Transcript PcG, trxG and the maintenance of gene expression

Polycomb, trithorax, and maintenance of gene expression
A key feature of development in metameric animals is the definition of body segments where
groups of cells with a specified fate will give rise to their relative body structures. Cell fates are
specified by particular combinations of homeotic gene products. During early embryogenesis, maternal
and segmentation genes regulate homeotic genes by binding to specific regulatory sequences located
in the promoter regions. Later in development, the expression pattern of homeotic genes as well as
other important developmental genes are maintained by a cell memory system dependent on two
groups of genes. The members of these two groups are able to recognize the active and inactive state
of expression and fix it to the cell progeny through many cell divisions. These components have been
classified in two genetic groups. The trithorax-group (trxG) maintain the active state of expression,
while the Polycomb-group (PcG) counteracts this activation with a stable repressive function.
There is strong evidence that the memory function encoded by these two groups of genes is
achieved through regulation of higher order chromatin structures. PcG gene products form large
multimeric protein complexes in Drosophila, mouse and human. PcG mediated gene silencing can be
directed by DNA elements in cis, defined as PcG response elements (PRE). On the other hand, several
trxG members act at elements defined as TRE (that overlap with PRE) via chromatin remodeling and
induction of histone modifications that increase chromatin accessibility to transcription factors.
In this set of slides, some of the features of PcG and of trxG factors are summarized and
examples of molecular and cell biological approaches to dissect their mechanisms of action are
presented.
Polycomb, trithorax, and maintenance of gene expression
Early development
Establishment of patterns
OFF
Ubx
ON
Maternal, Gap, Pair-rule, Segment polarity
OFF
ON
Maintenance phase
Polycomb-Group
trithorax-Group
Transmission of pattern
after disappearance of
early factors
OFF
Update: December 2004
ON
Ubx OFF
Ubx ON
wing
haltere
PcG and trxG proteins associate to multiple genomic loci
DAPI
Merge
PH
Polytene chromosome staining shows around 100 bands for each PcG protein
PcG and trxG proteins bind to specific DNA
elements, named PRE and TRE
PRE
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 homologous pairing of the transgenes
TRE
Bound by trxG proteins in vivo
Binding leads to maintenance of trxG-dependent activation of reporter genes
PRE and TRE often overlap in the same genomic region
Members of the PcG and of the trxG
PcG
Gene
protein motif
homologs
Polycomb (Pc)
chromo domain
(Binding to H3 methyl K9 or K27)
M33 (mouse); hPC (human)
polyhomeotic (ph)
one zinc finger
Mph1/Rae-28 (mouse); hph1; hph2 (human)
Posterior sex combs (Psc)
RING finger
bmi-1 (mouse/human); mel-18 (mouse)
Enhancer of zeste (E(z))
SET (H3MTase)
Ezh1; Ezh2 (human); clf (Arabidopsis)
extra sex combs (esc)
WD repeat
Eed (mouse); hEED (human)
Pleiohomeotic (pho)
Zinc-finger (DNA binding)
hYY-1 (human); mYY-1 (mouse)
Gene
protein motif
trithorax (trx)
SET (H3 HMTase)/ PHD-finger
MLL/ALL-1/HRX (human)
Ash-1
SET (H3/H4HMTase)/ PHD-finger
ASH-1 (human); NSD1 (mouse)
Brm complex
brahma (brm)
bromo domain
SWI2/SNF2 (yeast)
brg1 (mouse/human); Hbrm (human)
FACT complex
Trithorax-like (Trl)
BTB/POZ (dimerization)
zinc finger (DNA binding)
PRC1
complex
Esc/E(z)
Complex
trxG
TAC1 complex
(DNA dependent ATPase/helicase)
homologs
Action of PcG and trxG complexes on chromatin
Histone
acetylation and
methylation
(TAC1 and ASH1
complexes)
PRE
Ac
Nucleosome remodeling
(BRM complex)
trxG
ON
Maintenance of
active states
(open chromatin)
Me K4 H3
Target
gene
Deacetylation and
methylation
(ESC-E(Z) complex)
Me K27 H3
- Chromatin
compaction
- H2A Ubiquitination
(PRC1 complex)
OFF
PcG
Maintenance of
repressed states
(compact chromatin)
Ub H2A
Histone H3 methylation and Polycomb
Pc
H3 K9 triMe
Merge
Data from:
Ringrose et al. (2004)
Mol. Cell 16, 641
Pc
H3 K27 triMe
Merge
There is a strong but not absolute correlation between trimethylation of K27 (and K9)
trimethylation and Polycomb recruitment at target loci. i.e. there is more to Pc recruitment
What do « Polycomb » proteins do to chromatin ?
1. 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
What do « Polycomb » proteins do to chromatin ?
