Transcript Fab-7 1 + + - IGH

Introduction in:
Epigenetic regulation by Polycomb and
trithorax group proteins
Giacomo CAVALLI. Montpellier, December 2006
Cavalli
Lab
The chromatin Yin / Yang
Heterochromatin
- Heavily condensed
- Gene poor
- Silent genes
- Late replicating
- DNA hypermethylated
- Rich in histone H1
- Histones have
repressive post
translational marks
Euchrochromatin
- Less condensed
- Gene rich
- Active genes
- Early replicating
- DNA hypomethylated
- Poor in histone H1
- Histones have
specific, activating
post translational
marks
Cavalli
Lab
Where on the chromosomes are condensed and open
chromatin located?
Condensed chromatin
Constitutive
Heterochromatin
Telomeres
Centromeres
Open chromatin
Typical of the active
gene loci along the
chromosomal arms
Condensed
chromatin is also
found at several
silent genes located
in 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 expression
Early development
Establishment of patterns
OFF
Ubx
ON
Maternal, Gap, Pair-rule, Segment polarity
OFF
Polycomb-Group
OFF
ON
Maintenance phase
Transmission of pattern
after disappearance of
early factors
trithorax-Group
ON
PcG and trxG proteins associate to multiple genomic loci
DAPI
Merge
PH
Polytene chromosome staining shows around 100 bands for each PcG protein
PcG proteins bind to specific DNA elements,
named PREs
Bound by PcG proteins in vivo (in polytene chromosomes and by
cross-linking experiments)
Binding leads to maintenance of PcG-dependent repression of
reporter genes
Repression is enhanced by the presence of multiple PRE copies
Members of the PcG and of the trxG
PcG
Gene
protein motifs
PhoRC
Complex
Pleiohomeotic (pho)
Zinc-finger (DNA binding)
Enhancer of zeste (E(z))
SET (H3K27MTase)
extra sex combs (esc)
WD repeat
Polycomb (Pc)
chromo domain
(Binding to H3 methyl K9 or K27)
polyhomeotic (ph)
Zinc finger, SAM domain
Posterior sex combs (Psc)
RING finger
trxG
Gene
protein motifs
FACT
complex
Trithorax-like (Trl)
BTB/POZ (dimerization)
Zinc finger (DNA binding)
TAC1
complex
trithorax (trx)
SET (H3K4HMTase)/ PHD-finger
Ash-1
SET (H3/H4HMTase)/ PHD-finger
brahma (brm)
bromo domain
Esc/E(z)
Complex
PRC1
complex
Brm complex
(DNA dependent ATPase/helicase)
Action of PcG and trxG complexes on chromatin
Ac
Histone acetylation and
methylation
(TAC1 and ASH1
complexes)
Nucleosome remodeling
(BRM complex)
trxG
ON
Maintenance of active
states
(open chromatin)
Me K4 H3
Target gene
PRE
Deacetylation and
methylation
(ESC-E(Z) complex)
Me K27 H3
- Chromatin compaction
- H2A Ubiquitination
(PRC1 complex)
PcG
OFF
Maintenance of repressed
states
(compact chromatin)
Ub H2A
Histone H3 K27 methylation and Polycomb
Pc
H3K27me3
Merge
Data from:
Ringrose et al. (2004) Mol. Cell 16, 641
There is a strong correlation between trimethylation of K27 (and K9) trimethylation and Polycomb
recruitment at target loci.
What does Polycomb do to chromatin ?
Chromatin Condensation
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
Data from: Francis et al. (2004), Science 306, 1574
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
bxd5.1 UbxlacZ reporter construct
Bxd 5.1 PRE
Silencing of a Ubx-lacZ reporter mimicking
the wt behaviour of the Ubx gene, which is
silenced in parasegments 1 to 5
Ubx prom LacZ
mini-white
PcG dependent derepression of a UbxlacZ reporter in embryonic territories
where it is normally silenced
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-7
Fab-7
Pc -/+
Fab-7
Pc +/+
UAS-lacZ
white
Fab-7
trx -/+
Fab-7
trx +/+
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)
Fab-7
UAS-lacZ
white
Transgene :
transgene
24A
25E5
DAPI
Immunostaining
of PH protein
FISH
Immuno-FISH
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)
Cross-link cells or embryos with formaldehyde to
induce protein-DNA crosslinks
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
Use amplified DNA as probe to hybridize
DNA chips containing the genome.
Extract the distribution profile of the
proteins of interest
ChIP on chip
PH binding profiles at the embryonic developmental stage
X
sd
X chromosome tip
2L
Montpellier tiling paths
Yale tiling paths
Adh region
2R
en/inv
3R
3L
L82
Hh
Bx-C
4
ci
bi (1)
noc
ph
esg
mab2(3)
gt/z
2D
(2)
4C
5A 5D
elB
ct (4)
7B
34D
35B
stc
35D
36A
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
H3K27me3
PC
PH
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
Transcription factor family members
occupied by the PRC2 protein SUZ12 in human
ES cells
Overlap between PRC1 and PRC2 targets
in mouse ES cells
Eed
(831)
PRC2
66
IRX
BHLHB
CDX
PRC1
365
94
Suz12
(1271)
HOX
Rnf2
(1219)
MEIS/EVX
TBX
55
300
DLX
164
Phc
(922)
HES
FOX
ATOH
512
98
6
121
MYO
31
NEUROD
EBF
GATA
POU
RUNX
23
PAX
SOX
NKX
Boyer et al. Nature. 2006 May 18;441(7091):349-53. Epub 2006 Apr 19
SIX
LHX
Lee et al. Cell. 2006 Apr 21;125(2):301-13
5. Cross-talk between multiple copies
of PREs
Pairing Sensitive Silencing: enhanced silencing of the mini-white
reporter gene by multiple PRE copies
P
Fab-7
Transgenic Fab-7
heterozygous
w
mini-white
Chromosome X
P
Transgenic Fab-7
homozygous
w
w
---> weak silencing of the
mini-white reporter gene
---> strong mini-white silencing
PcG-mediated silencing is enhanced by the presence of
multiple copies of homologous PREs in the genome
Fab-X; Fab71
Fab-X
Fab-7
transgene
Endogenous Fab-7
sd
sd
X
X
BX-C
BX-C
III
III
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.
Homologous Fab-7 copies associate in the nucleus
Dapi
Sd
BX-C
Merge
WT
sd
BX-C
Fab-7
Fab-X
sd
Fab-7
BX-C
Fab-7
25
% of interaction
-
20
-
15
-
10
-
5
-
0
-
Fab-X; Fab-71
sd
Fab-7
BX-C
WT
3mm
Transgenic Fab-7 at sd (Chr.X)
-
Endogenous Fab-7 at the BX-C (Chr.III)
+
Fab-X Fab-X;
Fab-71
+
+
+
-
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.insermactualites.fr/index.php?id=562)
[email protected]