Download - The trouble with binding sites
The trouble with binding sites
Joe Pearson1/11/10
Long-Term Goal: Identify molecular mechanisms regulating midline gene expression
Choose midline genes
Identify midline CRMs
Identify novel motifsIdentify matches to known 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)
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
sim1.0pL
Sim Su(H)
Sim
Sim
Su(H)
Twi Twi
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 -GFP)– also required for expression
in subset of lateral cells• Twist sites not required?
Morel and Schweisguth, 2000
EnSimSema-1b:GFP
EnSimSema-1b:GFP
sema-1b I1f3sC gfp expression
w.t. SimMut Su(H)Mut TwistMut
st. 5
st. 9
sema1bI1F3sC
Sim
Su(H)
Sim Sim
Su(H)
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)
CCGCACGTGATT
Sim site #3
Twist(for)
Twist(rev)
BTNP ChIP-Chip,145 sites
*
*
CG13333 5’ enhancer
cg13333gfp
Motif A: AGGT(A/G)G
CAGGTAGTAGGTGG
CAGGTAGACGTG
GAGGTAG
A A A ASSimTgo: ACGTG
GFPSimEn
TAGTeamCAGGTAGTAGGTAGCAGGCAGTAGGTAA
CG13333 5’-GFP motif requirementswild-type MotifA1-4 ZldMut13
stage 11
stage 13
stage 14midline
CG13333 5’-GFP Sim En
zelda is required for cg13333 expression
Df(1)Exel6253/FM7cTwistGFP Df(1)Exel6253/Df(1)Exel6253
Encg13333
SimTauGFPzldtkr
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