cavalli lab introduction in: epigenetic regulation by polycomb and trithorax group proteins giacomo...
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CavalliCavalli LabLab
Introduction in:Epigenetic regulation by Polycomb and
trithorax group proteins
Giacomo CAVALLI. Montpellier, December 2006
CavalliCavalli LabLab
EuchrochromatinHeterochromatin
The chromatin Yin / Yang
- Less condensed- Gene rich- Active genes- Early replicating- DNA hypomethylated- Poor in histone H1- Histones have specific, activating post translational marks
- Heavily condensed- Gene poor- Silent genes- Late replicating- DNA hypermethylated- Rich in histone H1- Histones have repressive post translational marks
Where on the chromosomes are condensed and open chromatin located?
Condensed chromatin Open chromatin
Constitutive Heterochromatin
Telomeres
Centromeres
Condensed chromatin is also found at several
silent genes located in the chromosomal
arms
Typical of the active gene loci along the chromosomal arms
Polycomb group and trithorax group genes are important regulators of chromatin along the chromosomal arms
Polycomb (PcG) and trithorax (trxG) group proteins: epigenetic regulators of genome function
• Originally discovered in Drosophila as regulators of Homeotic genes, responsible for specification of the body plan, they also regulate many other targets involved in cell differentiation and proliferation
• PcG proteins silence genes, trxG proteins activate them
• Conserved throughout evolution
• In human, they maintain the fates of differentiated cells, but they are also required for cell proliferation and the maintenance of several types of stem cells including ES cells. Finally, they regulate X-inactivation in females as well as genomic imprinting
• Mutations in PcG and trxG genes induce many different cancers
PcG, trxG, and maintenance of gene expressionEarly development Establishment of patterns
Maternal, Gap, Pair-rule, Segment polarityONOFF
Ubx
Polycomb-Group trithorax-GroupMaintenance phaseTransmission of pattern after disappearance of
early factors
ON OFF
ON OFF
PcG and trxG proteins associate to multiple genomic loci
PH
DAPI
Polytene chromosome staining shows around 100 bands for each PcG protein
Merge
Binding leads to maintenance of PcG-dependent repression of reporter genes
Bound by PcG proteins in vivo (in polytene chromosomes and by cross-linking experiments)
Repression is enhanced by the presence of multiple PRE copies
PcG proteins bind to specific DNA elements, named PREs
protein motifs
Members of the PcG and of the trxG
GenetrxG
protein motifsGenePcG
Polycomb (Pc) chromo domain(Binding to H3 methyl K9 or K27)
polyhomeotic (ph) Zinc finger, SAM domain
Posterior sex combs (Psc) RING finger
PRC1 complex
Enhancer of zeste (E(z)) SET (H3K27MTase)
extra sex combs (esc) WD repeat
Esc/E(z)Complex
trithorax (trx) SET (H3K4HMTase)/ PHD-finger
Ash-1 SET (H3/H4HMTase)/ PHD-finger
TAC1 complex
brahma (brm) bromo domain
(DNA dependent ATPase/helicase)Brm complex
Trithorax-like (Trl)Zinc finger (DNA binding)BTB/POZ (dimerization)FACT
complex
Pleiohomeotic (pho) Zinc-finger (DNA binding)PhoRCComplex
OFF
ONtrxG
Maintenance of active states
(open chromatin)
PRETarget gene
Histone acetylation and methylation
(TAC1 and ASH1 complexes)
Deacetylation and methylation
(ESC-E(Z) complex)
Maintenance of repressed states
(compact chromatin)
PcG
- Chromatin compaction- H2A Ubiquitination
(PRC1 complex)
Nucleosome remodeling(BRM complex)
Ac
Me K27 H3
Action of PcG and trxG complexes on chromatin
Me K4 H3
Ub H2A
Histone H3 K27 methylation and Polycomb
Pc H3K27me3 Merge
There is a strong correlation between trimethylation of K27 (and K9) trimethylation and Polycomb recruitment at target loci.
Data from: Ringrose et al. (2004) Mol. Cell 16, 641
What does Polycomb do to chromatin ?Chromatin Condensation
Data from: Francis et al. (2004), Science 306, 1574
Recombinant PC-containing complexes can condense an array of 12 nucleosomes in vitro
Condensation requires PSC (not PH) protein, and involves histones but does not necessitate histone tails
Features of PREs and TREs, and examples of how they are studied in Drosophila
(No PREs characterized yet in vertebrates)
1. Example of PcG-dependent spatial specific silencing of homeotic genes
PcG dependent derepression of a Ubx-lacZ reporter in embryonic territories where it is normally silenced
Silencing of a Ubx-lacZ reporter mimicking the wt behaviour of the Ubx gene, which is silenced in parasegments 1 to 5
bxd5.1 UbxlacZ reporter construct
Bxd 5.1 PRE Ubx prom LacZ mini-white
Data from: Hodgson, J. W., Argiropoulos, B., and Brock, H. W. (2001). Site-specific recognition of a 70-base-pair element containing d(GA)(n) repeats mediates bithoraxoid polycomb group response element-dependent silencing. Mol Cell Biol 21, 4528-4543.
