Development of proteomics tools to study intranuclear organizationVasily Ogryzko

Group of “Proteomics & epigenetics’, UMR 8126 CNRS, Institut Gustave Roussy15 мая 2014

Программа визитов иностранных учёных в российские научные центры фонда "Династия"

Importance of proteomics

One genome

Two proteomes

Proteins:Structure Levels

Proteomics: High throughput Study of proteins

Questions: Amounts Localization Modifications Interactions

Methodology: 2D electrophoresis Mass spectrometry Epitope tagging

• Decipher mechanisms of epigenetic regulation(histone code, other self-perpetuating protein modifications)

•Predict function of newly discovered genes(protein-protein interaction partners)

can be addressed by proteomics

Challenges of postgenomic era:

• Study post-transcriptional steps in gene regulation(microRNA, etc)

Research Units(IRCIV)

Clinical Research Division(DRC)

Hospital Activities Research Division

Healthcare - Research - Education

International Scientific Advisory Board

Platforms

Jean-Yves Scoazec

Imaging / flow cytometry

Animal facility

Integrated biology

Translational researchTumor collection

BiotherapiesBioinformatics

Research DivisionEric Solary

Scientific Policy Committee

Clinical Research

Gilles Vassal

Steering Committe

e

Research Units

Eric Solary

Research Coordination and Management Service / Logistics Service

Steering Committe

e

Technology Transfert Company

Integrated Biology

Proteomicsplatform

Lipidomicsplatform

Metabolomicsplatform

Functional genomicsplatform

Bioinformaticsplatform

Biological resources center

Proteomics platform at IGR

Proteomics platform at IGR

Vasily OGRYZKO – DR2, INSERM

Emilie COCHET – Technicienne, IGR

Alain DEROUSSENT– IR, CNRS

Geographic location:

IGR, PR2, room 355

Personnel:

Main instrument:

Nano-HPLC/CHIP/ion-trap (Agilent)+

Agilent off-gel separator for preanalytic fractionation

+Small laboratory equipment

Proteomics platform at IGR

1. Protein footprinting: motivation

Epigenetic information can be encoded in macromolecular interactions

Proteins are much more interesting objects than DNA or RNA, i.e. not only their amounts but their conformation and interaction plays essential role

Protein footprinting: motivation

Biological system as:

Chemical reactor Mechanical device

Concentrations (quantities) are all what we need

Quantities will tell us nothing

Proteome footprinting: motivation

Comparing only protein amounts between proteomes might be looking at the tip of the

iceberg 1. Chemical reactor versus machine 2. Differences in protein amounts do not show immediately in many cases, unlike changes in conformations or interactions

Need to develop quantitative approaches to

monitor changes in protein surfaces in vivo

Protein/proteome footprinting: the principle

Goals:1. Monitor surface of a particular protein in vivo2. Detect changes in protein surfaces on

proteome-wide basis

Protein

Isolated

Interacting

Modification Identification of modified site

Protease Mass spec

Lysine containing peptide: mono-, di- and trimethylation by DMS

1methyl 2methyl 3methyl

14

Arginine containing peptide: mono- and dimethylation by DMS

14 1methyl 2methyl

Stable isotope DMSD6 produces a mass shift 17 instead of 14

14

17 CD3

CH3

Discrimination between in vitro and in vivo methylation using stable isotopes

KCH3

KCD3

KCH3

Footprinting of H2AZ expressed in bacteria

Total spectra

850 + 864,867

864 +867

Footprinting of H2AZ expressed in bacteria

1370850658

TTSHGR HLQLAIR ATIAGGGVIPHIHK

828.4

1168.6

850.5

1370.8

+CH3

+CD3

Footprinting of H2AZ/H2B dimer in vitro

DMSDMSD6Trypsin,

(affinity enrichment)LC-MS/MS

Denaturation

A

B

…

Ctrl

5

10

321

DMS

coomassie

1

2

4

3

…

Conclusions

1. DMS methylates proteins in vivo

Methodology:

2. Use of stable isotope DMSD6 allows to set up a quantitative approach to monitor reactivityof residues in vivo and in vitro

