Laboratory of Molecular Genetics, KNU
Protein – protein interaction
Laboratory of Molecular Genetics, KNU
Modular Organization of
Protein Interaction Network
Laboratory of Molecular Genetics, KNU
Biological Networks
Biological Systems
Made of many non-identical elements
connected by diverse interactions.
Biological Networks
Biological networks as framework for the study of biological systems
Laboratory of Molecular Genetics, KNU
A Section of Module Network of 30 Largest Modules
Laboratory of Molecular Genetics, KNU
METHODS FOR SHOWING INTERACTIONS
Between protein and another protein
• immunoprecipitation (in vivo)
• GST pull-down assay (in vitro)
• yeast two-hybrid system (in yeast)
Laboratory of Molecular Genetics, KNU
GST pull down assay
Laboratory of Molecular Genetics, KNU
GST pull down assay
Principle
a simple technique to test interaction between a tagged protein or the bait (GST, His6, biotin ...) and another protein
(test protein, or prey).
Method
Laboratory of Molecular Genetics, KNU
|BamH1 |
|EcoR1 ||SmaI
... ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG
... TAG CTT CCA GCA CCC TAG GGG TCC TTA AGG GCC
... Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg
| Factor Xa |
|SalI |
GTC GAC
CAG CTG
Val Asp
1. Engineered protease site allows
removal of fusion partner
XhoI |
TCG AGC
AGC TCG
Ser Ser
NotI |
GGC CGC ...
CCG GCG ...
Gly Arg ...
Laboratory of Molecular Genetics, KNU
GST pull-down assay
Sepharose
GSH
GST “X”
“Y”
Sepharose
GSH
GST
“Y”
Laboratory of Molecular Genetics, KNU
GST pull-down assay
Sepharose
GSH
Sepharose
GSH
GST “X”
“Y”
GST
Laboratory of Molecular Genetics, KNU
GST pull-down assay
Run Western blot
Sepharose
GSH
Input
anti-Y
GST “X”
“Y”
GST-X
GST
Laboratory of Molecular Genetics, KNU
Tracheae defective/Apontic is an MBF1 partner
Laboratory of Molecular Genetics, KNU
2. Addition of a few residues should have
minimal effect on recombinant protein
His6 Tag
• add 6 consecutive
His to either end
• binds metals
Epitope Tag
• 6-12 amino acids
• mAb for detection or purification
Laboratory of Molecular Genetics, KNU
Immunoprecipitation
Laboratory of Molecular Genetics, KNU
Immunoprecipitation
• affinity purification based on
isolation of Ag-Ab complexes
• analyze by gel electrophoresis
• initially based on centrifugation of
large supramolecular complexes
• [high] and equal amounts
• isolation of Ag-Ab complexes
• fixed S. aureus
• protein A-agarose
• protein G-agarose
Bacterial proteins that bind IgG (Fc):
• protein A (Staphylococcus aureus)
• protein G (Streptococcus)
• binds more species and subclasses
Laboratory of Molecular Genetics, KNU
Immunoprecipitation(면역침강)
Method
Cell lysate 준비
Cell lysate Preclearing
Immunoprecipitation과정 시 방해가 될 수 있는 chromosome이나 Proteins A or
Proteins G에 친화성이 강해 antigen-antibody의 침전과정과 별개로 직접 the bead에
붙을 수 있는 것들을 없애주기 위함
Immunoprecipitation
precleared lysate가 들어있는 tube에 1~10ug의 antibody을 넣어줍니다
Washing
Ab와 결합하지 않은 상층액을 제거후 Washing buffer를 이용하여 washing 하여 줌
SDS-PAGE loading 후 wastern blotting 으로 확인
DNase foot printing method
Figure. Immunoprecipitation
Laboratory of Molecular Genetics, KNU
Typical IP Protocol
1. Solubilize antigen
• usually non-denaturing
• SDS + excess of TX100
2. Mix extract and Ab
3. Add protein G-agarose, etc
4. Extensively wash
5. Elute with sample buffer
6. SDS-PAGE
7. Detection
• protein stain
• radioactivity
agarose
G
Laboratory of Molecular Genetics, KNU
Tracheae defective/Apontic is an MBF1 partner
