Protein – protein interaction

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Protein – protein interaction


Modular Organization of Protein Interaction Network


Biological Networks

Biological SystemsMade of many non-identical elements

connected by diverse interactions.

Biological Networks

Biological networks as framework for the study of biological systems


A Section of Module Network of 30 Largest Modules


METHODS FOR SHOWING INTERACTIONS

Between protein and another protein• immunoprecipitation (in vivo)• GST pull-down assay (in vitro)• yeast two-hybrid system (in yeast)


GST pull down assay


a simple technique to test interaction between a tagged protein or the bait (GST, His6, biotin ...) and another protein (test protein, or prey).

GST pull down assayGST pull down assay

Principle

Method


|BamH1 | |EcoR1 ||SmaI |SalI | XhoI | NotI |... ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ...... TAG CTT CCA GCA CCC TAG GGG TCC TTA AGG GCC CAG CTG AGC TCG CCG GCG ...... Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg ... | Factor Xa |

1. Engineered protease site allows removal of fusion partner


GST “X” “Y”

GST pull-down assay

SepharoseGSH

SepharoseGSH

GST

“Y”


GST pull-down assay

SepharoseGSH

GST “X” “Y”

SepharoseGSH

GST


GST pull-down assay

SepharoseGSH

GST “X” “Y”

Run Western blot

Input GST-X GST

anti-Y


Tracheae defective/Apontic is an MBF1 partner


His6 Tag

• add 6 consecutive His to either end

• binds metals

2. Addition of a few residues should have minimal effect on recombinant protein

Epitope Tag

• 6-12 amino acids• mAb for detection or purification


Immunoprecipitation


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


Immunoprecipitation(Immunoprecipitation( 면역침강면역침강 ))

Method

Figure. ImmunoprecipitationImmunoprecipitation

DNase foot printing method

Cell lysate 준비

Cell lysate Preclearing

Immunoprecipitation 과정 시 방해가 될 수 있는 chromosome 이나 Proteins A or Proteins G 에 친화성이 강해 antigen-antibody 의 침전과정과 별개로 직접 the bead 에 붙을 수 있는 것들을 없애주기 위함

Immunoprecipitation precleared lysate 가 들어있는 tube 에 1~10ug 의 antibody 을 넣어줍니다

Washing

Ab 와 결합하지 않은 상층액을 제거후 Washing buffer 를 이용하여 washing 하여 줌

SDS-PAGE loading 후 wastern blotting 으로 확인


Typical IP Protocol

1. Solubilize antigen• usually non-denaturing• SDS + excess of TX100

2. Mix extract and Ab 3. Add protein G-agarose, etc4. Extensively wash5. Elute with sample buffer6. SDS-PAGE7. Detection

• protein stain• radioactivity

Gagarose


Tracheae defective/Apontic is an MBF1 partner


Example

CD9 co-immunoprecipitates with aIIb 3 in Brij-35- (BRIJ), but not TX-100-solubilized platelets

500 : MIC-1 (ng/ml)20

E2F

IP :

R

b

Rb

E2FRb

E2FRbp p

+

ATP

gene expression

DNA replication

inactive

active

Detergent

Strong : TritonX-100, NP-40

Mild : Brij, CHAPS


Fusion Proteins • increase stability• affinity purification• detection/assay

• spectrophotometric• binding assays• antibodies

• export signals

partner target

Fusion Partner Affinity LigandGlutathione-S-Transferase glutathione

Thioredoxin phenylarsine oxide

Maltose Binding Protein amylose

Six Histidine Residues (His6) nickel

Flag, Myc, HA, GFP… antibody


Yeast two hybrid


The yeast two hybrid systemThe yeast two hybrid system

protein-protein interactions 을 알아 보기 위한 방법

Eukaryote 의 경우 complex 를 이루어서 signal 을 전달하는 경우가 많은데 이렇듯 complex 를 이루어서 작용하는 protein 을 찾아 낼 경우 사용하는 방법

protein 의 DNA binding domain 과 activation domain 분리하여 각각에 binding 할 것이라 생각되는 protein 을 붙인 후 yeast 의 형질 전환을 통해 protein 의 binding 을 예측 함

