000020 Common neighbor matrix of the bipartite graph

35. ABCDEFGHIJKLMN A01110000000000 B10110000000000 C11011100000000 D11101100000000 E00110110000000 F00111000000000 G00001001110000 H00000010110000 I00000011010000 J00000011100000 K00000000000100 L00000000001000 M00000000000001 N00000000000010 BiClus:Biclustering method incorporating DPClus This matrix represents a simple graph

36. BiClus:Biclustering method incorporating DPClus Simple graph derived from the common neighbor matrix. We can use DPClus to find clusters in the simple graph.

37. BiClus:Biclustering method incorporating DPClus Clustering by DPClus

38. BiClus:Biclustering method incorporating DPClus Clustering by DPClus

39. BiClus:Biclustering method incorporating DPClus Finally determined biclusters

40. Evaluation of BiClus -Using Synthetic data -Using real data

41. Synthetic data Artificially embedded biclusters with noise Evaluation of BiClus

42. Synthetic data Artificially embedded biclusters with overlap Evaluation of BiClus

43. Let M1, M2 be two sets of biclusters. The gene match score of M1 with respect to M2 is given by the function Evaluation of BiClus A systematic comparison and evaluation of biclustering methods for gene expression data Amela Prelic´, Stefan Bleuler, Philip Zimmermann, Anja Wille, Peter Bu¨ hlmann, Wilhelm Gruissem, Lars Hennig, Lothar Thiele and Eckart Zitzle BIOINFORMATICS, Vol. 22 no. 9 2006, pages 1122–1129

44. Evaluation of BiClus Synthetic data Artificially embedded biclusters with noise

45. Evaluation of BiClus Synthetic data Artificially embedded biclusters with overlap

46. Gasch,A.P. et al. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell, 11, 4241–4257. Gene expression data collected from the above work

47. Gene expression data can be represented as bipartite graphs gene/cond.cond0cond1cond2cond3cond4 YAL005C2.853.34000 YAL012W0.210.030.18-0.27-0.32 YAL014C-0.03-0.070.280.32-0.27 YAL015C-0.250.580.770.280.32 YAL016W0.110.040.750.820.21 YAL017W0.240.310.950.120.18 YAL021C-0.30.220.02-0.640.06 gene/cond.cond0cond1cond2cond3cond4 YAL005C11000 YAL012W00000 YAL014C00000 YAL015C00000 YAL016W00010 YAL017W00100 YAL021C00000 By transforming highest 5% values to 1 Before transforming, the data can be normalized Biclusters in gene expression data represents transcription modules

48. 0.0010.010.0030.002 Evaluation of BiClus Real gene expression data of yeast P-values represents statistical significance of functional richness of the modules P-Values calculated using FuncAssociate: The Gene Set Functionator from http://llama.med.harvard.edu/cgi/func/funcassociate