Laboratory of Molecular Genetics, KNU
Example
E2F
ATP
inactive
p
p
0
active
Rb
+
E2F
20
E2F
DNA replication
3
in Brij-35- (BRIJ), but not TX-100-solubilized platelets
Detergent
Strong : TritonX-100, NP-40
Mild : Brij, CHAPS
: MIC-1 (ng/ml)
Rb
gene expression
CD9 co-immunoprecipitates with aIIb
50
IP :
Rb
Rb
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Fusion Proteins
partner
• increase stability
• affinity purification
• detection/assay
• spectrophotometric
• binding assays
• antibodies
target
• export signals
Fusion Partner
Affinity Ligand
Glutathione-S-Transferase
Thioredoxin
Maltose Binding Protein
Six Histidine Residues (His6)
glutathione
phenylarsine oxide
amylose
nickel
Flag, Myc, HA, GFP…
antibody
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Yeast two hybrid
Laboratory of Molecular Genetics, KNU
The yeast two hybrid system
Principle
protein-protein interactions 을 알아 보기 위한 방법
Eukaryote의 경우 complex를 이루어서 signal을 전달하는 경우가 많은데 이렇듯 complex를 이루어서 작용하는 protein을 찾아 낼 경우 사용
하는 방법
protein의 DNA binding domain과 activation domain 분리하여 각각에 binding 할 것이라 생각되는 protein을 붙인 후 yeast의 형질 전환을 통
해 protein의 binding을 예측 함
두가지 type의 hybrid를 만듬
Method
DBD (DNA binding domain) – protein
AD (activation domain) – protein
: DBD와 AD가 동시에 존재할 경우만이
유전자의 promoter region에 binding
하여 유전자의 발현을 촉진 시킴
만들어진 두 가지 type의 protein을 세포
내에 도입시키고 유전자의 발현을 관찰
주로 reporter gene 을 통하여 간접적으
로 관찰함
yeast를 이용할 경우는 minimal medium
에서 자랄 수 없던 것이 형질이 전환되어
mm 에서도 자랄 수 있는 것과 같은 특징
이용 함
Figure.
Yeast two hybrid system
Laboratory of Molecular Genetics, KNU
The yeast two hybrid system
Laboratory of Molecular Genetics, KNU
DBD
AD
+
gene
DBD bait
AD
fish
+
Measurable
product
reporter
Laboratory of Molecular Genetics, KNU
Yeast 2-Hybrid Assay
HIS
his- leu- trpbait
DBD
trp
lacZ
AD fish
leu
reporter
his
nucleus
Measurable
product
Laboratory of Molecular Genetics, KNU
Example
Oncogene (2003) 22, 6151 - 6159 : Xiaoying Yin, Christine Giap and John S Lazo
Laboratory
of Molecular Genetics, KNU
Example
Figure.
PIAS 와 Smad 간의 물리적인 결합 확인
Purpose
PIAS가 Smad6, 7과 binding 하는지 알아 봄
yeast GAL4 DB - Smad7,6 MH2
yeast GAL4 AD - PIYS
Result
Smad 7과 PIYS가 함께 도입된 yeast 만이 생존
각각을 도입한 경우 자라지 않음.
즉 두 가지가Binding 함을 알 수 있음
mm medium 이용 – 두 가지의 결합 확인 가능
JBC. Vol, 278, pp 34253- 34258 : Swiyu Imoto, Kenji Sugiyama and Ryuta Muromoto
Laboratory of Molecular Genetics, KNU
Yeast two-hybrid assay
Laboratory of Molecular Genetics, KNU
protein - DNA interaction
Laboratory of Molecular Genetics, KNU
Gene expression이 왜 중요한가
세포의 성장
세포의 특성
세포의 변화
세포의 죽음
세포의 기능
세포의 역할
Signal
Signal
A cell
B cell
Behavior
Behavior
Behavior
Laboratory of Molecular Genetics, KNU
Structure of gene and its promoter
promoter
intron
exon 1
5'-nontranslating
region
5'
intron
exon 2
intron
exon 3
open reading frame(ORF)
AUG
Met
UGA
UAG
UAA
intron
exon 4
3'-nontranslating region
AAAAAAAAAA-3'
Laboratory of Molecular Genetics, KNU
rat GLUT2 gene structure (1995)
B SHS E E SS
4.3 kb
EE S
B S E SE
7.8 kb
BHE
H
S X
9.3 kb
X
EHS
SH B S X B S
E SBE
4.2 kb
8.0 kb
S EEHX
4.1 kb
10.0 kb
*
1a1b
1a
1b1c
1c1d
1d
2
4a
3
4b 5
7
6
8 9
10
transcription factors
J6
H6
P
response elements
A2
F&Pol4
D1
B3
E5
gene X
P
P
ATP7
TATA
spacer DNA
E
promoter
RNA transcript
Laboratory of Molecular Genetics, KNU
DEFINITION
Human mRNA for p53 cellular tumor antigen
ACCESSION
X02469 M60950
SOURCE
human
ORGANISM
Homo sapiens
AUTHORS
Zakut-Houri,R., Bienz-Tadmor,B., Givol,D. and Oren,M.