Principle

Method

Figure. Yeast two hybrid system

두가지 type 의 hybrid 를 만듬

DBD (DNA binding domain) – protein AD (activation domain) – protein

: DBD 와 AD 가 동시에 존재할 경우만이 유전자의 promoter region 에 binding

하여 유전자의 발현을 촉진 시킴

만들어진 두 가지 type 의 protein 을 세포내에 도입시키고 유전자의 발현을 관찰

주로 reporter gene 을 통하여 간접적으 로 관찰함

yeast 를 이용할 경우는 minimal medium

에서 자랄 수 없던 것이 형질이 전환되어 mm 에서도 자랄 수 있는 것과 같은 특징 이용 함


The yeast two hybrid systemThe yeast two hybrid system


ADDBD

gene

+

reporter

Measurableproduct

+

DBD bait AD fish

DBD

AD


nucleus

his- leu- trp-

Yeast 2-Hybrid Assay

Measurableproduct

DBD bait

trp

AD fishleu

reporter

DB

D

AD

his

HIS

lacZ


Oncogene (2003) 22, 6151 - 6159 : Xiaoying Yin, Christine Giap and John S Lazo

Example

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNUExample

JBC. Vol, 278, pp 34253- 34258 : Swiyu Imoto, Kenji Sugiyama and Ryuta Muromoto

Purpose

PIAS 가 Smad6, 7 과 binding 하는지 알아 봄

yeast GAL4 DB - Smad7,6 MH2

yeast GAL4 AD - PIYS

mm medium 이용 – 두 가지의 결합 확인 가능

Figure. PIAS 와 Smad 간의 물리적인 결합 확인

Result Smad 7 과 PIYS 가 함께 도입된 yeast 만이 생존각각을 도입한 경우 자라지 않음 . 즉 두 가지가 Binding 함을 알 수 있음


Yeast two-hybrid assay


protein - DNA interaction


Gene expression 이 왜 중요한가

A cell B cell

Signal

Behavior

Signal

Behavior

Behavior

세포의 성장세포의 특성세포의 변화세포의 죽음

세포의 기능세포의 역할


Structure of gene and its promoter

exon 1

in tronprom oter in tron in tron in tron

exon 2 exon 3 exon 4

A U G

5'-nontranslating region

3'-nontranslating regionopen reading frame(ORF)

U G AU A GU A A

M et

5 ' A A A A A A A A A A -3 '


4 .3 k b 7 .8 k b 9 .3 k b 4 .2 k b 8 .0 k b 4 .1 k b 1 0 .0 kb

1a1a 1b1b1c1c1d1d 2 3 4a 4b 6 7 8 9 105

BB BB X X X XEESS SSHH SS SS S S S S S S S SSEE EE E E E E E E EEEBH H H H HB B BEE

*

rat GLUT2 gene structure (1995)

P

P

TATA

prom oter

gene X

RNA transcrip t

transcrip tion fac tors

response e lem ents

spacer DNA

PD1

A 2

B 3F &P ol4

E 5

J6 H6

ATP 7


DEFINITION Human mRNA for p53 cellular tumor antigenACCESSION X02469 M60950SOURCE humanORGANISM Homo sapiensAUTHORS Zakut-Houri,R., Bienz-Tadmor,B., Givol,D. and Oren,M.TITLE Human p53 cellular tumor antigen: cDNA sequence and expression in COS cellsJOURNAL 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"


CDS 136..1317

ORIGIN 1 gtctagagcc accgtccagg gagcaggtag ctgctgggct ccggggacac tttgcgttcg 61 ggctgggagc gtgctttcca cgacggtgac acgcttccct ggattggcag ccagactgcc 121 ttccgggtca ctgccatgga ggagccgcag tcagatccta gcgtcgagcc ccctctgagt 181 caggaaacat tttcagacct atggaaacta cttcctgaaa acaacgttct gtcccccttg 241 ccgtcccaag caatggatga tttgatgctg tccccggacg atattgaaca atggttcact 301 gaagacccag gtccagatga agctcccaga atgccagagg ctgctccccc cgtggcccct 361 gcaccagcag ctcctacacc ggcggcccct gcaccagccc cctcctggcc cctgtcatct 421 tctgtccctt cccagaaaac ctaccagggc agctacggtt tccgtctggg cttcttgcat 481 tctgggacag ccaagtctgt gacttgcacg tactcccctg ccctcaacaa gatgttttgc 541 caactggcca agacctgccc tgtgcagctg tgggttgatt ccacaccccc gcccggcacc 601 cgcgtccgcg ccatggccat ctacaagcag tcacagcaca tgacggaggt tgtgaggcgc 661 tgcccccacc atgagcgctg ctcagatagc gatggtctgg cccctcctca gcatcttatc 721 cgagtggaag gaaatttgcg tgtggagtat ttggatgaca gaaacacttt tcgacatagt 781 gtggtggtgc cctatgagcc gcctgaggtt ggctctgact gtaccaccat ccactacaac 841 tacatgtgta acagttcctg catgggcggc atgaaccgga ggcccatcct caccatcatc 901 acactggaag actccagtgg taatctactg ggacggaaca gctttgaggt gcgtgtttgt 961 gcctgtcctg ggagagaccg gcgcacagag gaagagaatc tccgcaagaa aggggagcct 1021 caccacgagc tgcccccagg gagcactaag cgagcactgc ccaacaacac cagctcctct 1081 ccccagccaa agaagaaacc actggatgga gaatatttca cccttcagat ccgtgggcgt 1141 gagcgcttcg agatgttccg agagctgaat gaggccttgg aactcaagga tgcccaggct 1201 gggaaggagc caggggggag cagggctcac tccagccacc tgaagtccaa aaagggtcag 1261 tctacctccc gccataaaaa actcatgttc aagacagaag ggcctgactc agactga