3. H2AZ and H2B surfaces change after formingH2AZ/H2B dimer

2. New proteomics-based strategy to study protein-protein interactions in vivo

Proximity-Utilizing-Biotinylation (PUB)

3. LC-MS/MS Analysis of ratio Biotinylated/propionylatedpeptides

Biotinilated

propionilatedI

Retention time (min)

2. Purification of all HisTag proteinsOn Ni agarose beads, propionicanhydride treatment, trypsin digest

B

BAD

Р

BAD

24

Biotin ligase (wild type)

Biotin Accepting Domain

(Short peptide with HisTag)

BBiotin residue

PPropionyl residue

wtBirA

BAD

1. Interaction between protein A and B causes biotin transferand its covalent binding toLysine of BAD

Protein А

Protein ВB

BAD

wtBirA

Proximity-Utilizing-Biotinylation (PUB) for study interactions between known interaction partners

Biotinylation levels are interaction dependent

1. Protein oligomerisation

(TAP54a vs HP1g)

2. Binary protein-protein interaction

(KAP1 and HP1)

3. Different subnuclear domaines

(macroH2A vs H2A.BBD)

PentaHis-HRP Streptavidin-HRP

Protein oligomerisation (TAP54a vs HP1g)

51

28

39

BAP-TAP54a

1 2 3 4 1 2 3 4NS

BAP-HP1g

NS

1 - BAP.Tap54a+BirA.Tap54a

2 - BAP.Tap54a+BirA.HP1g

3 - BAP.HP1g+BirA.Tap54a

4 - BAP. HP1g+BirA. HP1g

51

28

39

BAP-TAP54a

1 2 3

NS

NS

BAP-HP1g NS

1 2 3

PentaHis-HRP Streptavidin-HRP

1 - control

2 - BAP.Tap54a + BAP.HP1g + BirA.Tap54a

3 - BAP.Tap54a + BAP.HP1 g + BirA.HP1g

1. TAP54a (RuvB-like 1) was shown to exist in oligomers2. The heterochromatin proteins HP1 (a, b, g) are also known to oligomerise3. But HP1 and Tap54 do not interact

Two BAP fusions (HP1 and Tap54) coexpressed with one Bira fusion (HP1 or Tap54)

PentaHis-HRP Streptavidin-HRP

Binary protein-protein interaction (HP1 and Kap1)