TITLE
Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells
JOURNAL
EMBO J. 4 (5), 1251-1255 (1985)
FEATURES Location/Qualifiers
source
1..1317
/organism="Homo sapiens"
/db_xref="taxon:9606"
CDS
136..1317
/note="p53 tumor antigen (aa 1-?)"
/codon_start=1
/protein_id="CAA26306.1"
/db_xref="PID:g35210"
/db_xref="GI:35210"
/db_xref="SWISS-PROT:P04637"
/translation="MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLM
LSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKT
YQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAM
AIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVV
PYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCA
CPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRG
RERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD"
Laboratory of Molecular Genetics, KNU
CDS
136..1317
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
gtctagagcc
ggctgggagc
ttccgggtca
caggaaacat
ccgtcccaag
gaagacccag
gcaccagcag
tctgtccctt
tctgggacag
caactggcca
cgcgtccgcg
tgcccccacc
cgagtggaag
gtggtggtgc
tacatgtgta
acactggaag
gcctgtcctg
caccacgagc
ccccagccaa
gagcgcttcg
gggaaggagc
tctacctccc
accgtccagg gagcaggtag ctgctgggct ccggggacac tttgcgttcg
gtgctttcca cgacggtgac acgcttccct ggattggcag ccagactgcc
ctgccatgga ggagccgcag tcagatccta gcgtcgagcc ccctctgagt
tttcagacct atggaaacta cttcctgaaa acaacgttct gtcccccttg
caatggatga tttgatgctg tccccggacg atattgaaca atggttcact
gtccagatga agctcccaga atgccagagg ctgctccccc cgtggcccct
ctcctacacc ggcggcccct gcaccagccc cctcctggcc cctgtcatct
cccagaaaac ctaccagggc agctacggtt tccgtctggg cttcttgcat
ccaagtctgt gacttgcacg tactcccctg ccctcaacaa gatgttttgc
5'-nontranslating
open reading frame(ORF)
3'-nontranslating region
agacctgccc
tgtgcagctg tgggttgatt ccacaccccc gcccggcacc
region
UGA
ccatggccat ctacaagcag tcacagcaca
tgacggaggt tgtgaggcgc
UAG
atgagcgctg
gatggtctgg
cccctcctca
gcatcttatc
AUG
UAA
5' ctcagatagc
AAAAAAAAAA-3'
gaaatttgcg tgtggagtat ttggatgaca gaaacacttt tcgacatagt
cctatgagcc gcctgaggtt ggctctgact gtaccaccat ccactacaac
acagttcctg catgggcggc
atgaaccgga ggcccatcct caccatcatc
Met
actccagtgg taatctactg ggacggaaca gctttgaggt gcgtgtttgt
ggagagaccg gcgcacagag gaagagaatc tccgcaagaa aggggagcct
tgcccccagg gagcactaag cgagcactgc ccaacaacac cagctcctct
agaagaaacc actggatgga gaatatttca cccttcagat ccgtgggcgt
agatgttccg agagctgaat gaggccttgg aactcaagga tgcccaggct
caggggggag cagggctcac tccagccacc tgaagtccaa aaagggtcag
gccataaaaa actcatgttc aagacagaag ggcctgactc agactga
Laboratory of Molecular Genetics, KNU
ATG?
promoter
exon 1
ATG?
