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The trouble with binding sites

The trouble with binding sites. Joe Pearson 1/11/10. Long-Term Goal: Identify molecular mechanisms regulating midline gene expression. Choose midline genes. Identify midline CRMs. Identify matches to known motifs. Identify novel motifs. Test Motifs. Test Motifs.

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The trouble with binding sites

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  1. The trouble with binding sites Joe Pearson 1/11/10

  2. Long-Term Goal: Identify molecular mechanisms regulating midline gene expression Choose midline genes Identify midline CRMs Identify matches to known motifs Identify novel motifs Test Motifs Test Motifs Confirm upstream regulator Identify upstream regulator Incorporate into midline development models

  3. Notch/Su(H) in midline gene regulation • Initial sim expression requires Su(H) • sim, hlh-m5/8 (mesectoderm), rst F6d (midline) expression is expanded with Notch 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) Twi Twi Sim Sim Sim sema1b I1F3sC Su(H) Sim Sim sim1.0pL

  5. Effects of mutating Sim, Su(H), Twist sites in midline CRMs • Sim • Required for Sema-1b and sim maintenance • Maintains rho expression in midline subset • Su(H) • Not required for Sema-1b or sim maintenance • Required for rho activation, “priming” for Sim maintenance • Twist • Not required for Sema-1b activation

  6. Problems with Sema-1b results • Sim sites required for all expression • including mesectoderm expression at stage 5? • Su(H) sites may cause variation of initial expression (difficult to see by a-GFP) • also required for expression in subset of lateral cells • Twist sites not required? Morel and Schweisguth, 2000 En Sim Sema-1b:GFP En Sim Sema-1b:GFP

  7. sema-1b I1f3sC gfp expression w.t. SimMut Su(H)Mut TwistMut st. 5 st. 9 Su(H) Su(H) Twi Twi Sim Sim Sim sema1b I1F3sC

  8. How I explain sema-1b results • Sim • Site #3 may also be Twist site (CACGTG) • Mutation abolishes initial and maintained expression • Su(H) • Subtle alteration of levels/expression sharpness? • Twist • I didn’t mutate 3rd Twist site (Sim site #3) Sim site #3 Twist (for) * CCGCACGTGATT * Twist (rev) BTNP ChIP-Chip, 145 sites

  9. CG13333 5’ enhancer Motif A: AGGT(A/G)G TAGTeam CAGGTAG TAGGTAG CAGGCAG TAGGTAA GFP gfp Sim cg13333 En SimTgo: ACGTG A A A S A GAGGTAG CAGGTAG CAGGTAG ACGTG TAGGTGG

  10. CG13333 5’-GFP motif requirements wild-type MotifA1-4 ZldMut13 stage 11 stage 13 stage 14 midline CG13333 5’-GFP Sim En

  11. zelda is required for cg13333 expression En cg13333 Df(1)Exel6253/FM7cTwistGFP Df(1)Exel6253/Df(1)Exel6253 SimTauGFP zld tkr J. Watson

  12. Resolving the CG13333 Cis-Trans conflict • zelda is required for CG13333 epidermal and midline expression • Zelda sites are not required for CG13333 midline expression • How can I resolve zelda-Zelda conflict? • Identify which MotifA site (2,4) sufficient for midline expression • Use S2 cell culture reporter assays/EMSAs to test whether Zelda binds these sites • Yes Zelda is directly regulating CG13333 midlineexpression • No Zelda is not directly regulating CG13333 midline expression

  13. zelda midline expression J. Watson

  14. CG13333-Zelda

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