1. Location analysis of transcription factor binding sites Guy Naamati Andrei Grodzovky

2. A brief history What about today? What about today? Two weeks ago Masha and Michal told us about gene expression and gene clusters. Last week, Lior and Ofer told us about tfbs, and how to identify them.

3. Today! A revolutionary new method that identifies where and when in the genome a binding factor actually binds! A revolutionary new method that identifies where and when in the genome a binding factor actually binds! We will talk about the method that reveals the genome wide localization, and provide several important examples from the world of yeast cells. We will talk about the method that reveals the genome wide localization, and provide several important examples from the world of yeast cells.

4. The star of the show

6. What can location analysis give us that micro- arrays alone can ’ t? What can location analysis give us that micro- arrays alone can ’ t? Micro-arrays identifies changes in mRNA levels, but can not distinguish direct from indirect effects. Motivation no 1

7. Motivation no 2 What advantage does localization have over try to identify the binding site? What advantage does localization have over try to identify the binding site? Right! We don’t have to handle many case in which it “looks” like we identified binding site, but in vivo it’s not.

8. The Method

9. Developed by the group of Richard A. Young in Cambridge. Developed by the group of Richard A. Young in Cambridge. A combination of location and expression profile. A combination of location and expression profile. Allows protein-DNA interactions to be monitored across the entire yeast genome. Allows protein-DNA interactions to be monitored across the entire yeast genome.

10. The Method A modified ChIP, combined with micro-array analysis. A modified ChIP, combined with micro-array analysis. DNA was taken from a cell, and broken with sound waves (sonication). DNA was taken from a cell, and broken with sound waves (sonication). Proteins of interest where tagged with myc. Proteins of interest where tagged with myc. Fragments cross linked to those proteins were enriched by immunoprecipitation (IP). Fragments cross linked to those proteins were enriched by immunoprecipitation (IP).

11. What now? Cross links were reversed, and the enriched DNA was amplified and labeled (Cy5). Cross links were reversed, and the enriched DNA was amplified and labeled (Cy5). Cy5 labeled DNA was hybridized to a micro- array, together with non-enriched DNA labeled with Cy3. Cy5 labeled DNA was hybridized to a micro- array, together with non-enriched DNA labeled with Cy3. Gene expression was also analyzed. Gene expression was also analyzed. Three independent experiments, for accuracy. Three independent experiments, for accuracy.

14. Handling noise A single-array error method was used. A single-array error method was used.

15. How accurate is it? This method can identify factors binding to DNA, but cannot recognize the exact location of the binding site. Why? This method can identify factors binding to DNA, but cannot recognize the exact location of the binding site. Why? The sonication breaks the DNA into fragments 500-1000 bases long. Not very specific.

16. Testing if it works Used to identify sites bound by Gal4 in the yeast genome. Used to identify sites bound by Gal4 in the yeast genome. Found seven genes previously reported to be regulated by Gal4. Found seven genes previously reported to be regulated by Gal4. In addition, 3 more genes were found! In addition, 3 more genes were found!

17. An important reminder The consensus binding site for Gal4 was found in many places in the gene where Gal4 did not bind. Why is that? The consensus binding site for Gal4 was found in many places in the gene where Gal4 did not bind. Why is that? Previous studies of Gal4 have suggested that chromatin structure also has a big role.

19. Confirmation

20. The next investigation Ste12 functions in the response of haploid yeast to mating pheromones. Ste12 functions in the response of haploid yeast to mating pheromones. More than 200 genes are activated in a Ste12 dependent fashion. Which are directly regulated? More than 200 genes are activated in a Ste12 dependent fashion. Which are directly regulated? By this method, only 29!

22. What ’ s next? This method can identify the global set of genes that are regulated directly in vivo. This method can identify the global set of genes that are regulated directly in vivo. Gives us accurate information about where and when transcription factors bind. Gives us accurate information about where and when transcription factors bind. Opens a new pathway into regulation analysis … Opens a new pathway into regulation analysis …

23. Transcriptional regulatory networks in yeast Lee et al.

24. Just as there are networks of metabolic pathways … There are networks of regulator- gene interactions

25. But the network consists of building blocks : Those are …

26. How we identify them ? Using genome wide location analysis Using genome wide location analysis Identification of a set of promoter regions that are bound by specific regulators allowed us to predict sequence motifs that are bound by these regulators Identification of a set of promoter regions that are bound by specific regulators allowed us to predict sequence motifs that are bound by these regulators

27. Provides reduced response time to environmental stimuli

28. Offers the potential to produce bistable systems that can switch between two alternative states.

29. Provides a form of multi step ultra sensitivity as small changes in the level of activity of the master regulator at the top of the loop might be amplified at the ultimate target.