intron
exon 2
intron
intron
exon 3
intron
exon 4
5’ end of 1st exon
missing when cDNA cloning
can be determined by
1) Primer extension
2) RNase protection assay (RNase mapping)
3) S1 nuclease assay
4) 5’-RACE (Rapid Amplification of cDNA End)
Laboratory of Molecular Genetics, KNU
transcription factors
J6
H6
P
response elements
A2
F&Pol4
D1
B3
E5
gene X
P
P
ATP7
TATA
spacer DNA
promoter
RNA transcript
Trancription machinery components
General (Basal) factors
Upstream factors
ubiquitous
not regulated
initiation efficiency 
Inducible factors
similar to upstream factors
regulatory role at specific time and specific tissue
binding site is called "response element"
Definition of promoter and enhancer
Promoter : responsible for only initiation (~200 bp)
Enhancer : enhance initiation, closely packed array (~100 bp)
Laboratory of Molecular Genetics, KNU
Promoter Characterization
It is not possible to predict the DNA sequence recognized by proteins
1) Upstream factors
Module
Consensus
Factor
Comments
TATA box
TATAAAA
TBP
-25 ; initiation precision
CAAT box
GGCCAATCT
CTF/NF1
CP1, 2, 3, 4
C/EBP, ACF
-75 ; either orientation
GC box
GGGCGG
Sp1
-90 ; either orientation, often multicopy
Sp1 is a monomer, 105 kDa
Octamer
ATTTGCAT
Oct-1
Oct-2
Oct-1 is ubiquitous
Oct-2 is lymphoid specific factor
B
GGGACTTTCC
NFB
ATF
GTGACGT
ATF
Basal factor (TATA, Inr) : initiation location
Upstream factor (GC, CAAT) frequency of initiation (assembly)
Conserved element does not inevitably imply binding of protein
Laboratory of Molecular Genetics, KNU
2) Response elements
Module
Consensus
Factor
Regulatory agent
HSE
CNNGAANNTCCNNG
HSTF
heat shock
GRE
TGGTACAAATGTTCT
Receptor
glucocorticoid
TRE
TGACTCA
AP1
phorbol ester
SRE
CCATATTAGG
SRF
serum
May be located in promoters or enhancers
Active protein is available only under certain condition
Any one of several elements can independently activate the gene
Laboratory of Molecular Genetics, KNU
3) Character of transcription factors
Structure
① DNA-binding domain (usually basic)
② Activation domain (usually acidic)
③ Dimerization domain
Domains are independent, interchangable
Laboratory of Molecular Genetics, KNU
Transactivation
direct interaction or with coactivators
contact with TFIID (most common, esp TAFs), TFIIB, TFIIA
can influence to initiation complex by looping at a distance
F&Pol4
D1
TATAA
Laboratory of Molecular Genetics, KNU
Categories of Transcription Activators
according to DBD, [Gene VII]
1)
2)
3)
4)
Helix-turn-helix (HTH) motif
Zn finger motif
Leucine zipper (Zip) motif, usually basic (bZip)
Helix-loop-helix (HLH) motif, usually basic (bHLH)
1) Helix-Turn-Helix motif
two or three  helices and short chain (turn)
can form dimer
Homeodomain proteins
found in proteins related to development
originally found in Drosophila
determine the identity of body structure
also found in higher eukaryote
Oct proteins ; 75 aa, called Pou domain
C
N
Laboratory of Molecular Genetics, KNU
2) Zinc Finger motif
 helix - Zn -  sheet
 helix contacts DNA
H
H
Classic zinc finger proteins : Sp1 (3 fingers)
Steroid receptors : 2 fingers
steroid hormone : MR, AR, PR, ER
-sheet -helix
thyroid hormone : T3R
retinoic acid (vitamin A) : RAR, RXR
bind to a specific receptor that activates gene transcription
"receptor" may be a misnomer
recognize special consensus sequence, like GRE
consist of
central DNA binding domain
N-terminal activation domain
C-terminal ligand binding domain
C
Zn
C
H
C
Zn
C
H
C
Zn
C
H
H
Laboratory of Molecular Genetics, KNU
Additional) -barrels
two  sheets contacts DNA
Papilloma virus activator E2
3) Leucine Zipper motif
two  helices
DNA binding and protein dimerization by same motif
homodimer or heterodimer
expand the repertoire of DNA-binding specificities
basic region is DNA binding domain, bZip
Leu
Leu Leu
Leu Leu
Leu
Leu
Leu Leu
Leu Leu
Leu
Leu
Leu
C/EBP
Jun/Fos, JunB, JunD, Fra (Fos-related Antigen)
Fos cannot homodimerize
Jun/Fos can bind with an activity more 10 folds than Jun/Jun
++++
++++
DNA
Laboratory of Molecular Genetics, KNU
4) Helix-Loop-Helix motif
short  helix and long  helix
homodimer or heterodimer
highly basic region can bind to DNA ; bHLH
E12, E47 (Ig gene enhancer)
MyoD, myogenin, Myf-5 (myogenesis), Myc (oncogene)
bHLH fall into 2 groups
class A : ubiquitously expressed (E12/E47)
class B : tissue-specific manner (MyoD)
some HLH protein lacks long helix
can dimerize, unable to bind
dominant negative way (Id proteins)
DNA
Laboratory of Molecular Genetics, KNU
dominant negative fashion like HLH proteins
repressor
F&Pol4
D1
TATAA
Laboratory of Molecular Genetics, KNU
PROMOTER ANALYSIS
1) Oocyte system
2) Transfection system
CAT assay
Luciferase assay
3) Transgenic system
4) in vitro system
EMSA (electrophoretic mobility shift assay)
DNase I footprinting assay
In vitro transcription assay
5) Transcription factor characterization
Affinity chromatography
Two hybrid
6) in vivo system
in vivo DNase I footprinting assay
ChIP assay (chromatin immunoprecipitation)
Laboratory of Molecular Genetics, KNU
How to measure gene activation in eukaryotic cells ?