PentaHis-HRP

Streptavidin-HRP

BAP-HP1

BAP-GFP

1 2 3 4

NS1 2 3 4

1 - BAP.HP1g+BirA.wtKap1

2 - BAP. HP1g+BirA.mutKap1

3 - BAP.GFP+BirA.wtKap1

4 - BAP. GFP+BirA.mutKap1

BAP-KAP1

- Biotin + KAP1wt

+ KAP1mut

Competitor

BAP-KAP1

-

1 2 3 4 5 6 7 8

BAP.HP1g + BirA.Kap1 system

BAP.Kap1 + BirA.HP1g system

1,3,5,7 - BAP.wtKap1

2,4,6,8 - BAP.mutKap1

A

BAP-TAP54a

a-His-HRP Streptavidin-HRP a-His-HRP Streptavidin-HRP

BAP-HP1

BAP-GFP

B

BAP-TAP54a

1 2 3 4 5 6 7 8

1 2 3 4 5 6

1 2 3 4 5 6 7 8

BAP-KAP1

a-His-HRP

Streptavidin-HRP

- Biotin + KAP1wt + KAP1mut Competitor

BAP-KAP1

-

1 2 3 4 5 6 7 8

NS

NS

NS

NS

BAP-HP1g

BAP-HP1g

NS

NS

- + - +

DICBiotin

BirA-PCNA+

BAP-H3.1

BirA-PCNA+

BAP-CenpA

C

BirA-TAP54

BirA-HP1

BAP-TAP54

BAP-HP1

+ - + -- + - + + + - -- - + +

+ - + -- + - + + + - -- - + +

- + -- - + - + +- + +

BirA-TAP54

BirA-HP1

BAP-TAP54

BAP-HP1

- - + - + +- + +

- + -

+ - + -- + - + + + - -- - + +

BirA-KAP1BDwt

BirA-KAP1BDMut

BAP-GFP

BAP-HP1

+ - + -- + - + + + - -- - + +

aHis-HRP

Streptavidin-HRP

1 2 3 4

BirA-GFP

BirA-PCNA

BAP-H3.1

BAP-CenpA

+ + - - - - + + + - + -

BAP-H3.1

BAP-CenpA

BAP-H3.1

BAP-CenpA

Expt1 Expt2

0.2

0.4

0.6

0.8

00

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1 2 3 4 5

1

2

3

4

5

H3.1 CenpA H3.1 CenpA

Bir

A:

PC

NA

/GF

P

BAP:

Proximity-Utilizing-Biotinylation (PUB) for study interactions between known interaction partners

Advantage of PUB

Possibility to use mass spectrometry instead of western blotting to detect biotinylation

Can use multiplexing

Can use stable isotopes

30

Structure of different types of BAD domains

BAD1070: M G H H H H H H H G L T R I L E A Q K I V R G G L E BAD1118: M G H H H H H H H G L T R I L E A Q K I F R G G L E BAD1135: M G H H H H H H H G L T R I L E A Q K I Y R G G L E

BAD

trypsin

H2Az

B

BAD

Interaction strength

1 2 3 4

% o

f b

ioti

ny

lati

on

100%

50%

Linear region

BAD1070 Biotinylated

b-seria

y-seria

B

I L E A Q K I V R

BAD1070Propionylated

P

I L E A Q K I V R

N-terminus С-terminusB

BAD 1070

Р

BAD 1070

N-terminus С-terminus

MRM of ions with m/z 648 and 563

32

B

I L E A Q K I F R

BAD1118 Biotinylated

1185.7

B

I L E A Q K I Y R

BAD1135 Biotinylated

MRM of ions with m/z 672 (BAD1118) and 680 (BAD1135)

H2A.BirA + H2AZ.BAD

1 2 3 1 2 3 1 2 3

Input FlowThrough Elution

b-H2AZ

Ubi-b-H2AZ

FlowThrough Elution

1 2 3 1 2 3

b-H2AZ

Ubi-b-H2AZ

Anti-His-HRPStreptavidin-HRP

Figure 3

d

Coomassie Blue Streptavidin-HRP

In FT El In FT ElM

H2AZ

Ub-H2AZ

Ni-NTA purification

AGAATCCTGGAAGCTCAGAAGATCGTGAGAGGAGGCCTCGAG…

R I L E A Q K I V R G G L E AGAATCCTGGAAGCTCAGAAGATCTTCAGAGGAGGCCTCGAG…

R I L E A Q K I F R G G L E AGAATCCTGGAAGCTCAGAAGATCTACAGAGGAGGCCTCGAG…

R I L E A Q K I Y R G G L E

BAP1070

BAP1118

BAP1135

Experimental scheme

pOz.

H2A

.Bir

A

c. Incubation on Ni2+-NTA agarose

CMV.H2Az.BAP1070

CMV.H2Az.BAP1118

CMV.H2Az.BAP1135

a. 4hr biotin pulse before harvest

b. Mix 3 samples

c. Wash, ON trypsin and LC-MS/MS

MS2(623.3)

MS2(708.4)

2 4 6 8 10 12 Time [min]

MS2(563.2)

MS2(648.8)

MS2(587.2)

MS2(672.8)

BAP1070 propionylated

BAP1070 biotinylated

BAP1118 propionylated

BAP1118 biotinylated

BAP1135 propionylated

BAP1135 biotinylated

I L E A Q K(Pr) I V Ry8 y7

b2 b3

y6

b4

y4

b6

y3

b7

y2

b8

y5Intens.

b7

y7

b8 y8

y6

b6

y5y4b4

b2 y3y2

+MS2(563.2), 6.2min

0.0

1.0

7x10

400 600 800 1000 m/z

b3

200

I L E A Q K(Biot) I V Ry8 y7

b2 b3

y6

b4

y4

b6

y3

b7

y2

b8

y5

b7

y7

b8

y8

y6

b6y5

y4

b4

b2

y3y2

y8++y6++

+MS2(648.8), 6.6min

0.0

1.0

2.0

5x10

Intens.

