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In vivo protein-DNA interactions. Giacomo Cavalli. Institut de Génétique Humaine, CNRS Montpellier, France. UE Méthodologie, 11 April, 2014. Compaction by higher order determinants. 10,000 nm. DNA compaction in the nucleus. 11 nm. 30nm. 1bp (0.3nm). Compaction of DNA by histones. - PowerPoint PPT Presentation
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In vivo protein-DNA interactions
Giacomo Cavalli
UE Méthodologie, 11 April, 2014Institut de Génétique Humaine, CNRS
Montpellier, France
DNA compactioncompaction in the nucleus
1bp (0.3nm)
10,000 nm
30nm
11 nm
Compaction of DNA by histones Compaction by higher order determinants
Signaling genes
TFs of developmental
networks
Cell cycle genes
Cellular Memory System
PcG proteins
Implication of PcG proteins in dynamic gene regulation
HOXgenes
Cancer development
ProliferationCell cycle Control
Stem cell plasticity
Developmental pathways
DifferentiationCell fate
determination
PRE
Su(z)12EscE(z)
Nurf-55
Core PRC2
H3 H3 H3 H3
Me3K27
Me3K27
Histone Methyl Transferase
Target gene
Schematic mechanism of Polycomb mediated silencingSchematic mechanism of Polycomb mediated silencing
PRE
Su(z)12EscE(z)
Nurf-55
Core PRC2
H3 H3 H3 H3
Me3K27
Me3K27
Histone Methyl Transferase
Target gene
Schematic mechanism of Polycomb mediated silencingSchematic mechanism of Polycomb mediated silencing
H3 H3 H3 H3
Me3K27
Me3K27
Core PRC1Pc
Ph
Psc
dRing
Scm
PRE
Su(z)12EscE(z)
Nurf-55
Core PRC2
H3 H3 H3 H3
Me3K27
Me3K27
Histone Methyl Transferase
Target gene
Schematic mechanism of Polycomb mediated silencingSchematic mechanism of Polycomb mediated silencing
H3 H3 H3 H3
Me3K27
Me3K27
Core PRC1Pc
Ph
Psc
dRing
Scm
Ub E3 ligase
UbK119
UbK119
PRE
Su(z)12EscE(z)
Nurf-55
Core PRC2
H3 H3 H3 H3
Me3K27
Me3K27
Histone Methyl Transferase
Target gene
Schematic mechanism of Polycomb mediated silencingSchematic mechanism of Polycomb mediated silencing
Core PRC1Pc
Ph
Psc
dRing
Scm
Ub E3 ligase
H3 H3 H3 H3
Me3K27
Me3K27
H2AH2AH2AH2A
UbK119
UbK119
ATP-dependent chromatin remodeling
PcG and trxG proteins associate to multiple genomic loci
Polytene chromosome staining shows around 100 bands for each PcG protein
Genome-wide identification of downstream PcG target genes
« ChIP-on-chip » approach
DNA chip
ChIP
Control IP
Protein IP
The ChIP on chip approach
Produce fluorescent labeled probes
Hybridize to the DNA chip Obtain the profile
1st generation microarraysProduce 2 KB PCR fragments of overlapping
genomic DNA fragments
IP step
Produce soluble
chromatin
Cross-link chromatin
2nd generation microarrayswhole genome coverage with 1,000,000 long oligonucleotides, i.e. 1 Oligo per 120 bp of
euchromatin
Dynamic function of Polycomb proteins and cell proliferationDynamic function of Polycomb proteins and cell proliferation
200Kb H3K27me3
PC
Ph
S2 cells data-Schwartz et al 2006
H3K27me3
PC
Psc
Embryos -Schuettengruber et al 2009
http://www.purl.org/NET/polycomb
ChIP on chip validation: Comparing ChIP on chip data with a chromatin profiling using an independent technology called DamID
In DamID, the chromatin protein of interest is fused to the bacterial Dam-methylase and the construct is transfected into the cells of interest. The protein of interest drives the Dam partner to its targets, and the methylase puts a methyl mark at the “A” of GATC sequences. Methylated DNA is then isolated and hybridized onto microarrays of interest
Correspondence between ChIP on chip and DamID data
N=13
N=11
N=54
N=8
N=21
100 %
Maternal genes
Gap genes
Pair-rule genes
Segment polaritygenes
Homeotic genes
Direct Hox gene targets
76.9%
23.1%
27.3%
72.7%
40.7%
59.3%
PcG target
No target
N=53
52.4%
47.6%
26.4%
73.6%
ey / PAX6
eyg / PAX6(5A)
Optix / SIX3/6 shf / WIF1
eya / EYA1-4 so / SIX1/2
dac / DACH1-2
Eye specification
toy / PAX6
Signaling pathways interacting with RDGN genes:
FLY MOUSE HUMANtoy2 Pax6 PAX6ey 1 Pax6 PAX6eyg (toe)1 - -Optix1 Six6 SIX6shf Wif1 WIF1eya2 Eya1-4 EYA1-4so1 Six1 SIX1dac1 Dach1 DACH1hh1 Shh SHHdpp1 Bmp2 BMP2
Additional factors involved in eye development:oc1 Otx1 OTX1ato3 Atoh1-8 ATOH1-8tsh2 - -bi1 Tbx2 TBX2
PcG target genes regulate genes at multiple layers of
transcriptional cascades
ChIP-Seq Library constructionH3K36
ChIP
100bp
200bp
300bp
400bp
500bp
600bp
Polish ends
5’3’
Taq extend
A A
Ligate Solexa Linkers
~5-10ng
1K
b+
10
0b
p
The evolution of ChIP: massive sequencing of the immunoprecipitated chromatin DNA
Illumina sequencing
Amplify to form clusters
Sequenceone base at a time
T
A C
G
Laser
Linker ligated DNA
Flow cell imaging by microscopy60 X objective: thousands tiff images / hundred thousands of images per run.