Transfection assays
RAR
reporter
RXR
nucleus
Measurable
product
Laboratory of Molecular Genetics, KNU
How to measure gene activation in eukaryotic cells ?
Transfection assays
Hormone (steroid)
RAR
reporter
RXR
nucleus
Measurable
product
Laboratory of Molecular Genetics, KNU
0
2
Relative CAT activity (fold)
4
6
8
10
12
+189
no C/EBP
C/EBP
C/EBP
C/EBP and 
C A T
-503
H
G
F
E
D
C
B
A
+189
1x
C A T
+56
A
+189
H
2.9x
2.6x
C A T
+56
A
9.1x
+189
C A T
+56
F
A
2.2x
1.6x
3.8x
14
Laboratory of Molecular Genetics, KNU
+1
LUC
-890
LUC
-389
LUC
-283
LUC
-166
LUC
-57
d-283/-166
Control
RXRPPAR
40
35
30
25
20
15
10
5
-1112
-890
-389
-283
45
LUC
LUC
Relative luciferase activity (fold)
-1112
Relative luciferase activity (fold)
45
-166
Control
RXRPPAR
40
35
30
25
20
15
10
5
-1112
-389
d-283/-166
-57
Laboratory of Molecular Genetics, KNU
DNA foot printing
Laboratory of Molecular Genetics, KNU
DNase foot printing
Principle
protein과 DNA interaction을 알아 보기 위한 방법
protein이 DNA에 binding한 경우 protein에 의해 DNA가 보호 받으므로 DNase I를 처리한다 하여도
DNA가 잘리지 않고 그 결과 gel loading 시 DNA band가 생기지 않는 부위가 생김
이 부위를 foot print라 함
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Fig. DNase foot printing
Laboratory of Molecular Genetics, KNU
Method
특정 protein binding site를 포함하고 있는 Restriction
fragment 한쪽 끝을 방사선 동위 원소(주로 32P사용)
이용하여 labeling 힘
방사선 동위원소로 표지 된 DNA만 존재 하는 조건과
DNA Protein이 함께 존재하는 조건에 DNase I를 처리
DNase I : DNA상에 single brake를 생성함
주의 : 이 경우 DNase I은 mild하게 처리해야 함.
평균 하나의 strand 당 하나의 nick이 생성 됨
DNase I의 작용을 stop 시킴. DNA는 denatured 시킴
이것을 denaturing polyacrylamide gel running 시킴
위와 같이 running 시킨 DNA band는 autoradiography
또는 Phosphorimager를 이용하여 확인 함
결과로 나타난 band의 ladder는 DNasae I에 의해 잘린
다양한 site 를 의미 함
Control과 비교하여 Band가 생성되지 않은 부위가 바로
Protein이 DNA에 binding 한 부위이며 이곳의 sequence
및 size는 여러 sequencing 방법을 통해 알 수 있음
Laboratory of Molecular Genetics, KNU
Example
HA-NFATp binds DNA alone and cooperatively with cJun/cFos. DNase I footprinting assays were performed to
investigate the binding of HA-NFATp to a region of the human IL-2 promoter in the absence (lanes 1-5) and
presence (lanes 6-10) of recombinant human cJun/cFos (3.2 nM). HA-NFATp was added to reactions at the
following final concentrations: 3 nM, lanes 2 and 7; 9 nM, lanes 3 and 8; 27 nM, lanes 4 and 9; and 54 nM, lanes 5
and 10. Footprinting reactions were resolved by denaturing PAGE and analyzed with a Molecular Dynamics
PhosphorImager. Positions relative to the transcriptional start site (+1) of the human IL-2 promoter are indicated
on the left. Locations of the previously characterized high affinity -45 NFAT site and composite element are
indicated on the right.
Laboratory of Molecular Genetics, KNU
EMSA(electrophoretic mobility gel shift assay )
Principle
Protein-DNA complex
Protein-DNA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or
agarose gel electrophoresis
Laboratory of Molecular Genetics, KNU
Method
Figure.
