Stories arising… Joe Pearson 5/10/2009

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 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?

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

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

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

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?

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

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?

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?

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?

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