Chromatin Immunoprecipitation Tag Sequencing
After obtaining the sequences, they are positioned on the genome by automated algorythms (like Blast but quicker) and each tag is thus assigned its position on the genome.
These profiles can then be quantified and analyzed just like normal ChIP on chip profiles
PC
H3K27me3
PH
PC
H3K27me3
PH
•PcGtargets (PC/PH/H3K27me3)
eye discs
(+)
(-)
embryos
Identification of new PcG target genes
0
0
0
0
0
0
145
181
353
275
350
305
dan danr fd96Ca fd96Cb
maintained New domain
Anna Delest
wgWnt4
PH Mel
PH Yak
PC Mel
PC Yak
K27 Mel
K27 Yak
PHO Mel
PHO Yak
DSP1 Mel
DSP1 Yak
K4 Mel
K4 Yak
PRE position is highly conserved in Drosophila species
D.Melanogster vs D.Yakuba
→ species-specific differences can be used to study PRE sequence features
Bernd Schüttengruber
• Exploiting In vivo protein-DNA interactions to learn about the three dimensional conformation of chromatin
PcG proteins
PRE
?Mecanisms
PREs are sometimes located at positions overlapping the proximal gene promoter, but in other instances they can be at tens of kilobases away from it.
How can PcG proteins repress transcription in all these cases?
> 30 kb28 kb
"SPREADING versus LOOPING"
PRE
PcG proteins
"SPREADING"
PR
E
PcG proteins
"LOOPING"
Two models have been proposed in order to explain how PcG proteins repress their target genes:1. They might spread from the PRE into flanking chromatin, covering the whole domain including the target promoter2. Alternatively, they might reach the promoter via direct looping of the PRE and establishment of protein-protein contacts.
Interestingly, at some endogenous target genes PREs are located at very large distance from the promoter and they are flanked by elements called: "chromatin insulators"
Insulators
Enhancer blockers
Chromatin boundaries
Insulatorsthat can be"bypassed"
• Insulators are divided into three classes depending on their abilities
Ins. En.En. Gene
Ins. Ins.En. Gene
Ins. Ins.En. Gene
• One insulator can have many of these properties
Domain B
● DNA element isolated from the drosophila gypsy retrotransposon
● This sequence contains 12 binding sites for the Su(Hw) protein, that is required for insulator function
Insulator bypass model
The gypsy insulator
Domain A
Insulating proteins Gerasimova et al, Mol. Cell, 2000
Model of nuclear chromosomal architecture based on insulators interaction
Ins. Ins.En. Gene
Bypass of the gypsy insulator by a PRE
red
brown
orange
yellow
white
Expression of whitered
brown
orange
yellow
white
Ins
. Ins
.
Expression of white
PRE
PRE
Insulator
Gene
Insulator
PRE Enhancer Gene
yellow
Insulator
Insulator
yellow white
Insulator Insulator
Yes! the PRE can bypass 2 insulators
PRE PRE
white
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
yellow white
Fo
ld e
nric
hm
en
t
1kb
InsulatorPRE Insulator
ChIP analyisis of the molecular landmarks of insulator bypass
pupal stage ● PcG proteins bound to the PRE can reach a downstream promoter without coating an insulated chromatin domain
● Two insulators build a chromatin domain fully shielded from invasion by PcG proteins
● PcG proteins are able to spread from a PRE into a neighboring region of several kb. This spreading is blocked by one insulator
Fo
ld e
nric
hm
en
t
1kb
PC
PHpupal stage
The data shown before provide good evidence for a spreading process
Can we get direct evidence for looping?
