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