rhomboid MLE regulation by Sim:Tgo and Su(H) 8/24/09
Jan 23, 2016
rhomboidMLE regulation by Sim:Tgo and Su(H)
8/24/09
Evidence for Sim Target Regulation
• expression is lost in sim mutants
• Sim:Tgo sites are required for midline expression
• Sim over-expression expands target expression in neuroectoderm– What restricts to neuroectoderm?– Maybe Su(H), says Scott
Nambu, 1991
heat-shock Simcontrol
sim
rho
cdi
sli
Circumstantial evidence for Sim-Su(H) cooperation
• sim initation requires Su(H)• sim, m5/8 (mesectoderm), rst F6d (midline) expression is
expanded with Su(H) 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
Element Sim:Tgo Su(H)
sim 3.7pE 4(5) 6 (10)
sim 1.0pL 2 1
sim Sandmann 3 0
rho E-Ss 2 1
tl 950 4 2
rst F6d 2 2
Element Sim:Tgo Su(H)
btl 23 3 0
Sema1b I1F3sC 3 2
hlh m5/8 1 4
Kr StH0.6 1 0
Kr PP3.0 3 2
• Sim:Tgo=ACGTG• Su(H)=T/C A/G TG A/G/T GAA
Test cases for Sim-Su(H) cooperation
rhoMLE
Sim
Su(H)
Sim
sema1bI1F3sC
Sim
Su(H)
Sim
sim1.0pLSim
Su(H)
Sim
Sim
Su(H)
rho embryonic expressionst. 5 st. 6 st. 9
st. 11 st. 13 st. 17
• Initially broad neuroectodermal expression• Refines to mesectoderm before gastrulation• In all midline thru st. 11
rho cis-regulatory locus
• Ip ‘92- 2.2 kb upstream controls neuroectoderm and midline expression– NEE activated by Dorsal, Twist, repressed by Snail– MLE contains 2 Sim:Tgo binding sites
• “1- to 2-hr delay in onset of (rho2.2-lacZ) expression in the mesectoderm relative to the endogenous rho gene”
rho embryonic cis-regulation
Ip, 1992
Zelzer, 2000
rhoMLE (Midline Element) motifs
Sim/SuH sites
Repeats 1/2
rho MLE expression
SU
S
w.t.
SU
S
SimMut
X X
SU
S
Su(H)Mut
X
St. 11 St. 13
GFPSim GFPSimGFP GFP
12xSu(H)-LacZ expression
Wheeler et al.,2008
• Expression in AMG, PMG, MP5, MP6, MNB• VUM expression gradient • Similar to rhoMLE SimMut
How is rhoMLE regulated?
• Sim enhances AMG, MP1, MP4 expression– Sim not required for posterior midline expression – Sim not required for midline restriction
• Activated Su(H) enhances AMG, MP1 (With Sim) PMG and Posterior neurons (without Sim)– Inactive Su(H) represses MP3/4?
• rhoMLE is not mesectoderm element– Where is it?
sim
• 6(10)Su(H), 4(5) Sim:Tgo sitessim3.7
modified from genome.ucsc.edu
hlh m5/8
5/8
modified from genome.ucsc.edu
• 4 Su(H), 1 Sim:Tgo sites
Sema-1b
• 3 Su(H), 2 Sim:Tgo sites
I1F3sC
modified from genome.ucsc.edu
Where’s the rho mesectoderm element?• Multiple regions bound by Dl, Sna, Twi• Upstream region I: 1 conserved Su(H), 3 Sim:Tgo sites (0
conserved• Upstream region II : 0 Su(H), 4 Sim:Tgo sites (3 conserved)• Upstream region III: 1 Su(H), 2 Sim:Tgo sites
I II III
modified from genome.ucsc.edu
Midline Primordum Elements with Sim:Tgo and Su(H) consensus sites
Element Sim:Tgo Su(H)
sim3.7 4 6
sim1.0 2 1
simSandmann 3 0
rho E-Ss 2 1
tl950 4 2
rst F6d 2 2
Element Sim:Tgo Su(H)
btl 23 3 0
Sema1b I1F3sC 3 2
hlh m5/8 0 0
Kr StH0.6 1 0
Kr PP3.0 3 2
• Sim:Tgo=ACGTG• Su(H)=T/C A/G TG A/G/T GAA
Sim-Su(H) hypothesis
• Sim requires a co-activator in neuroectoderm• Su(H) activation (from Notch) detected by
Su(H)-LacZ expantion thru neuroectoderm• Su(H) required for Sim initiation in
mesectoderm• Many Sim target elements have conserved
Su(H) sites• Sim and Su(H) sites can overlap, evolutionary
tuning mechanism?
rho MLE expression: St. 13
SU
S
SU
S
SU
S
w.t.
SimMut
Su(H)Mut
X X
X
rho MLE expression: St. 14
Where’s the rho mesectoderm enhancer?
Sema-1b
sim
hlh m5/8