1. Stories arising…
Joe Pearson
5/10/2009

2. CG13333 5’Frag widely expressed, including in midline
Repeated AGGTRG identified by PhyloGibbs
Mutating AGGTRG eliminates all expression
Mutating ACGTG specifically reduces midline expression

3. CG13333 5’ motifs Dmel Dsim Dsec Dyak Dere Dana Dpse Dper Dwil Dgri
Dmoj
Dvir
CAGGTAG
CAGGTAC
TAGGTGG
TAGGTAG
CAGGTAG
CAGGTAA
CACGT
CTCGT
GAGGTAG
GAGGAAG
When comparing the orthologous sequences from the 12 sequenced drosophila genomes,
I found a motif that is repeated 4 times in 300 bp, which is pretty statistically significant.
In fact, two instances of this motif are more conserved than the putative Sim binding site.
I am currently testing this motif for function.
PhyloGibbs identified AGGTRG as a repeated, conserved motif
Perfectly conserved instances extend to CAGGTAG
Zelda binds CAGGTAG to promote widespread pre-blastoderm expression (Liang et al., 2008)
Is Zelda acting as ubiquitous activator in late embryo?

4. Next Steps: CG13333 Mutate “Zelda” sites to block Zelda binding
CG13333 expression in vielfaltig/zelda mutants
vfl
Staudt et al., 2006

5. argos I1-1 I1-3 Intron1-1: Sporadic PMG, some AMG Intron1-3: AMG only
midline expression
no midline expression
untested
I1-1
I1-3
Intron1-1: Sporadic PMG, some AMG
Intron1-3: AMG only
Phylogibbs identified SCGGAAGTS in I1-3, similar to ETS consensus CMGGAWRY
4 of 5 fragments transformed.
3 of 5 fragments tested.
1 fragment shows midline expression.
This element gives sporadic expression in one or more PMG, which are interesting in that they all die by the end of embryogenesis,
and it isn’t clear what they do before that.
Also see some AMG expression, especially in anterior segments and as development continues.
GFP
SIM
EGFR
MAPK
Pnt
Argos

6. argos
100%
SCGGAAGTS
CMGGAWRY
ACGTG 0%
subA
subB
subC
subD
SubA
I1f3
subB
subC
SubD

7. Next Steps: argos Mutate ETS-like sites Mutate Sim site
Test argos role in AMG development?
Cell autonomous inhibition of EGFR signaling?
Inhibition of EGFR signaling in other AMG?
Inhibition of EGFR in other cells?

8. Glec locus dissection 5’-2 vs. 5’2sA Separable enhancers
Derepression in MP1
5’-3 vs. 5’3sA
Derepression in anterior midline
5’2 vs. 5’3sA
Repeated 300bp fragment with 2 Sim:Tgo sites
5’-2 vs 5’2sA
5’-1 vs 5’2sA
5’-3 vs 5’3sA
5’2 vs 5’3sA

10. repeated glec upstream sequence
Evolutionary history
Promoter-proximal (5’-3sA) instance ancestral
Highly conserved through D. pseudoobscura
Degenerate match through D. virilis
Unclear when duplication occurred
Repeated Anterior midline motif?
Two Sim:Tgo sites, both conserved
Why repeated at same gene?

11. Next Steps: glec Test remaining 5’-2 and 3’-1 subfragments
Mutate Sim:Tgo, CREB binding sites in 5’-3
Test repeated motif for midline expression function
Test 5’-2 and 5’-3 in pBPGw-UCP (GAL4 reporter)
GAL4 amplification?
hsp70 vs. UCP?

12. No midline primordium expression
VUM expression
Not 100%
Missing motif?
Not complete element?
No Sim:Tgo site
No midline primordium expression
Shared enhancer with ect3?

13. 600 bp fragment drives expression in all midline primordium (mesectoderm?)
3 Sim:Tgo sites
2 Su(H) sites
2 Twist sites (also bound in vivo)
Caveat: overexpression of sim does not expand sema-1b expression