Transcript The trouble with binding sites Joe Pearson 1/11/10 Long-Term Goal: Identify molecular mechanisms regulating midline gene expression Choose midline genes Identify midline CRMs Identify matches to.

The trouble with binding sites
Joe Pearson
1/11/10
Long-Term Goal: Identify molecular mechanisms
regulating midline gene expression
Choose midline genes
Identify midline CRMs
Identify matches to known motifs
Identify novel motifs
Test Motifs
Test Motifs
Confirm upstream regulator
Identify upstream regulator
Incorporate into midline development models
Notch/Su(H) in midline gene regulation
• Initial sim expression requires Su(H)
• sim, hlh-m5/8 (mesectoderm), rst F6d (midline) expression is
expanded with Notch 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)
Element
Sim:Tgo Su(H)
sim 3.7pE
4(5)
6 (10)
btl 23
3
0
sim 1.0pL
2
1
Sema1b I1F3sC
3
2
sim Sandmann
3
0
hlh m5/8
1
4
rho E-Ss
2
1
Kr StH0.6
1
0
Tl 950
4
2
Kr PP3.0
3
2
rst F6d
2
2
Sim:Tgo=ACGTG
Su(H)=(T/C) (A/G) TG (A/G/T) GAA
Test cases for Sim-Su(H) cooperation
Su(H)
Sim
Sim
rhoMLE
Su(H) Su(H)
SimSim Sim
Twi
Twi
sema1b
I1F3sC
Su(H)
Sim
Sim
sim1.0pL
Effects of mutating Sim, Su(H),
Twist sites in midline CRMs
•Sim
•Required for Sema-1b and sim maintenance
•Maintains rho expression in midline subset
•Su(H)
•Not required for Sema-1b or sim maintenance
•Required for rho activation, “priming” for Sim maintenance
•Twist
•Not required for Sema-1b activation
Problems with Sema-1b results
• Sim sites required for all
expression
– including mesectoderm
expression at stage 5?
• Su(H) sites may cause
variation of initial
expression (difficult to see
by a-GFP)
– also required for expression
in subset of lateral cells
Morel and Schweisguth, 2000
En
Sim
Sema-1b:GFP
• Twist sites not required?
En
Sim
Sema-1b:GFP
sema-1b I1f3sC gfp expression
w.t.
SimMut
Su(H)Mut
TwistMut
st. 5
st. 9
Su(H) Su(H)
SimSim Sim
sema1b
I1F3sC
Twi
Twi
How I explain sema-1b results
• Sim
– Site #3 may also be Twist site
(CACGTG)
– Mutation abolishes initial and
maintained expression
• Su(H)
– Subtle alteration of
levels/expression sharpness?
• Twist
– I didn’t mutate 3rd Twist site
(Sim site #3)
Sim site #3
Twist
(for)
*
CCGCACGTGATT
*
Twist
(rev)
BTNP ChIP-Chip,
145 sites
CG13333 5’ enhancer
Motif A: AGGT(A/G)G
SimTgo: ACGTG
A A
CAGGTAG
GFP
Sim
En
A S
A
CAGGTAG
TAGGTGG
GAGGTAG
ACGTG
gfp
cg13333
TAGTeam
CAGGTAG
TAGGTAG
CAGGCAG
TAGGTAA
CG13333 5’-GFP motif requirements
wild-type
stage 11
stage 13
stage 14
midline
CG13333 5’-GFP Sim En
MotifA1-4
ZldMut13
zelda is required for cg13333 expression
En
cg13333
Df(1)Exel6253/FM7cTwistGFP
Df(1)Exel6253/Df(1)Exel6253
SimTauGFP
zld
tkr
J. Watson
Resolving the CG13333 Cis-Trans conflict
• zelda is required for CG13333 epidermal and midline
expression
• Zelda sites are not required for CG13333 midline expression
• How can I resolve zelda-Zelda conflict?
– Identify which MotifA site (2,4) sufficient for midline expression
– Use S2 cell culture reporter assays/EMSAs to test whether Zelda binds
these sites
• Yes  Zelda is directly regulating CG13333 midline expression
• No Zelda is not directly regulating CG13333 midline expression
zelda midline expression
J. Watson
CG13333-Zelda