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 novel motifsIdentify matches to known motifs Test Motifs Confirm upstream regulatorIdentify 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 ElementSim:TgoSu(H) sim 3.7pE4(5)6 (10) sim 1.0pL21 sim Sandmann30 rho E-Ss21 Tl 95042 rst F6d22 ElementSim:TgoSu(H) btl 2330 Sema1b I1F3sC32 hlh m5/814 Kr StH0.610 Kr PP3.032 Sim:Tgo=ACGTG Su(H)=(T/C) (A/G) TG (A/G/T) GAA

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

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  -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.SimMutSu(H)MutTwistMut st. 5 st. 9 sema1b I1F3sC Sim Su(H) Sim Su(H) Twi

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 3 rd Twist site (Sim site #3) CCGCACGTGATT Sim site #3 Twist (for) Twist (rev) BTNP ChIP-Chip, 145 sites * *

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

10. CG13333 5’-GFP motif requirements wild-typeMotifA1-4ZldMut13 stage 11 stage 13 stage 14 midline CG13333 5’-GFPSimEn

11. zelda is required for cg13333 expression Df(1)Exel6253/FM7cTwistGFPDf(1)Exel6253/Df(1)Exel6253 En cg13333 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 midline expression No  Zelda is not directly regulating CG13333 midline expression

13. zelda midline expression J. Watson

14. CG13333-Zelda