2. Silencing of a transgenic reporter gene depends on PcG and trxG proteins
Fab-7Pc +/+
Fab-7Pc -/+
Fab-7trx +/+
Fab-7trx -/+
Fab-7 UAS-lacZ white
3. Recruitment of PcG and trxG proteins to PREs: analysis in Fab-7 by a combination of immunostaining and FISH in polytene chromosomes (immuno-FISH)
24A
25E5
transgene
DAPI Immunostainingof PH protein
FISH Immuno-FISH
Fab-7 UAS-lacZ white
Transgene :
4. Recruitment of PcG and trxG proteins to PREs: analysis by ChIP
Recruitment of PcG proteins at PREs: chromatin analysis by chromatin immunoprecipitation (ChIP) and DNA microarrays (chips)
Sonicate and purify chromatin (average size = 1 kb)
Add antibody and purify antibody-chromatin complexes on Protein A Sepharose, purify DNA and amplify by Linker-mediated PCR
Cross-link cells or embryos with formaldehyde to induce protein-DNA crosslinks
Use amplified DNA as probe to hybridize DNA chips containing the genome. Extract the distribution profile of the proteins of interest
ChIP on chip
sd
en/inv
Bx-CHh
ci
Adh region
X
2L 2R
3L 3R
4
Montpellier tiling paths
Yale tiling paths
L82
X chromosome tip
bi (1)
(2)
mab2(3)
ct (4)
ph
gt/z
34D 35B 35D 36A2D 4C 5A 7B5D
elB
noc
stc
esg
PH binding profiles at the embryonic developmental stage
Negre N, Hennetin J, Sun LV, Lavrov S, Bellis M, White KP, Cavalli G. Chromosomal Distribution of PcG Proteins during Drosophila Development.PLoS Biol. 2006 Apr 20;4(6):e170
Example of a high-resolution description of PcG binding using ChIP and oligonucleotides arrays with one oligo every 100 bp
PH
PC
H3K27me3
GATA
ATOH
LHX
POU
IRX
DLX
SIX
NEUROD
BHLHB
PAX
FOX
HOX
SOX
TBX
NKX
HES
EBF
RUNX
MYO
CDX
MEIS/EVX
Transcription factor family membersoccupied by the PRC2 protein SUZ12 in human
ES cells
Overlap between PRC1 and PRC2 targets in mouse ES cells
Eed(831)
Suz12(1271)
Rnf2(1219)
Phc(922)
PRC2 PRC166
94
300
98 6
164
55
365
121 31
512
23
PcG proteins are required for the maintenance of ES cell fate, as well as for the fate of hematopoietic and neuronal stem cells and of differentiated cell types
Genome-wide Chip on chip in ES cells
Lee et al. Cell. 2006 Apr 21;125(2):301-13Boyer et al. Nature. 2006 May 18;441(7091):349-53. Epub 2006 Apr 19
5. Cross-talk between multiple copies of PREs
w w
w
Chromosome X
Pairing Sensitive Silencing: enhanced silencing of the mini-white reporter gene by multiple PRE copies
---> strong mini-white silencing---> weak silencing of the mini-white reporter gene
Transgenic Fab-7heterozygous
Transgenic Fab-7homozygous
PP mini-whiteFab-7
PcG-mediated silencing is enhanced by the presence of multiple copies of homologous PREs in the genome
Fab-X; Fab71
XX
III III
Fab-X
sd sd
BX-C BX-C
Fab-7 transgene
Endogenous Fab-7
Endogenous Fab-7 deletion, named Fab-71
Data from: Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G. (2003). Inheritance of Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev 17, 2406-2420.
0
5
10
15
20
25
+ + -Endogenous Fab-7 at the BX-C (Chr.III)
3m
-
-
-
-
-
-Fab-X Fab-X;
Fab-71
WT
WT
Fab-X
Fab-X; Fab-71
+ +-Transgenic Fab-7 at sd (Chr.X)
Fab-7
Fab-7
Fab-7
Fab-7
sd
BX-C
sd
BX-C
sd
BX-C
Dapi Sd BX-C Merge
Homologous Fab-7 copies associate in the nucleus
% of interaction
Two identical Fab-7 can interact in the nucleus, leading to an increase of PcG-dependent silencing
Nucleus in a Rabl configuration
For more info…
http://www.igh.cnrs.fr/equip/cavalli
http://www.epigenome-noe.net
In french…
Inserm Actualités N. 201, Octobre 2006 (http://www.inserm-actualites.fr/index.php?id=562)