30. Single-input motifs are potentially useful for coordinating a discrete unit of biological function, such as a set of genes that code for the subunits of a biosynthetic apparatus or enzymes of a metabolic pathway. Single Input Motif

31. This motif offers the potential for coordinating gene expression across a wide variety of growth conditions.

32. The chain represents the simplest circuit logic for ordering transcriptional events in a temporal sequence.

33. FHL1 – Ribosomal proteins regulator. Forms a single input regulatory motif consisting of essentially all ribosomal protein genes Genome wide location analysis Single Input Motif Example

34. Assembling motifs into network structures An algorithm based on genome wide An algorithm based on genome wide location data and expression data from over 500 experiments was developed in order to identify group location data and expression data from over 500 experiments was developed in order to identify group of genes that are both coordinately of genes that are both coordinately bound and expressed. bound and expressed.

35. Network assembly algorithm 1-Define a set of genes G bound by a set of regulators S. 1-Define a set of genes G bound by a set of regulators S. 2- Find a subset of G with a similar expression pattern. 2- Find a subset of G with a similar expression pattern. 3- Go over the genes in G and drop genes with a significantly different expression pattern. 3- Go over the genes in G and drop genes with a significantly different expression pattern. 4- Scan the remaining genome for genes with similar expression profile and check if they ’ re bound by factors from S. 4- Scan the remaining genome for genes with similar expression profile and check if they ’ re bound by factors from S.

36. What have we got ? The resulting sets of genes and regulators are multi input motifs. The resulting sets of genes and regulators are multi input motifs. But they are refined for common expression But they are refined for common expression

37. MIM-CE ’ s: What are they good for ? Using MIM-CE ’ s the yeasts cell cycle Using MIM-CE ’ s the yeasts cell cycle networks was constructed using an networks was constructed using an automated method, without prior automated method, without prior knowledge of the regulators that knowledge of the regulators that control transcription. control transcription.

38. The process Check for MIM-CE ’ s significantly enriched in genes whose expression oscillates during the cell cycle. Check for MIM-CE ’ s significantly enriched in genes whose expression oscillates during the cell cycle. Align MIM-CE ’ s around the cell cycle on the basis of peak expression of the genes in the MIM-CE. Align MIM-CE ’ s around the cell cycle on the basis of peak expression of the genes in the MIM-CE.

39. The outcome Yeasts cell cycle transcriptional regulatory network.

40. Features of the network model: Correlation of the computational positioning of regulators with previous studies. Correlation of the computational positioning of regulators with previous studies. Regulators whose function was not known before could be positioned in the network on the basis of direct binding data. Regulators whose function was not known before could be positioned in the network on the basis of direct binding data. Third, and most important, reconstruction of the regulatory architecture was automatic and required no prior knowledge of the regulators that control transcription during the cell cycle. Third, and most important, reconstruction of the regulatory architecture was automatic and required no prior knowledge of the regulators that control transcription during the cell cycle.

41. Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle Simon et al.

42. Many transcriptional regulatory networks in yeast … Why the cell cycle network ?

43. Cyclins regulate the cell cycle Regulation of the cell cycle clock is effected through activity of the cyclin-dependent kinase (CDK) family of protein kinases.

44. But who regulate the regulators ? Nine Nine transcriptional transcriptional regulators were regulators were identified identified

45. The method Using genome wide location analysis to Using genome wide location analysis to identify the binding sites for each of the factors in vivo. identify the binding sites for each of the factors in vivo.

46. The results ChIP Micro Array

47. These results confirm the stage specific regulation of gene expression by those factors.

48. The results also confirm that genes encoding several of the cell cycle transcriptional regulators are themselves bound by other cell cycle regulators In this way a full regulatory network is formed.

