Stories arising…

Joe Pearson5/10/2009

CG13333

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

CG13333 5’ motifs

CAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTACCAGGTAGCAGGTAC

DmelDsimDsecDyakDereDanaDpseDperDwilDgriDmojDvir

TAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTGGTAGGTAG

CAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAGCAGGTAACAGGTAG

CACGTCACGTCACGTCACGTCTCGTCACGTCACGTCACGT

GAGGTAGGAGGAAGGAGGAAG

•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?

Next Steps: CG13333

• Mutate “Zelda” sites to block Zelda binding• CG13333 expression in vielfaltig/zelda

mutants

vflStaudt et al., 2006

argos

GFP SIM

midline expression no midline expression untested

•Intron1-1: Sporadic PMG, some AMG•Intron1-3: AMG only•Phylogibbs identified SCGGAAGTS in I1-3, similar to ETS consensus CMGGAWRY

I1-1 I1-3

EGFR MAPK Pnt Argos

100%

0%

CMGGAWRYSCGGAAGTS

ACGTG

subC

subC subB subDsubA

SubA SubDI1f3 subB

argos

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?

•5’-2 vs. 5’2sA•Separable enhancers

•5’-1 vs. 5’2sA•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

Glec locus dissection

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?

1 1 CAGCAGTTAAACTCATGCGCAGCGCGTGCTCCATTTCCATTGATCGGTTGCGTTGGgttc-GGATTCATTCATTTCACTATCATTCAATGGTTTCTGACC 2 1 CAGCAGTTAACCTTATGCGCAGCGCGTGCGACAGTTCCATTGATCGGTTGCGTTGGctcctGGGTTCATTCATTTCATTATCATTCAATGGTTTCTGACC 1 100 TTTTGAGCCGGGGTTACATGGTACGTGGGCCATAAACTAATTTCTGCCAGCCAGTCGGgaaaaaAAAACAGCTTGAGTTTTCCGCAGACAAGCTGCGTGC 2 101 TTTTGAGCCGGGGTTACATGGTACGTGGCCCATAAACTAATTTCTGCCAGCCAGTCGGcgg---AAAACAGCTTGAGTTTTTCGCAGACAAGATGCGTGC 1 200 GGCGGAAGTGACGGCGGAAAAGTAGCCCATCAATAGACCCAGACACGTACCCAGAAAACATCTaAAAGATACAAAAATACACGCCGACATGCATTCaaat 2 198 GGCGGAAGTGACGACGGAAAAGTAGCCCATCAGTAGACCCAGACACGTACCCAGAAAACATCTgAAAGATACAAATATACGCGACGACATGCATTCga-- 1 300 aatgccCAGTGAAAAATCAGCGCAGCTACGAGATATCAAAATATAAGCAAAGCTGAA 2 295 ------CAGTGAAAAATCAGCGCAGCTACGAGATATTAAAAAAAAAACAAAGCTGAA

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?

• VUM expression– Not 100%

• Missing motif?• Not complete

element?• No Sim:Tgo site

• No midline primordium expression– Shared enhancer

with ect3?

•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