Identification and Comparison of Midline Cis-Regulatory Elements

Does common expression indicate common regulation?

A

B

C

DC D

A B

A

B C D

C DA B

w x y z

Project Goals

• Identify Large Set of Midline Primordium Enhancers

• Compare enhancers for shared motifs

• Experimentally confirm required motifs (activators/repressors)

• Contribute to knowledge of Midline Development

Known Midline Enhancers

• Sim 2.8 pE

• Sim (Sandmann)

• sli380

• rho E-Ss

• Rst F6d

• Kr PP3.0Hz

• Kr StH0.6Hz

• btl-23

• tl950

Candidate Midline Enhancer Sources(midline database search)

Midline Primordia

Midline Glia

1913 69

CG13333

St. 11 Midline Genesargosab bib bnb

btl

cdi cenB1A CG31145

CG32594 CG3409 CG7224 CG8291

ct dve glec

hbs HGTX mfas oc

rhoSema-1b

sog sty vvlTkr

CG9634

rst sim Tl

Midline/Glial Expressing Genes

• mfas• oc• rho• sim• sty• wrapper• alan-shepard• slit• GH22170

• argos• cdi• CG13333• CG31145• CG8291• CG9634• cut• dve• glec• hbs

Midline Primordium Genes

• CG7224

• cenB1A

• (Sema-1B)

• bib

• bnb

• CG3409

• CG32594

• HGTX

• sog

• ab

• Tkr

• ct

• vvl

AttL2

Enhancer Cloning Pipeline

Entry VectorReporter Vectors

AttL1AttR2AttR1

Reporter

C31

At

tB

Entry Vector Issues

• pENTR/D-TOPO does not like big fragments

• Enhancer hunts do like big fragments

• Solutions:– Use Restriction/Ligation into pENTR/D-TOPO– Use pCR8/GW/TOPO vector (TA cloning)

NotI/EagI PstISbfI

NgoMIV/NaeIFseI NcoI DraI

PmeISpeI AscI

Compatible ends:

NotI/EagI SbfI/PstI->(NsiI) NgoMIV->(AgeI)/(XmaI)

NcoI->(BspHI)/(PciI) SpeI->(AvrII)/(NheI)/(XbaI) AscI->(MluI)/(BssHII)

pGEM-T sites: NcoI, NotI (both sides), NsiI, PstI, SpeI

pCRII sites: NotI, NsiI (both sides), SpeI, XbaI

Rare MCS pENTR vector

AttL2AttL1

pENTR/D-Topo

Reporter Vector Issues

• pBPGw has GAL4

• pBPGw doesn’t have a promoter

• pBPGw is named pBPGw

pMintGate

mintGate9679 bp

pBR322 origin

GFP-NLS stop

SV40 3'UTR

EGFP 5'UTR

ChlorR

ccdB

EGFP

tra NLS

mini-white

AmpR

AttR

AttR

phiC31 AttB

Apa I (4225)

Bgl II (1804) Kpn I (1847)

Nde I (7564)

Sac I (5688)

Xho I (1869)Bam HI (1863)

Eco RI (1826)

Spe I (3165)

Asc I (1836)

Asc I (3427)

Asc I (7554)

Xba I (1812)

Xba I (2934)

Xba I (3171)

pBPJPhGFPw

pBPJPhGFPw

Test Case:Sim2.8 pE

MintGate

GF

P.nls

C31

At

tB

CinnamonGate

DsR

ed.nlsC

31

AttB

pBPGw

GA

L4

C31

At

tB

Preliminary results

• Cloned Sim2.8 pE into pMintGate, pCinnamonGate, pBPGw

• One Insertion isolated for Sim2.8 pMintGate (so far)

(10/29: 2 lines CinnamonGate, 1 line pBPGw)

Applications of Enhancers

• GAL4-UAS– Cell-specific gene

rescue– RNAi

• Cell labeling

• Enhancer Dissection– Common Activation?– Common Repression?– Evolutionary

Conservation?

Future Experiments

• Clone/Test Putative Enhancer Fragments– Best case: 30+ midline enhancers with similar

expression– compare bioinformatically– Identify required motifs

• Replace current FPs with newer/better FPs– mCherry, mPlum, Cerulean, CFP, etc– Live, in vivo comparisons of multiple midline enhancers

• Genome-wide midline enhancer tests– Chip-Seq– FAIRE

ChIP-Seq

+

Fix chromatin, IP with SIM

FACS

Solexa Sequence

FAIRE

FAIRE

+

Fix chromatin, Phenol ExtractFree DNA

FACS

Label DNA,Custom Microarray

geneA

geneB

geneC

geneA

geneB

geneC

Midline cells Midline Subtracted

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ABCDEFGHIJKL

FAIRE