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rhomboid MLE regulation by Sim:Tgo and Su(H)

rhomboid MLE regulation by Sim:Tgo and Su(H). 8/24/09. Evidence for Sim Target Regulation. expression is lost in sim mutants Sim:Tgo sites are required for midline expression Sim over-expression expands target expression in neuroectoderm What restricts to neuroectoderm?

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rhomboid MLE regulation by Sim:Tgo and Su(H)

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  1. rhomboidMLEregulation by Sim:Tgo and Su(H) 8/24/09

  2. Evidence for Sim Target Regulation • expression is lost in sim mutants • Sim:Tgo sites are required for midline expression • Sim over-expression expands target expression in neuroectoderm • What restricts to neuroectoderm? • Maybe Su(H), says Scott heat-shock Sim control sim rho cdi sli Nambu, 1991

  3. Circumstantial evidence for Sim-Su(H) cooperation • sim initation requires Su(H) • sim, m5/8 (mesectoderm), rst F6d (midline) expression is expanded with Su(H) over-expression • Su(H)/Notch required for multiple stages of midline development (Wheeler et al. ’08) • Frequent Su(H) binding sites in midline primordium elements • Sim:Tgo=ACGTG • Su(H)=T/C A/G TG A/G/T GAA

  4. Test cases for Sim-Su(H) cooperation Su(H) Sim Sim rhoMLE Su(H) Su(H) Sim Sim Sim sema1b I1F3sC Su(H) Sim Sim sim1.0pL

  5. rho embryonic expression st. 5 st. 6 st. 9 • Initially broad neuroectodermal expression • Refines to mesectoderm before gastrulation • In all midline thru st. 11 st. 11 st. 13 st. 17

  6. rho cis-regulatory locus • Ip ‘92- 2.2 kb upstream controls neuroectoderm and midline expression • NEE activated by Dorsal, Twist, repressed by Snail • MLE contains 2 Sim:Tgo binding sites • “1- to 2-hr delay in onset of (rho2.2-lacZ) expression in the mesectoderm relative to the endogenous rho gene”

  7. rho embryonic cis-regulation Ip, 1992 Zelzer, 2000

  8. rhoMLE (Midline Element) motifs Sim/SuH sites Repeats 1/2

  9. rho MLE expression St. 13 St. 11 U S S w.t. U S X X S SimMut U X S S Su(H)Mut GFP GFP Sim GFP GFP Sim

  10. 12xSu(H)-LacZ expression • Expression in AMG, PMG, MP5, MP6, MNB • VUM expression gradient • Similar to rhoMLE SimMut Wheeler et al.,2008

  11. How is rhoMLEregulated? • Sim enhances AMG, MP1, MP4 expression • Sim not required for posterior midline expression • Sim not required for midline restriction • Activated Su(H) enhances AMG, MP1 (With Sim) PMG and Posterior neurons (without Sim) • Inactive Su(H) represses MP3/4? • rhoMLE is not mesectoderm element • Where is it?

  12. sim • 6(10)Su(H), 4(5) Sim:Tgo sites sim3.7 modified from genome.ucsc.edu

  13. hlh m5/8 • 4 Su(H), 1 Sim:Tgo sites 5/8 modified from genome.ucsc.edu

  14. Sema-1b • 3 Su(H), 2 Sim:Tgo sites I1F3sC modified from genome.ucsc.edu

  15. Where’s the rho mesectoderm element? • Multiple regions bound by Dl, Sna, Twi • Upstream region I: 1 conserved Su(H), 3 Sim:Tgo sites (0 conserved • Upstream region II : 0 Su(H), 4 Sim:Tgo sites (3 conserved) • Upstream region III: 1 Su(H), 2 Sim:Tgo sites I II III modified from genome.ucsc.edu

  16. Midline Primordum Elements with Sim:Tgo and Su(H) consensus sites • Sim:Tgo=ACGTG • Su(H)=T/C A/G TG A/G/T GAA

  17. Sim-Su(H) hypothesis • Sim requires a co-activator in neuroectoderm • Su(H) activation (from Notch) detected by Su(H)-LacZexpantion thru neuroectoderm • Su(H) required for Sim initiation in mesectoderm • Many Sim target elements have conserved Su(H) sites • Sim and Su(H) sites can overlap, evolutionary tuning mechanism?

  18. rho MLE expression: St. 13 U S S w.t. U S X X S SimMut U X S S Su(H)Mut

  19. rho MLE expression: St. 14

  20. Where’s the rho mesectoderm enhancer?

  21. Sema-1b

  22. sim

  23. hlh m5/8

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