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Project III

“Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class Switch”, Muto et al., EMBO, 2010 Review the evidence for the interaction network after rendering it in BioTapestry .

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Project III

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  1. “Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class Switch”, Muto et al., EMBO, 2010 • Review the evidence for the interaction network after rendering it in BioTapestry. • Interpret the ODE’s in physical terms, and show under what parameter conditions does the model work. • Is this reasonable or completely ad hoc? (This problem will require simulations) Magdalena Koziol Molly Megraw Mitra Mojtahedi Jiyang Yu Project III

  2. B cell development B cell Pax5 Antigen (LPS) Absence of Antigen B cell Class switch recombination (CSR) Pax5 Pax5 Antibody-secreting cells (ASC) Isotope switched Plasma cell Blimp1 Plasma cell Blimp1

  3. Prior to this paper, people focused on this network: Signal

  4. EVIDENCE FOR GRN Bach2: -B cell specific TF -Express high in B cells -Down regulate in plasma cells -It is downstream of Pax5 -It is activated by Pax5 -It represses Blimp1 Pax5 Bach2: qPCR analysis of expression of Pax5 in Bach2-/- Bach2 Blimp1: Blimp1 GFP reporter mice Blimp1 Bach2: Rescue of Bach2-/- phenotype by Blimp1-/- Blimp1 Pax5: qPCR analysis of expression of Pax5 in Blimp1-/- Bach2 CSR: Bach2-/- cells do not undergo CSR

  5. B Cell State

  6. Class Switch State

  7. Plasma Cell State

  8. Network model The authors focus on Pax5, Bach2, Blimp-1 interaction They observe that the presence of Bach2 causes a delay in Pax5 downregulation They use a set of ordinary differential equations to model the system- does model simulation address the time delay?

  9. Their Kinetic Model

  10. They removed Blimp-1 in the model

  11. Reproducing their simulation

  12. Wait a second… Put mRNA expression of those three genes in Figure 5B

  13. What if Blimp1=1 at t=0?

  14. Different initial conditions

  15. Different initial conditions Their model only works when Blimp1 is > Bach2 at t=0!

  16. Their steady state analysis: Parameters not realistic

  17. More Problems • Two sets of parameters before and after 24h • Some of the parameter values are questionable, i.e. C2=2.441e-7, Ns=12 before 24h So, their model is PROBLEMATIC!

  18. Is there a way to make the model correct?

  19. Is there a way to make the model correct?

  20. Our Proposed Model A Equation (3’) is from

  21. It’s working with correct initial conditions Put mRNA expression of those three genes in Figure 5B

  22. Steady State Analysis 3.2 Blimp-1=1 3.2 Blimp-1=1 3.6 2.0 2.0 1.5 1.5 3.6

  23. Robustness of Our ModelSensitivity of Parameters: Nr

  24. Conclusions • Bach2 is required for CSR, and causes a time-delay of plasma cell differentiation upon antigen stimulations • Their mathematical model isPROBLEMATIC • Only works when Blimp1 > Bach2 at time 0 • Inconsistent parameters with steady state analysis of Pax5 • Two parameter sets before and after 24h • Our proposed model A explains all the observations • The GRN of the negative feedback loop via Bach2 is REASONABLEfor the time-delay of Blimp-1 induction

  25. If you need the simulation codes, send me an email: jy2322@c2b2.columbia.edu.Thanks for THE BEST EVER class of GRNs !!!

  26. Back up slides

  27. Simulation Outline • Model in the paper: negative feedback via Bach2 • Our proposed Model A • Double Negative Feedback Loop • Double Positive Feedback Loop • Our proposed Model B • Our proposed Model C

  28. W/ Antigen: a Negative Feedback Loop via Bach2

  29. Correct Steady State Plots for their Model Blimp-1=1 1.5 3.2 2.0 3.6

  30. Our proposed model A: Blimp1 will not go to Infinity

  31. Robustness of Our ModelSensitivity of Parameters: C2

  32. Double Negative Feedback Loop in B Cell (Toggle Switch)

  33. Double Positive Feedback Loop in Plasma Cell

  34. What’s wrong with Toggle Switch to regulate CSR? • Pax5 knock-down has no effects on CSR

  35. Alternative Model B This model is similar to our Model I as restricting maximum Blimp1 level and will work similarly. Model is coded up, but we need a few more hours to optimize parameters and run simulations !

  36. Alternative Model C to Test No direct evidence to support Blimp1 Bach2, but the model with it works well To test: Removing it might work as well Or other factors connecting Blimp1 and Bach2

  37. Detailed evidences for their GRN interactions

  38. Bach2 represses Blimp1 Evidence: Blimp-1 GFP reporter Increase % of plasma cells in Bach2-/-

  39. Bach2 seems to delay Blimp1 activation Evidence: In Bach2-/- B cells, expression of Blimp1 increases faster Closed triangle: Bac2-/- Open circle: Bac2+/+

  40. Bach2 seems to prevent plasma cells Evidence: Relationship between cell division and plasma cells (marker CD138) Triangle: Bach2-/-

  41. Rescue of Bach2-/- phenotype by Blimp1-/- Evidence: IgG1 & IgG3 are markers for isotope switch Isotope switch inhibited in Bach2-/- Isotontpe switch rescued by Blimp1 double muta

  42. Regulatory network Further evidence: what Regulates what, and if positive or negative regulation mRNA level mRNA level mRNA level

  43. Network components transcription during B cell to plasma transition Evidence: Level of network components during B cell to plasma cell transition

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