A U G

5'-nontranslating region

3'-nontranslating regionopen reading frame(ORF)

U G AU A GU A A

M et

5 ' A A A A A A A A A A -3 '


exon 1

in tronprom oter in tron in tron in tron

exon 2 exon 3 exon 4

ATG? ATG?

5’ end of 1st exon

missing when cDNA cloningcan be determined by

1) Primer extension2) RNase protection assay (RNase

mapping)3) S1 nuclease assay4) 5’-RACE (Rapid Amplification of cDNA

End)


General (Basal) factorsUpstream factors

ubiquitousnot regulatedinitiation efficiency

Inducible factorssimilar to upstream factorsregulatory role at specific time and specific

tissuebinding site is called "response element"

P

P

TATA

prom oter

gene X

RNA transcrip t

transcrip tion fac tors

response e lem ents

spacer DNA

PD1

A 2

B 3F &P ol4

E 5

J6 H6

ATP 7

Trancription machinery components

Definition of promoter and enhancer

Promoter : responsible for only initiation (~200 bp)Enhancer : enhance initiation, closely packed array

(~100 bp)


Module Consensus Factor Comments

TATA box TATAAAA TBP -25 ; initiation precision

CAAT box GGCCAATCT CTF/NF1CP1, 2, 3, 4C/EBP, ACF

-75 ; either orientation

GC box GGGCGG Sp1 -90 ; either orientation, often multicopySp1 is a monomer, 105 kDa

Octamer ATTTGCAT Oct-1Oct-2

Oct-1 is ubiquitousOct-2 is lymphoid specific factor

B GGGACTTTCC NFB

ATF GTGACGT ATF

Promoter Characterization

It is not possible to predict the DNA sequence recognized by proteins1) Upstream factors

Basal factor (TATA, Inr) : initiation locationUpstream factor (GC, CAAT) frequency of initiation (assembly)Conserved element does not inevitably imply binding of protein


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 enhancersActive protein is available only under certain conditionAny one of several elements can independently activate the gene

2) Response elements


3) Character of transcription factors Structure

① DNA-binding domain (usually basic) ② Activation domain (usually acidic) ③ Dimerization domain

Domains are independent, interchangable


Transactivation

direct interaction or with coactivatorscontact with TFIID (most common, esp TAFs),

TFIIB, TFIIAcan influence to initiation complex by looping at a

distance

F& Pol4

TATA AD1


Categories of Transcription Activators

according to DBD, [Gene VII]

1) Helix-turn-helix (HTH) motif2) Zn finger motif3) Leucine zipper (Zip) motif, usually basic (bZip)4) Helix-loop-helix (HLH) motif, usually basic (bHLH)

1) Helix-Turn-Helix motif

two or three helices and short chain (turn)

can form dimerHomeodomain proteins

found in proteins related to development

originally found in Drosophiladetermine the identity of body

structurealso found in higher eukaryote

Oct proteins ; 75 aa, called Pou domain

NC


2) Zinc Finger motif

helix - Zn - sheet helix contacts DNA Classic zinc finger proteins : Sp1 (3 fingers)Steroid receptors : 2 fingers

steroid hormone : MR, AR, PR, ERthyroid hormone : T3Rretinoic acid (vitamin A) : RAR, RXRbind to a specific receptor that activates gene

transcription"receptor" may be a misnomer

recognize special consensus sequence, like GREconsist of

central DNA binding domainN-terminal activation domainC-terminal ligand binding domain

C C CH H H

C C CH H H

Zn Zn Zn

-sheet -he lix


3) Leucine Zipper motif

two helicesDNA binding and protein dimerization by same motifhomodimer or heterodimer

expand the repertoire of DNA-binding specificitiesbasic region is DNA binding domain, bZip C/EBPJun/Fos, JunB, JunD, Fra (Fos-related Antigen)Fos cannot homodimerizeJun/Fos can bind with an activity more 10 folds than

