Data from:

CavalliCavalli LabLab

Analysis of cis action of PRE/TRE in the regulation

of cellular memory

An example of a functional molecular study

Data from: Dejardin, J., and Cavalli, G. (2004). Chromatin inheritance upon Zeste-mediated Brahma recruitment at a minimal cellular memory module. Embo J 23, 857-868.Déjardin, J., Rappailles, A., Cuvier, O., Grimaud, C., Decoville, M., Locker, D., and Cavalli, G. (2005). Recruitment of Drosophila Polycomb Group proteins to chromatin by DSP1. Nature, (2005) 434, 533-538

Fab-7 UAS-lacZ white

Hsp 70 GAL4

Driver construct

Reporter construct

The GAL4 system for the study of PRE/TRE function: mimicking the developmental pathway leading to

maintenance of homeotic gene expression

Check Eye colorLight Eye color white repressed

Embryo

Larva

Pupa1st 2nd3rd instar

HsGAL4 pulse during early development

Experimental approach

?

Maintenance of active states

GAL4

Transient transactivator

If there is maintenance of cellular memory: sustained gene expression after decay of the primary transactivator

Expression levels

embryogenesis

L1 L2 L3 pupa adulthood

If no memory: loss of transient activation is expected

Chromosomal elements maintaining memory of active and repressed chromatin states are defined as “Cellular

Memory Modules”

larva

Fab-7: a cellular memory module (CMM) that maintains active as well as silenced chromatin throughout

development

+HS

-HS

lacZ whiteFab-7

G

3,6 kb

Gal4hsp70

GAL4HS

UAS

Beta-gal stains to study lacZ expression

lacZ whiteUAS

Ab-Fab(217 bp)

The 219 bp Ab-Fab fragment is a minimal CMM

Embryonic pulse of GAL4

Activated state correlates with Zeste recruitmentActivated state correlates with Zeste recruitment

DAPI FISH Zeste Merge Merge split

Ab-FabNo GAL4

Ab-FabLow GAL4

Ab-FabGAL4 pulse in embryos

Ab-Fab minimal CMM

Still a CMM ?

Still a PRE ?

Z-Fab

ggzzz p pp z

Molecular swicth for activation/repression: the role of Zeste proteinMolecular swicth for activation/repression: the role of Zeste protein

PREs/TREs contain binding sites for the Zeste protein.

Zeste was suggested to be a trxG member on the basis of its ability to recruit the Brm chromatin remodeling complex in vitro and of genetic evidence on the effect of z mutations on PREs in transgenes

… but Zeste was also shown to be a component of PcG complexes in vitro

Is Zeste involved in silencing or in activation at PREs/TREs in vivo?

PH

Mutation of Zeste binding sites does not impair PcG recruitmentMutation of Zeste binding sites does not impair PcG recruitment

Ab∆Z-Fab lines

GafPho

Embryonic Pulse of GAL4

Zeste binding site mutation abolishes maintenance of active chromatin statesZeste binding site mutation abolishes maintenance of active chromatin states

no HS

HSE

Zeste stabilizes PH recruitment at Zeste stabilizes PH recruitment at Fab-7Fab-7

PH

PH

HSEPH

Wild type

Deletion of Zeste sites

zestezeste gene behaves like a trxG gene if Zeste sites gene behaves like a trxG gene if Zeste sites are present, The action of are present, The action of brmbrm parallels the parallels the

effect of effect of zestezeste

Z-Fab

WT trxE2 brm2

Ab-Fab

za

Activated state correlates with BRM recruitmentActivated state correlates with BRM recruitment

+brm2

+WT

-WT

Embryonic activation

Z-Fab;hsGAL4

31A31F1

32A

33A

30A

DAPI FISH BRM MERGE Split merge

Ab14

Ab-Fab

;hsGAL4

43A

44A

42A

42F1

43A 42F1

44A

42A

43A

42F1

42A

44A

+

+

-

Analysis of recruitment of PcG proteins at PREs

Jérome Déjardin

Collaborator:Daniel Locker

University of OrléansFrance

Minimal DNA sequence requirements for inheritance

of silencing

Abd-B abd-A

PRE Boundary

d p

3,6 kb

Fab7

In Fab-7 there are 3 binding sites for Pho, 3 sites for zeste and two sites for GAF within a small region of about 200 nucleotides: is this sufficient to convey memory of

repressed chromatin states?

