1. Identification and Comparison of Midline Cis-Regulatory Elements

2. Does common expression indicate common regulation? A B C D CD AB A B CD CDAB wxyz

3. Project Goals Identify Large Set of Midline Primordium Enhancers Compare enhancers for shared motifs Experimentally confirm required motifs (activators/repressors) Contribute to knowledge of Midline Development

4. Known Midline Enhancers Sim 2.8 pE Sim (Sandmann) sli380 rho E-Ss Rst F6d Kr PP3.0Hz Kr StH0.6Hz btl-23 tl950

5. Candidate Midline Enhancer Sources (midline database search) Midline Primordia Midline Glia 191369

6. CG13333 St. 11 Midline Genes argos ab bib bnb btl cdi cenB1A CG31145 CG32594CG3409 CG7224 CG8291 ctdveglec hbsHGTX mfas oc rhoSema-1b sog sty vvlTkr CG9634 rstsimTl

7. Midline/Glial Expressing Genes mfas oc rho sim sty wrapper alan-shepard slit GH22170 argos cdi CG13333 CG31145 CG8291 CG9634 cut dve glec hbs

8. Midline Primordium Genes CG7224 cenB1A (Sema-1B) bib bnb CG3409 CG32594 HGTX sog ab Tkr ct vvl

9. AttL2 Enhancer Cloning Pipeline Entry Vector Reporter Vectors AttL1 AttR2 AttR1 Reporter  C31 AttB

10. Entry Vector Issues pENTR/D-TOPO does not like big fragments Enhancer hunts do like big fragments Solutions: –Use Restriction/Ligation into pENTR/D-TOPO –Use pCR8/GW/TOPO vector (TA cloning)

11. NotI/EagI PstI SbfI NgoMIV/ NaeI FseI NcoI DraI PmeI SpeI AscI Compatible ends: NotI/EagISbfI/PstI->(NsiI)NgoMIV->(AgeI)/(XmaI) NcoI->(BspHI)/(PciI)SpeI->(AvrII)/(NheI)/(XbaI)AscI->(MluI)/(BssHII) pGEM-T sites: NcoI, NotI (both sides), NsiI, PstI, SpeI pCRII sites: NotI, NsiI (both sides), SpeI, XbaI Rare MCS pENTR vector AttL2 AttL1 pENTR/D-Topo

12. Reporter Vector Issues pBPGw has GAL4 pBPGw doesn’t have a promoter pBPGw is named pBPGw

13. pMintGate pBPJPhGFPw

14. Test Case:Sim2.8 pE MintGate GFP.nls  C31 AttB CinnamonGate DsRed.nls  C31 AttB pBPGw GAL4  C31 AttB

15. Preliminary results Cloned Sim2.8 pE into pMintGate, pCinnamonGate, pBPGw One Insertion isolated for Sim2.8 pMintGate (so far) (10/29: 2 lines CinnamonGate, 1 line pBPGw)

16. Applications of Enhancers GAL4-UAS –Cell-specific gene rescue –RNAi Cell labeling Enhancer Dissection –Common Activation? –Common Repression? –Evolutionary Conservation?

17. Future Experiments Clone/Test Putative Enhancer Fragments –Best case: 30+ midline enhancers with similar expression –compare bioinformatically –Identify required motifs Replace current FPs with newer/better FPs –mCherry, mPlum, Cerulean, CFP, etc –Live, in vivo comparisons of multiple midline enhancers Genome-wide midline enhancer tests –Chip-Seq –FAIRE

18. ChIP-Seq + Fix chromatin, IP with SIM FACS Solexa Sequence

19. FAIRE

20. + Fix chromatin, Phenol Extract Free DNA FACS Label DNA, Custom Microarray

21. geneA geneB geneC geneA geneB geneC Midline cellsMidline Subtracted ABCDEFGHIJKLABCDEFGHIJKL ABCDEFGHIJKLABCDEFGHIJKL FAIRE