49. And of course the cell cycle regulators Cyclin ’ s/CDK ’ s are also regulated by those factors.

50. Functional redundancy Each of the factors binds a critical cell cycle gene. Each of the factors binds a critical cell cycle gene. Deletion mutants with one of the factors deleted survive … Deletion mutants with one of the factors deleted survive … Why ? Why ?

51. What for Insures that the cell cycle completes Insures that the cell cycle completes efficiently. efficiently. On the other hand devoting the two members of the pair to distinct functional group of genes enables coordinated regulation of those functions.

52. The Genome-Wide Localization of Rsc-9 Damelin et al., 2002

53. A bit of background Recent studies identified common set of genes that are repressed/induced in response to stress (in yeast). Recent studies identified common set of genes that are repressed/induced in response to stress (in yeast). Generalized the roles of Msn2 and Msn4 in the stress response. Generalized the roles of Msn2 and Msn4 in the stress response. Do they account for all the observed changes in transcription response to stress? Do they account for all the observed changes in transcription response to stress?

54. Evidently not Must account for extensive gene repression as well as activation. Must account for extensive gene repression as well as activation. Previous evidence (Gasch et al, 2000): many genes involving Msn2/4 are activated only in some stress conditions. Previous evidence (Gasch et al, 2000): many genes involving Msn2/4 are activated only in some stress conditions. Tempting to consider a role for general transcription factors in the stress response.

55. Along came RSC Regulation of gene expression is closely connected to change in Chromatin structure. Regulation of gene expression is closely connected to change in Chromatin structure. RSC: a 15 protein complex that uses ATP energy to reposition nucleosomes. RSC: a 15 protein complex that uses ATP energy to reposition nucleosomes. Rsc9: a stable component of the RSC complex. Rsc9: a stable component of the RSC complex.

56. Genome wide localization The exact method we talked about was used for Rsc-9. The exact method we talked about was used for Rsc-9. Two categories with significant enrichment: 1. Genes coding the cytoplasmatic and mitochondrial ribosomal proteins. 2. Genes involved with stress response. Two categories with significant enrichment: 1. Genes coding the cytoplasmatic and mitochondrial ribosomal proteins. 2. Genes involved with stress response.

57. What kind of stress? Both set of genes are are affected by many types of stress. Both set of genes are are affected by many types of stress. The question is raised whether Rsc9 responds to specific or general stress. How do we find out? The question is raised whether Rsc9 responds to specific or general stress. How do we find out? Localization to the rescue!!

58. Two Stress Treatments Hydrogen-peroxide (elicits a transcriptional response similar to many other stress). Hydrogen-peroxide (elicits a transcriptional response similar to many other stress). Rapamycin (cell response is similar to starvation). Rapamycin (cell response is similar to starvation). Similar changes of Rsc9 localization after both treatments suggest a general stress response.

59. A question Right. A genome wide localization was used after treatment with the mating pheromone alpha factor. The results were: Right. A genome wide localization was used after treatment with the mating pheromone alpha factor. The results were: How would we know that the changes wouldn’t occur from an unrelated treatment?

61. We have seen how genome wide localization helps us recognize regulation motifs and networks Also we’ve seen a computational method to create a whole regulatory network without prior knowledge of the factors involved. Conclusion

62. Conclusion The changes in Rsc9 localization suggests that the genome itself is conditioned during widespread transcriptional regulation. The changes in Rsc9 localization suggests that the genome itself is conditioned during widespread transcriptional regulation. Raises new and interesting questions for transcriptional regulation. Raises new and interesting questions for transcriptional regulation.

63. Bibliography Lee et al. Transcriptional Regulatory Networks in Saccharomyces cerevisiae. Science. 2002 298:799-804 Damelin et al. The Genome-Wide Localization of Rsc9, a Component of the RSC Chromatin-Remodeling a Component of the RSC Chromatin-Remodeling Complex, Changes in Response to Stress. Mol Cell. Complex, Changes in Response to Stress. Mol Cell. 2002 9:563-573 2002 9:563-573 Simon et al. Serial Regulation of Transcriptional Regulators in the Yeast Cell Cycle. Cell 2001 106:697-708 in the Yeast Cell Cycle. Cell 2001 106:697-708 Ren et al. Genome-Wide Location and Function of DNA Binding Proteins. Science 2000 290:2306-2309

64. Hope you had fun! Hope you had fun!