EMSA
protocol
Laboratory
of Molecular Genetics, KNU
Nuclear extract preparation
1. Drug treatment
2. Washing 1 times with cold PBS (7ml)
3. Scrape with cold PBS 1ml
4. Centrifuge at3,000 rpm for 1min
5. Resuspend with buffer A 400ul
6. Incubate on ice for 15min
7. Add 25ul of 10% NP-400 (final conc’ 0.6%)
8. Vortexing or pipetting for 30sec (강하게)
9. Centrifuge at 14,000rpm for 2-5min in 4’C
10. Resuspend with buffer C 50ul
11. Incubate on ice for 30min
12. Centrifuge at 14,000rpm for 5min in 4’C
13. Transfer 50ul of the soup to fresh tube
14. Storage at -70’c in deep freezer
Laboratory of Molecular Genetics, KNU
Buffer A
-10mM HEPES pH 7.9, 10mM KCL, 0.1mM EDTA,
0.1mM EGTA, 1mM DTT, 0.5mM PMSF,
0.5mM Leupeptin
Buffer C
-20mM HEPES pH 7.9, 0.4M NACL 1mM EDTA, 1mM
EGTA, 1mM DTT, 1mM PMSF,
1mM Leupeptin
protocol
Laboratory
of Molecular Genetics, KNU
Laboratory
of Molecular Genetics, KNU
protocol
Laboratory of Molecular Genetics, KNU
protocol
Laboratory of Molecular Genetics, KNU
Free probe
Laboratory of Molecular Genetics, KNU
Example
wt
wt
-
-
wt
+
mt
wt
+
+
+
+
-
-
+
: probe (E2F)
: Cold probe
: Ab (α-E2F1)
: MIC-1
-
E2F
+ IgG
E2F
-
wt
wt
mt
-
+
+
+
-
wt
-
+
wt
-
+
wt
-
+
wt
-
+
wt
-
+
wt
-
+
Fra-2
wt
Fra1
wt
Fos
B
-
c-fos
-
Jun
D
-
Jun
B
-
cJun
Free probe
Free probe
wt
-
+
: Ab
: Probe
: Cold probe
: FN
*
AP-1 +
IgG
AP-1
Laboratory of Molecular Genetics, KNU
ChIP(chromatin immunoprecipitation)
Principle
Protein 이 어느 유전자 부위에 결합하는지를 알아보는 방법
Protein과 specific DNA region을 알아보기 위해 보고자 하는 protein Ab로 immunoprecifitation실시 후 primer를 이용, PCR을 수행하는 방법
Laboratory of Molecular Genetics, KNU
Chromatin immunoprecipitation
• Used to determine whether a given protein
binds to a given DNA sequence in vivo
• Like all protein analysis involving antibodies
(including westerns) a specific antibody is
required
• If there is no specific antibody, then epitope
tagging can be employed (FLAG, MYC, HIS)
• An epitope is a portion of a molecule to
which an antibody binds
Laboratory of Molecular Genetics, KNU
Method
포름알데하이드로 단백질(DNA에 결합된 단백질)과 DNA를
결합시킵니다.
Sonication 시켜서 세포와 DNA를 잘게 부순다.
Immunoprecipitation 과정 수행
DNA를 분리 정제
보고자 하는 유전자 의 프로모터 주변에서 primer를 디자인 하여
PCR을 수행.
만약 어떤 전사인자가 A라고 하는 유전자의 프로모터에
결합한다면 위의 과정을 거치면 대부분의 DNA는 제거되고
A 유전자 프로모터 부위는 남게됨, 그러면 A유전자 프로모터에
결합하는 primer를 이용, PCR을 수행하면 밴드를 얻게되고.
그래서 밴드가 진하게 나오면 결합하는 것이고 그렇지 않으면
결합하지 않는다고 봄
Figure.
Yeast two hybrid system
Chromatin ImmunoPrecipitation (ChIP)
- Purification of nuclei from 0-16 hrs embryos
- UV cross-linking
- Sonication (0.1 - 3 Kb)
- in vivo IP with anti-EN Ab
-Addition of linkers
PCR amplification
- in vitro IP with anti-EN Ab
PCR amplification.
HindIII digestion and cloning in Ks+
603 individual clones isolated and sequenced
315 independent clones
Laboratory of Molecular Genetics, KNU
Example
Snail promoter 지역에는 MTA3와 MBD3분자가
Association되어 있음.
