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