400 600 800 1000 m/z

b3

200

a b

c

e

3

AK052209000012.D: EIC 1070-1071 +MS2(648.8)

1

AK052209000012.D: EIC 905-906 +MS2(566.2)

2

AK052209000012.D: EIC 899-901 +MS2(563.2)

0

1

2

3

4x10Intens.

0.0

0.5

1.0

1.5

5x10

0

1

2

3

4

5x10

6 8 10 12 14 16 18 20 22 Time [min]

227.1

247.1274.1

328.1

350.1

387.3

418.2

472.2 523.3554.7

608.3 638.1 673.3

699.4

715.4

770.5

853.5882.6

899.5

951.6

+MS2(563.2), 6.2min #1593

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

200 300 400 500 600 700 800 900 m/z

256.1

274.1

320.3

342.2

387.3

427.1446.9

480.4

510.3

534.1

577.4

602.7

621.3

723.3

745.4

776.5

811.3

860.5

905.6

957.6

+MS2(566.2), 6.2min #1594

0.00

0.25

0.50

0.75

1.00

1.25

5x10Intens.

200 300 400 500 600 700 800 900 m/z

LIGHT propionylated peptide from BAD1070

HEAVY propionylated peptide from BAD1070

HEAVYpropionylated

LIGHTPropionylated

10’ biotin pulse

LIGHTbiotinylated

y7

y7

y6

y6

y5

y5

y4

y4

y3

y3

y2

y2

b8

b8

b7

b7

Identification of Light and Heavy peptides

BAP-PCNA

Ub-BAP-PCNA

Bir

A-G

FP

Bir

A-P

olH

wt

Bir

A-P

OL

HΔ

Δ

UV+ - + - ++ + + + +

BirA-POLHΔΔ

BirA-POLH.UBZ

BirA-POLH.PIP

BirA-POLHwt

aPCNA

6XHis-HRP

BirA-POLH

+

BAP-PCNA

5 min biotin

48 hr 6 hr

UV 20 J/m2 Streptavidin

pulldown

FT

Elu

A

C

PCNA BAP-PCNA

Ub-BAP-PCNA

Bir

A-G

FP

Bir

A-P

OL

Hw

t

Bir

-AG

FP

Bir

A-P

OL

Hw

t

FT Elu

B

UT

1 2 3 4 5

1 2 3 4 5 6 7 8 9 10

BAP-PCNA

BAP-PCNAmut

+ ++

+

+

+ + + +

+

- - -

- - -

- - -

- - -

-

-

-

-

- -- -

Analysis of a specific sub-population of BAP-fusion

Proximity Utilizing Biotinylation (PUB) &

Native Chromatin Immunoprecipitation (NChIP)

3- PUB-NChIP

NChIP

using α-Histone PTM

No need to crosslink

use the DNA-histone interactions

Any DNA could be damaged

Current Approaches to Study Histone PTMs in Proximity to DNA Damage & Repair

Classic ChIP

using DDR implicated chromatin protein

Crosslinking is necessary

Protein part is damaged

PUB-NChIP

In Vivo biotinylation approach to study chromatin in proximity of a protein of interest

YBAP

X

BirA

BirA: Biotin Ligase

BAP: Biotin Accepting Peptide

Biotin

3- PUB-NChIP

Proximity Utilizing Biotinylation (PUB)

Kulyyassov A, Shoaib M, et al. J Proteome Res. 2011 Sep 2

Y

X

X

BirA

YBAP

BAP.Histone (Biotinylated)

BirA.X

Biotin

BAP.Histone

BirA.XCotransfection with

......

........

....

....

........

......

....

....

..

........

.. .... ........

..

....

..

....

....

....

........

....

....

........

........

..

..

........

.. .... ........

..

....

..

..

.. ..Biotinylated chromatin can be purified

PUB-NChIP

....