MTA3와 MBD3분자는 Snail promoter
Start 지점의 500bp 정도 떨어진
upstream 부분에서 강하게 Binding한다.
ChIP analysis on estrogen target genes.
Mol. Cell Biol. (2004)
Knockdown of RIZ1 by siRNA affects expression
and methylation of pS2 gene.
Mol. Cell Biol. (2004)
Laboratory
of Molecular Genetics, KNU
protocol
Part A. Optimization of DNA Shearing
Establish optimal conditions required for shearing cross-linked DNA to 200-1000 base pairs in length by following steps 1- 9 below.
Vary the power setting and/or the number of 10-second pulses during sonication of the samples. Be sure to keep the sample on ice at
all times (the sonication generates heat which will denature the DNA). Check the size of sonicated DNA by gel electrophoresis after
reversion of cross-links. Our experience shows DNA is sheared to the appropriate length with 3-4 sets of 10-second pulses using a
Cole Parmer, High Intensity Ultrasonic Processor/Sonicator, 50 watt model equipped with a 2mm tip and set to 30% of maximum
power. Once sonication conditions have been optimized, keep cell number consistent for subsequent experiments. The protocol
below for the optimization of DNA Shearing is for one Chip assay (~1 x 106 cells per condition).
Note: Steps 3 - 7 should be done on ice
1.
Stimulate or treat 1 x 106 cells on a 10cm dish as appropriate. (Cells should be treated under
conditions for which transcriptional activation of the gene of interest has been demonstrated). Include
one extra dish (1 x 106 ) to be used solely for estimation of cell number.
2. Cross link histones to DNA by adding formaldehyde directly to culture medium to a final concentration
of 1% and incubate for 10 minutes at 37C. (For example, add 270 microliters 37% formaldehyde into
10mof growth medium on plate).
3. Aspirate medium, removing as much medium as possible. Wash cells twice using ice cold
PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1microgram/ml
aprotinin and 1microgram/ml pepstatin A). Note: Add protease inhibitors to PBS just prior to use.
PMSF has a half-life of approximately 30 minutes in aqueous solutions.
4. Scrape cells into conical tube.
Laboratory of Molecular Genetics, KNU
5. Pellet cells for 4 minutes at 2000 rpm at 4ºC. Warm SDS Lysis Buffer (Catalog # 20-163) to
room
temperature to dissolve precipitated SDS and add protease inhibitors (inhibitors: 1mM PMSF,
1microgram/ml aprotinin and 1microgram/ml pepstatin A).
6. Resuspend cell pellet in 200 microliters of SDS Lysis Buffer (Catalog # 20-163) and incubate
for 10
minutes on ice. Note: The 200 microliters of SDS Lysis Buffer is per 1 X 106 cells; if more cells
are
used, the resuspended cell pellet should be divided into 200 microliters aliquots so that each
200ml
aliquot contains ~1 X 106 cells.
7. Sonicate lysate to shear DNA to lengths between 200 and 1000 basepairs being sure to keep
samples ice cold (Note: Once sonication conditions have been optimized following steps 1 to 9,
proceed to Part B, step 1 below).
8. Add 8 microliters 5M NaCl (Catalog # 20-159) and reverse crosslinks at 65ºC for 4 hours.
9. Recover DNA by phenol/chloroform extraction and run sample (example 5 microliter, 10
microliter,
and 20 microliter samples) in an agarose gel to visualize shearing efficiency.
Laboratory
of Molecular Genetics, KNU
protocol
Part B. Experimental protocol.
If sonication conditions have been optimized (Part A), complete steps 1 through 7 and continue with the protocol below.
For a negative/background control, prepare a sample to use as a no-antibody immunoprecipitation control in step 5 below.
Additionally, transcriptionally unactivated DNA samples should be prepared as controls for PCR in section II.
1.
Centrifuge samples (part A, step 7) for 10 minutes at 13,000 rpm at 4°C, and add 200 microliters of the sonicated cell
pellet suspension to a new 2ml-microcentrifuge tube.
2. Dilute the sonicated cell pellet suspension 10 fold in ChIP Dilution Buffer (Catalog # 20153), adding protease inhibitors as above. This is done by adding 1800 microliters ChIP
Dilution Buffer to the 200microliter sonicated cell pellet suspension for a final volume of 2ml in
each immunoprecipitation condition. Note: If proceeding to PCR a portion of the diluted cell
pellet suspension 1% (~20 microliters) can be kept to quantitate the amount of DNA present in
different samples at the PCR protocol, Part B, section II, step 6. This sample is considered to
be your input/starting material, andneeds to have the Histone-DNA crosslinks reversed by
heating at 65C for 4 hours (see section II, step3.)
