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Yeast Transcription Networks: Structure, Dynamics, and Evolution

Yeast Transcription Networks: Structure, Dynamics, and Evolution. Croucher Advanced Study Institute Opportunities and Challenges for Physicists in quantitative and systems biology December 5, 2006. the transcription networks of a cell. ER stress. Cellular conditions. Amino acid starvation.

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Yeast Transcription Networks: Structure, Dynamics, and Evolution

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  1. Yeast Transcription Networks: Structure, Dynamics, and Evolution Croucher Advanced Study Institute Opportunities and Challenges for Physicistsin quantitative and systems biology December 5, 2006

  2. the transcription networks of a cell ER stress Cellular conditions Amino acid starvation Low phosphate Ire1 signal transduction pathways Pho85 Gcn2 msn2/4 hac1 transcription factors pho4 gcn4 genes proteins

  3. motif2 motif3 motif1 typically ~10 binding sites per promoter Chin, Chuang, and Li,Genome Research, 2005 Cis-regulatory logic output: transcript level for all genes input: the activity of TFs combinations of sites define complex regulatory logic

  4. yeast transcriptional sub-networks static, less than 5% of the existing connections

  5. How is the network structured? Genomic Reconstruction of the network identifying binding sites and targets of transcription factors identifying conditions under which a TF is activated/repressed combinatorial regulation by multiple TFs What are the functional constraints? dynamics of sub-networks controlling specific biological processes How does it evolve? evolution of transcription factors and their targets

  6. dynamics and design features of a basic regulatory module in metabolic pathways

  7. gcn4 leu3 met4 MET1 MET16 MET22 …. LEU4 LEU1 ILV3 ….. a combinatorial transcriptional circuit controlling amino acid starvation response

  8. Multilayered control in the leucine synthesis pathway • Combinatorial control by two transcription factors GCN4 and LEU3 • Feedback inhibition by the end product • Regulation of Leu3 by the intermediate Negative Feedback Postive Feedback Co-activator of Leu3

  9. network architecture combinatorial regulation dynamical response What are the basic constraints and design principles?

  10. Gcn4 Gcn4+Met4 Gcn4+Leu3 microarray expression data lacks desired accuracy and temporal resolution mRNA expression time course during nitrogen depletion

  11. need to measure the kinetic with high precisionand high temporal resolution Solution: • GFP tagging + flow cytometer • High temporal resolution needs automation

  12. GFP DNA stop Host gene Stop codon (End of a gene) GFP tagging • GFP: Green Fluorescent Protein • Excitation at 488 nm / Emission at 509 nm, optimized for flow cytometry measurement • Can be fused to a host protein to monitor the abundance of the protein in vivo

  13. Detector for monitoring GFP -- Flow Cytometer Flow cell: hydrodynamic focusing Multicolor Optics

  14. Summary • differential dynamics for genes up and downstream of the control point • differential control by two TFs --> differential dynamics • features of pathway design --> fast dynamics for intracellular leucine recovery?

  15. Evolution of a transcriptional circuit controlling mating types in yeast

  16. Why study evolution of transcriptional circuit?

  17. Evolution by changes in gene regulation • Morphological diversity linked to changes in transcriptional regulation. Carroll SB, et al. From DNA to Diversity. 2005

  18. regulatory complexity --> complexity of the organism? ~ 6,000 ~ 14,000 ~ 18,000 ~ 30,000

  19. Evolution of transcriptional circuit Evolution of complex traits involving multiple genes interacting with each other fundamental constraints? basic mode of changes?

  20. a cell  cell 1 1 1 1 2 2 2 2 a1 a2 a2 a1 Mating in two yeasts S. cerevisiae C. albicans a cell  cell MAT MATa a1 MATa MAT a1 a/ cell a/ cell

  21. Mating-type regulation in two yeasts S. cerevisiae C. albicans a1 a2 a1 a cells a2 a-specific genes on by default a-specific genes 2 1 2 1  cells 2 a-specific genes off by default a-specific genes

  22. What is the ancestral mode of asg regulation? Negative regulation newly acquired a2 lost ancestral mode positive regulation

  23. x asgs a2 2 The transition from positive to negative regulation involves: Lost of a2 asg expression became constitutively expressed in a cell asgs came under negative control by 2 in  cell

  24. Identifying asgs in both lineages Aga2 Asg7* Bar1* Mfa1 Mfa2 Ste2* Ste6* S. cerevisiae (Galgoczy et al.) Axl1 Asg7* Bar1* Ram2 Ste2* Ste6* C. albicans

  25. Regulation of mating type requires Mcm1 In an a cell: a-specific gene Mcm1 In an  cell: x 2 a-specific gene 2 Mcm1 Mcm1

  26. S. cerevisiae Motif 2 a2 Mcm1 Change of cis regulatory elements for asgs The site flanking the Mcm1 site in C. albicans is similar to an experimentally-defined consensus site for a2 in S. pombe: C. albicans Motif CATTGTT

  27. Experimental verification of the a2 site • Mutated the putative a2 site of an asg promoter-driven GFP. The putative a2 site is required for asg expression.

  28. Asg cis-regulatory sequences across species

  29. 2 a2 Mcm1 Changes to the cis regulatory element 2-Mcm1 intxn strengthened? a2 lost 2-Mcm1 intxn gained?

  30. Evolution of transcription factor

  31. A model for sequential changes

  32. What have we learned in general? A series of concerted subtle changes -> profound change of the wiring diagram (positive -> negative regulation) Logic output maintained throughout the transition Transition through intermediate with redundant control Combinatorial regulation -> evovability?

  33. Acknowledgement Collaborators: Annie Tsong, UC Berkeley Alexander Johnson, UCSF Joe DeRisi, UCSF Lab members: Brian Tuch Chenshan Chin Victor Chubukov

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