Chromosome Conformation Capture (3C) technology:
Formaldehyde-fixed nuclei preparation
1
2
Chromatin digestion
Main steps of 3C technology
5
DNA purification andquantitative PCR analysis
3
Extensive dilution
4
Ligation
Biological material
3C technology allows to convert chromosomal interaction events into DNA ligation events that can be analyzed by PCR
Inte
rac
tio
n l
ev
el
in p
erc
en
tag
e o
f in
pu
t
(P)(S)YSW-22E lines - AdultH3C - distal gypsy insulator anchor
0.0%
0.1%
0.2%
0.3%
0.4%
0.5%
tRNA:CR31939-RA
tRNA:CR31940-RAtRNA:CR31669-RA
tRNA:CR31944-RA
tRNA:CR31943-RA
CG4238-RF CG15353-RA
Nplp4-RA CG33543-RC
0-5kb-10kb-15kb +5kb +10kb +15kb
yellow mini-white
(P)(S)YSW transposon
Dist.Ins.
Prox. Ins.
PRE
Anchor
Two gypsy insulators build a chromatin loop
PRE
PR
E
"SPREADING" "LOOPING"
PRE close to its target promoter
b w br
yellow mini-white
Dist.Ins.
Prox.Ins.
PRE yellowb w br
yellow mini-white
Dist.Ins.
PRE yellow
PRE distant from its target promoter
PcG proteins
In summary, both spreading and looping models could be correct, each one accounting for a particular context
+
Comet et al, Dev. Cell 2006
How PcG proteins and insulators might work in the cell nucleus
PcG bodiesNucleus
Insulator-binding protein complexes
Insulator bodies
High-resolution 3C is appropriate to study chromatin conformation
Analysis of Hox gene contacts by 4CAnalysis of Hox gene contacts by 4C
• We developed a new 4C method based on “biotinylated primer extension”
streptavidin bead
GGGGGCCCCC
Biotinylated Primer
• The amplified material is then hybridized to a Microarray (Roche Nimblegen)
• We used the Fab-7 PRE sequence as a bait, which negatively regulates the Abd-B gene in the BX-C
Itys COMET
Modification and control of the 4C procedureModification and control of the 4C procedure
G GG G G
G GG G G
“In situ” linker synthesis
Quantitative amplificationby real-time PCR
Genomic DNA-Chip Hybridization
Affinity purification on streptavidin beads
1.Biotinylated-primer extension
3C preparation
UnknownpartnerAnchor
fragment
2.
3.
4.
5.
101214161820
02468
2 Ins. / 1 Ins.
4C 4CBEFORE
amplificationAFTERamplification
2 Ins. / 1 Ins.
3C 4C
101214161820
02468
INPUT BEFOREamplification
Primer dimers
Anchor fragment
Unknown partnersligated to the anchor fragment
100bp
200bp
300bp
400bp
500bp
1kb
Denaturing 4% agarose gel
Cop
ies
num
ber
ratio
Cop
ies
num
ber
ratio
4C data analysis by generation of domainograms4C data analysis by generation of domainograms
Normalized profile intensities for each probe i are transformed into rank based scores Qi, which are combined into Siw multiscale scores and transformed as Piw probabilities using Fisher's Chi square law.
probabilities atscale = 3 probes
probabilities atscale = 1 probe
Piw at scale =N probes
Legend:N is the total number of probes ri is the rank of probe i
Piw at Log scale =N probes
The Piw values represent probabilities of 4C events as a function of chromatin domain size
Piw in false color
Benjamin LEBLANC
Major Fab-7 4C hits are Polycomb bound regionsMajor Fab-7 4C hits are Polycomb bound regions
4C Domainogram
Polycomb ChIP Domainogram
10-5000 10-500 10-50 10-10 10-1
ANT-C NK-Cpnt
Fab-7BX-C
1Mb
prospero E5-emsgrn hth
srp-pnr ss
Drop
3R3R 1Mb
Simplified Hi-C procedure
•Fix nuclei of 16-18 hr embryos
•Digestion with 4-cutter DpnII
•Ligation and DNA purification as 3C
•Sonication and selection of ~800 bp
•Deep paired-end sequencing
Hi-C efficiently reproduces known 3C contacts Hi-C efficiently reproduces known 3C contacts
Chromatin contact features
2. Matrix diagonal is not homogeneous
Polycomb-mediated interactions
Bantignies et al., 2011
Bernd SCHÜTTENGRUBER
Nicolas NEGRE
Benjamin LEBLANC
Anna DELEST
Itys COMET
Tom SEXTONERCEU - 7FPCNRS, ARCFrench ministry of research
http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html
References: Schüttengruber et al. (2009) PLoS Biol 7(1): e1000013; Comet et al. Dev Cell 11, 117-124 and PNAS , 108(6):2294-9; Bantignies et al. Cell 144, 214-26, Sexton et al. Cell 148, 458-472