evelopment of proteomics tools to study intranuclear organizatio Vasily Ogryzko Group of “Proteomics & epigenetics’, UMR 8126 CNRS, Institut Gustave Roussy 15 мая 2014 Программа визитов иностранных учёных в российские научные центры фонда "Династия"
Jan 05, 2016
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