..

....

.. ....

....

..

....

Histone

BirA-Rad18BAP-H3.1BAP-H2A

- - + +- + - +-+-+

αHis-HRP

Streptavidin HRP

Rad18 Proximal Chromatin is Specifically Biotinylated

PUB-nChIP 38/50

αH2A biotin combined

biotinαRad18 combined

BirA.Rad18 + BAP.H2A

HEK

– 2

93T

cells

6 h after UVC (20 J/m2)15 min Biotin Pulse

3- PUB-NChIP

BirA: Biotin Ligase

BAP: Biotin Accepting Peptide

Coomassie Blue Staining

12 kDa

20 kDa

BAP.H2AH3H2BH2AH4 + Streptavidin

MN

ase

Supe

rnat

ant

Pelle

t

Inpu

t

Flow

thro

ugh

Eluti

on

EthBr Staining

300 bp500 bp

200 bp MonoDiTri

WB: Streptavidin-HRP

BAP.H2A12 kDa

30 kDa

Chromatin Purification in PUB-NChIP

PUB-nChIP 39/503- PUB-NChIP

15 min Biotin Pulse before harvesting

Harvest cells and prepare Nuclei

Micrococcal Nuclease Digestion

0.4 M salt extraction of nucleosomes

3h binding of nucleosomes in Sepharose-Streptavidin beads

Elution of Biotinylated H2A along with other histones

BirA-HP1α

α -H3

α - γH2AX

BirA-RAD18

BirA-HP1α

BAP-H2A

+ -- ++ +

γH2AX biotin combined

BirA-RAD18

BAP-H2A

+

3 h after Ionizing Radiation (10 Gy)15 min Biotin Pulse

HEK

293

T ce

lls

PUB-nChIP 40/503- PUB-NChIP

Chromatin Purified by PUB-NChIP is Enriched in Expected PTMs

PUB-nChIP

PUB-NChIP Reveals a Specific Pattern of H4 Acetylation in Rad18 Proximal Chromatin

41/50

1Ac 2Ac 3Ac 4AcUM

H/L

ratio

s

MS analysis of Histone H4 peptide 4-17 (GKGGKGLGKGGAKR)

1- GFP+H2A (H) / GFP+H2A (L)2- Rad18+H2A (H) / GFP+H2A (L)

H/L ratios

1 2

3- PUB-NChIP

BirA.GFP cotransfected with BAP.H2A controlBiotinylates everything

GFP Biotin

BirA.GFP + BAP.H2A

HEK

293

T ce

lls

BirA.GFPBAP.H2A

L

BirA.GFPBAP.H2A

HBirA.Rad18

BAP.H2A

H

1 : 1 1 : 11 2

SILAC Experimental Design

HEK-293T cells

PUB-nChIP

Proximity of Biotinylated Chromatin with Rad18 is Diminished after 6h Chase

42/503- PUB-NChIP

Chase

Zoom

Biotin OverlapRad18

Pulse

Biotin OverlapRad18

Puls

e

Chas

e

BAP.H3 BAP.H2A25kDa

α6XHis-HRP

Streptavidin-HRP25kDa

BirA.Rad18

Puls

e

Chas

e

HEK

– 2

93T

cells

BirA.Rad18 + BAP.H2A

6h after UVC (20 J/m2)15 min Biotin Pulse

Fixed 6h laterCHASE SAMPLE

Fixed ImmediatelyPULSE Sample

Rad18 Specific Pattern Changes after Proximity with Rad18 is Diminished

H/L

ratio

s

MS analysis of Histone H4 peptide 4-17 (GKGGKGLGKGGAKR)

1 2 3 4

UM1Ac 2Ac 3Ac 4Ac

1

0.6

0.2

0

1.4

1- GFP+H2A (H) / GFP+H2A (L)2- Rad18+H2A (H) / GFP+H2A (L)

3- GFP+H2A (H) / GFP+H2A (L)4- Rad18+H2A (H) / GFP+H2A (L)