2. H2A Ubiquitination
Data from:
Wang et al. (2004)
Nature 431, 873
Purified human PRC1-type complexes can Ubiquitinate H2A in vitro, and the drosophila counterpart of
the same complex induces a dRing-dependent H2A Ub at the Ubx PcG target gene
Features of PREs and TREs, and
examples of how they are studied
in drosophila
The Bithorax-complex is a target locus for PcG and trxG proteins
bx
bxd
iab2/3 Mcp Fab-7&8
iab-4
Regulatory regions in the Bithorax Complex are shown in red
Target elements for PcG and/or trxG proteins are shown in green
1. Spatial specific maintenance of silencing of homeotic genes
Silencing initiates correctly, thanks to early repressors like Hunchback (Hb), but
degenerates in the absence of PcG proteins when these repressors disappear
Wild type embryo
Mutant PcG embryo
No effect on initiation of silencing
in anterior parasegments
A
HbZYG
Ubx
P
HbZYG
A
Ubx
Gastrulation stage (3h)
---> Anterior derepression,
antero-posterior transformation
Ubx
Ubx
Extended germ band stage (6h)
P
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. Pairing Sensitive Silencing
During embryonic development, chromosome homologs pair in diptera.
Pairing brings homolog sequences in close physical proximity. This
pairing correlates with the strength of PcG and trxG mediated regulation
PRE Heterozygous
PRE Homozygous
Weak PcG mediated
repression
Strong PcG mediated
repression
The Fab-7 element of the BX-C
The Fab-7 element is a 3.6 Kb region that regulates expression of the homeotic gene
Abdominal-B (Abd-B), located in the locus named Bithorax Complex (BX-C) in
chromosome III of Drosophila. This element is partitioned in a PRE and a so-called
“chromatin boundary”, i.e. an element that might segregate independent
chromosomal domains from each other.
Locus BX-C
Fab-7
Ubx
abd-A
Abd-B
Boundary
PRE
Chr. III
3,6 Kb
2. An example of Pairing Sensitive Silencing: silencing of the mini-white
reporter gene by the Fab-7 element in the Fab-X transgenic line
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
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.
2. Silencing of mini-white 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 proteins at PREs: chromatin analysis by
Formaldehyde cross-linking and chromatin immunoprecipitation (ChIP)
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 on a
Southern of a genomic walk, quantify by
PhosphorImager
Genomic walk
Example: analysis of PC and GAGA factor binding to Fab-7 by ChIP
Mock PC Ip
Mock GAGA Ip
Quantification of the signals
Boundary PRE
p
1230 bp
50
Boundary PRE
d
p
d
200
1 Kb
50
200
30
30
120
120
10
10
40
40
1 Kb
778 bp
422 bp
356 bp
H
PC
GAF
H
PF E
PC
Xb Xb Xh
H
H
PF E
Xb Xb Xh
GAF
Data from: Cavalli, G., and Paro, R. (1998). The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93,
505-518.
5. Maintenance of active as well as repressed states: PREs and
TREs form elements named as Cellular Memory Modules
The GAL4 system for the study of PRE/TRE function: mimicking the developmental
pathway leading to maintenance of homeotic gene expression
Hsp 70 GAL4
Driver construct
GAL4
Fab-7
UAS-lacZ
white
Reporter construct
Experimental approach
HsGAL4 pulse during early development
Embryo 1st 2nd 3rd instar
Pupa
?
Larva
Light Eye color
white repressed
Check Eye color
Fab-7: a cellular memory module (CMM) that maintains active as well as
silenced chromatin throughout development
hsp7
0
Fab-7
Gal4
HS
UAS G lacZ
GAL4
white
3,6 kb
-HS
+HS
Beta-gal stains to study lacZ expression
Data from: Cavalli, G., and Paro, R. (1998). The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93,
505-518.
Chromatin states can be inherited through
meiosis by the following generations
Meiotic inheritance of derepressed states
Repressed
Derepressed G0
GAL4 pulse in embryos
Derepressed
G1, G2, G3...
Re-cross red eyed flies
Big open questions
What are the precise developmental cues that recruit PcG or trxG proteins to PREs of
homeotic genes? Do they also apply for other PREs/TREs?
What are the molecular mechanisms for recruitment of PcG and trxG proteins to
PREs and TREs? What are the effects of these recruitments on chromatin?
Once recruited, how can these proteins maintain chromatin states through DNA
replication and through mitosis (and meiosis)?
What is the basis of the Pairing Sensitive Effects and of long distance interactions?
How does this phenomenon contribute to inheritance of chromatin states?
What is the genome-wide profile of PcG and trxG binding? What is the identity of the
corresponding target genes? Are they all regulated like homeotic genes, or are there
different categories of regulatory mechanisms?