Midi-Fab

Ab-Fab

Ab- Pho

Ab- Gaga

Syn-Fab

PH FISH mergeEye color

Ab-Fab line

CONSTRUCT SILENCING PH BINDING

YESYES

NONO

YES YESNO

NO

NO

NO

NO

Fab-73.6 Kb

Mini-Fab

NO

A minimal Fab-7 silencer

C T G C CT C T G A A A A C A T G G C C T C T Tmcp core PRE(2)

bxd (1)

C A A A AT C C G A A A A T G A G C C A T A A Abxd (2)

En core PRE

G C C G A G C T G A A A A T G A A G A A G A A Giab7core PRE

G G C C A G A A A A T G G C T G C C T T GT A G

C A A T G T C G G A A A A A G A G G C C A T G Tmcp core PRE(1)

A C A A G C C C G A A A A A G A A G A A G A A G

A A A A A G A GC T CG A T A AG C CA A A GTbxd (3) Pho site at 12 bp

Pho site at 34 bp

Pho site at 4 bp

T G G C A G A A A A A A G A A G A T G A AG A C

A A A A A G A A A A G G A A A C G A G A CG C APho site at 4 bp

C A A G A G A A A A G C G C A T G C T C GG G A

Ph-p PRE418 (1)

Ph-p PRE418 (2)

Ph-p PRE418 (3)

Pho site at 8 bp

C G C A T G A A A A T T T A T G C G G C CT C A

G G A G C G A A A A G A A C A A C A A C CG T G APho site at 89 bpAntp-P1 PRE (1)Antp-P1 PRE (2)

A T

A T

The GA motif is present in all known PRE, often close to Pho sitesThe GA motif is present in all known PRE, often close to Pho sites

Testing the GA motif by adding it to the synthetic sequenceTesting the GA motif by adding it to the synthetic sequence

Synthetic-Fab

SynGA-Fab

Mini-Fab

Ab-Fab

‘ PRE activity ’

yes

no

no

?

Constructs

GA motif (15 nucleotides added)

Ab-Fab

Synthetic-Fab

SynGA-Fab

PRE activity

yes

no

yes (weak)

Constructs

The GA motif restores patterned variegationThe GA motif restores patterned variegation

The GA motif recruits PH to SynGA-Fab The GA motif recruits PH to SynGA-Fab in vivoin vivo

Signal weaker than the one observed for Ab-Fab

Other unknown determinants in Ab-Fab may be important for PRE strength

SynGA-Fab

Syn26

SynGA23

w1118

DAPI PH

PH+ FISH

89B5

89B5

72BSyn-Fab

No Pairing Sensitive Silencing

Mutation of the GA motif disrupts PcG silencing and Mutation of the GA motif disrupts PcG silencing and recruitmentrecruitment

DAPI FISH Merge

Ab-Fab

AbGAmut

Polyhomeotic

AbGAmut

AbGAmut-Fab

Ab-Fab

The DSP1 protein is recruited at Ab-The DSP1 protein is recruited at Ab-FabFab in a GA motif -dependent manner in a GA motif -dependent manner

DAPI DSP1 FISH Merge

Ab-Fab

AbGAm

In conclusion, we have reconstituted a synthetic PRE and have defined minimal requirements of PcG proteins. However, part of the

silencing function is still missing, and we still need to understand how are the trxG activators recruited…

GAF GAF GAF

GAF

GAF

GAF

GAF

Summary mechanism of CMM action an equilibrium Summary mechanism of CMM action an equilibrium between silencing and activation dependent on early between silencing and activation dependent on early

developmental cuesdevelopmental cues

CBP

CMMdRing PH

PC

Pho

PRC1/other PcG related complexes

PSC ZZ Z

Z

PRE TRE

BAP111osa

mor

BRM-C

Brm

?

trx

TAC-1

?Z

When PRE is favored during early development…When PRE is favored during early development…

dRing PHPC

Z

PSC

dRing PHPC

Z

PSC

CMMdRing PH

PCPSC

PRECBP

Z ZZ Z

Z

BAP111osa

morBrmtrx

TRE

Pho

CavalliCavalli LabLab

… … and when TRE function is activatedand when TRE function is activated

CBPtrx

CMM

dRing PHPC

Z

PSC

Z Z Z

Z ZZZ

PRE

TRE

BAP111osa

morBrm

CBPtrx

BAP111osa

morBrmBAP111osa

morBrm

Pho