3. To reduce nonspecific background, pre-clear the 2ml diluted cell pellet suspension with 80
microlitersof Salmon Sperm DNA/Protein A Agarose-50% Slurry (Catalog # 16-157) for 30
minutes at 4ºC withagitation.
4. Pellet agarose by brief centrifugation and collect the supernatant fraction.
Laboratory of Molecular Genetics, KNU
5. Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2ml
supernatant fraction and incubate overnight at 4ºC with rotation. For a negative control,
perform a no-antibody immunoprecipitation by incubating the supernatant fraction with 60
microliters of Salmon SpermDNA/Protein A Agarose- 50% Slurry (Catalog # 16-157) for one
hour at 4ºC with rotation and proceedto step 7.
6. Add 60 microliters of Salmon Sperm DNA/Protein A Agarose Slurry (Catalog # 16-157) for
one hour at4ºC with rotation to collect the antibody/histone complex.
7. Pellet agarose by gentle centrifugation (700 to 1000 rpm at 4ºC, ~1min). Carefully remove
thesupernatant that contains unbound, non-specific DNA. Wash the protein A
agarose/antibody/histonecomplex for 3-5 minutes on a rotating platform with 1ml of each of the
buffers listed in the order asgiven below:
a) Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one wash
b) High Salt Immune Complex Wash Buffer (Catalog # 20-155), one wash
c) LiCl Immune Complex Wash Buffer (Catalog # 20-156), one wash
d) 1X TE (Catalog # 20-157), two washes
After step 7 above, the sample is now a protein A/antibody/histone/DNA complex ready for either an
Immunoprecipitation/Immunoblot assay (Section I) or Polymerase Chain Reaction (PCR) assay (Section II):
Laboratory of Molecular Genetics, KNU
protocol
Section I. Immunoprecipitation/Immunoblot protocol to detect histone.
1 Following washing of the beads in part B, step 7, immunoprecipitated histones can be
analyzed by immunoblot analysis. Add 25 microliters of 1X Laemmli buffer per sample and boil for 10 minutes. Load 20
microliters per lane and perform immunoblot procedure as described per appropriate antibody.
Section II. PCR protocol to amplify DNA that is bound to the immunoprecipitated histone.
1. Freshly prepare elution buffer (1%SDS, 0.1M NaHCO3).
2. Elute the histone complex from the antibody by adding 250 microliter elution buffer to the pelleted protein A
agarose/antibody/histone complex from step 7d above. Vortex briefly to mix and incubate at room temperature for 15 minutes with
rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to another tube and repeat elution. Combine
eluates (total volume= approximately 500 microliters.)
3. Add 20 microliters 5M NaCl (Catalog # 20-159) to the combined eluates (500 microliters) and reverse histone-DNA crosslinks
by heating at 65ºC for 4 hours. At this step the sample can be stored and -20°C and the protocol continued the next day.
Laboratory of Molecular Genetics, KNU
Note: Include the input/starting material (the sample saved from Part B, step 2, which has had the Histone-DNA
crosslinks reversed) as well as a transcriptionally-unactivated DNA sample as negative and background controls
for the PCR reaction. Previously, a 5 microliter sample has been used in a nested PCR reaction. However, the
amount of sample used per reaction must be determined empirically (e.g., titrate the sample at this step by using
1, 2, 5, or 10 microliters per PCR reaction). If PCR results are poor, complete steps 4, 5 and 6 below to purify
the DNA sample. NOTE: Handle the samples carefully, some DNA may be lost during the purification steps.
4. Add 10 microliters of 0.5M EDTA (Catalog # 20-158), 20 microliters 1M Tris-HCl, pH 6.5 (Catalog # 20-160)
and 2 microliters of 10mg/ml Proteinase K to the combined eluates and incubate for one hour at 45ºC.
5. Recover DNA by phenol/chloroform extraction and ethanol precipitation. Addition of an inert carrier,such as 20
micrograms glycogen or yeast tRNA, helps visualize the DNA pellet. Wash pellets with 70%ethanol and air dry.
6. Resuspend pellets in an appropriate buffer for PCR or slot-blot reactions. PCR or slot-blot conditionsmust be
determined empirically.
Laboratory of Molecular Genetics, KNU
ChIP on Chip
Chromatin immunopreciptation on a microarray Chip
DNA from binding site
Fluorescently
label
http://www.chiponchip.org/