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