Pulse

Chase

3- PUB-NChIP

HEK-293T cells

BirA.GFPBAP.H2A

LBirA.GFPBAP.H2A

HBirA.Rad18

BAP.H2A

H

BirA.Rad18BAP.H2A

HBirA.GFPBAP.H2A

H

1 : 1 1 : 1

1 : 1

1 2

4

SILAC Experimental Design

UVC: 20 J/m2

15 min Biotin Pulse after 6 h of UVC

Chase samples, Biotin was removed, cells washed, reincubated in normal medium, harvested after 6h

Pulse samples, Biotin was removed, cells washed and harvested immediately

1 : 1

3

Puls

e Sa

mpl

esCh

ase

sam

ples

PUB-NChIP to Study Alternative Chromatin States

StreptavidinHRP

BirA-Rad18

BAP-H2ABBD

+ - - + - - + + - - - - - - - + + - BAP-mH2A

BAP-mH2A

BAP-H2ABBD

BirA-Rad18

BAP-H2AZ

- + - +- - + +

BAP-H2AZ

BAP-H2AZub

StreptavidinHRP

3- PUB-NChIP

BAP-H2ABBD

BAP-MacroH2A

BAP-H2AZ

BirA-Rad18 +CombinedαRad18 streptavidin

HEK

– 2

93T

cells

6 h after UVC (20 J/m2)15 min Biotin Pulse

Pattern of H4 Acetylation near Rad18 is Different in H2AZ Containing Chromatin

3- PUB-NChIP

1 2 3 4

UM1Ac 2Ac 3Ac 4Ac

H/L

ratio

s

1- GFP+H2A (H) / GFP+H2A (L)2- Rad18+H2A (H) / GFP+H2A (L)

3- GFP+H2AZ (H) / GFP+H2A (L)4- Rad18+H2AZ (H) / GFP+H2A (L)

MS analysis of Histone H4 peptide 4-17 (GKGGKGLGKGGAKR)

H2A

H2AZHEK-293T cells

BirA.GFPBAP.H2A

LBirA.GFPBAP.H2A

HBirA.Rad18

BAP.H2A

H

BirA.Rad18BAP.H2AZ

HBirA.GFPBAP.H2AZ

H

1 : 1 1 : 1

1 : 11 : 1

1 2

3 4

SILAC Experimental Design

UVC: 20 J/m2

15 min Biotin Pulse after 6h of UVC

H6

H2

R1(49) R2(17) R3(24)

H3(81) H4(80) H5(83)

H1(41)

0.6 1.0 1.4 1.8

H6(54)

H2(31)

VMR (Fano factor) –

variance to mean ratio

The statistics of distances between

successive mutations in experimental samples is

compared with simulated random

mutations.

Parkhomchuk D et al. Use of high throughput sequencing to observe genome dynamics at a single cell level.

Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20830-5

Cellular variability

Heritable Nonheritable

EpigeneticGenetic Stochasticity at the level of individual cells

Tumor heterogeneity requires Single-Cell analysis

Position Type

129499013104501359180139791214189421442258145444514712901472324154691816036361611796166196217143011714977173064117833701799682180292518463971859442193389520370052037101207471720980542135475

G A G AG AG AG AG AG AG AG AG AG AG AG AC TC TG AG AC TC TC TC TC TC TC TC TC TC T

PUB allows to study the protein of interest at defined time after the interaction took place

Emerin-GFP Nurim-GFP

BirA-Emerin+ BAP-H2A BirA-Nurim

+ BAP-H2A

DAPI

biotin

biotinGFP

GFP

The chromatin domains that were proximal to nuclear envelope in the interphase appear

as discrete bands on mitotic chromosomes

Pulse labeling with biotin

Pulse – chase setup:Cells are labeled with biotin for 5’,

then washed and allowed to enter mitosis

A

B

BiotinGFP

Use of PUB to study epigenetic variability

Acknowledgements

Undine MecholdMartine ComissoAntoine ViensShoaib MuhammadEvelyne SaadeDamien VertutArman KulyyassovChloe Robin

Group members: Collaborators:

Pasquale MoioFranck BroouillardPatricia Kannouche