Jun/Jun

Le u Le uLe u Le uLe u Le uLe u Le uLe u Le uLe u Le u

+ ++ ++ ++ +

Le u Le u

D N A

Additional) -barrels

two sheets contacts DNA Papilloma virus activator E2


4) Helix-Loop-Helix motif

short helix and long helixhomodimer or heterodimerhighly 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 helixcan dimerize, unable to binddominant negative way (Id proteins) D N A


dominant negative fashion like HLH proteins

F& Pol4

TATA AD1

repressor


PROMOTER ANALYSIS 1) Oocyte system2) Transfection system

CAT assayLuciferase assay

3) Transgenic system4) in vitro system

EMSA (electrophoretic mobility shift assay)

DNase I footprinting assayIn vitro transcription assay

5) Transcription factor characterizationAffinity chromatographyTwo hybrid

6) in vivo systemin vivo DNase I footprinting assayChIP assay (chromatin

immunoprecipitation)


How to measure gene activation in eukaryotic cells ?

Transfection assays

nucleus

reporter MeasurableproductRXR

RAR


How to measure gene activation in eukaryotic cells ?

Transfection assays

nucleus

reporter MeasurableproductRXR

RAR

Hormone (steroid)


H

H

G F

F

E D C B A

A

A

A

- 503

+56

+56

+56

+189

+189

+189

+189

C A T

C A T

C A T

C A T

0 2 4 6 8 10 12 14Relative C AT ac tivity (fo ld)

no C / E BPC /EB PC /EB PC /EB P and 1x

2.9x2.6x 9.1x

2.2x

3.8x1.6x


LUC

LUC

LUC

LUC

LUC

LUC

LUC

-111 2

-89 0

-38 9

d -2 83 /-1 66

-28 3

-16 6

-57

+1

-1112 -890 -389 -283 -166 -57

5

10

15

20

25

30

35

40

45

Re

lativ

e lu

cife

rase

act

ivity

(fo

ld) R X R PPA R

C o ntro l

-1112 -389 d-283 /-166

5

10

15

20

25

30

35

40

45

Re

lativ

e lu

cife

rase

act

ivity

(fo

ld) R X R PPA R

C o ntro l


DNA foot printing


DNase foot printingDNase foot printing

protein 과 DNA interaction 을 알아 보기 위한 방법

protein 이 DNA 에 binding 한 경우 protein 에 의해 DNA 가 보호 받으므로 DNase I 를 처리한다 하여도 DNA 가 잘리지 않고 그 결과 gel loading 시 DNA band 가 생기지 않는 부위가 생김 이 부위를 foot print 라 함

Principle


Fig. DNase foot printingFig. DNase foot printing


Method

특정 protein binding site 를 포함하고 있는 Restrictionfragment 한쪽 끝을 방사선 동위 원소 ( 주로 32P 사용 )이용하여 labeling 힘

방사선 동위원소로 표지 된 DNA 만 존재 하는 조건과 DNA Protein 이 함께 존재하는 조건에 DNase I 를 처리 DNase I : DNA 상에 single brake 를 생성함주의 : 이 경우 DNase I 은 mild 하게 처리해야 함 . 평균 하나의 strand 당 하나의 nick 이 생성 됨

DNase I 의 작용을 stop 시킴 . DNA 는 denatured 시킴이것을 denaturing polyacrylamide gel running 시킴

위와 같이 running 시킨 DNA band 는 autoradiography 또는 Phosphorimager 를 이용하여 확인 함

결과로 나타난 band 의 ladder 는 DNasae I 에 의해 잘린 다양한 site 를 의미 함

Control 과 비교하여 Band 가 생성되지 않은 부위가 바로 Protein 이 DNA 에 binding 한 부위이며 이곳의 sequence및 size 는 여러 sequencing 방법을 통해 알 수 있음

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNUExample

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.


EMSA(EMSA(electrophoretic mobility gel shift assayelectrophoretic mobility gel shift assay ) )

Protein-DNA complexProtein-DNA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis

Principle


Method

Figure. EMSA


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

protocol


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

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNUprotocol


protocol


: MIC-1+ + + + -

wtwt wt mt

: Ab (α-E2F1) - - - - +

wt : probe (E2F) - - - - +

E2F + IgG

E2F

- - -: Probe: Ab

- + + + + + + + + + : FN+

-

- : Cold probewt- - - - - - - -Fre

e p

rob

e

wt-

wt wt wt wt wt wt wt wt wtmt

AP-1 + IgG

AP-1

c- Jun

Jun

B Jun

D c-fo

s

Fo

sB F

ra-

1 Fra

-2

*

Example

Fre

e p

rob

e

: Cold probe

Free probe


ChIP(chromatin immunoprecipitation)ChIP(chromatin immunoprecipitation)

Protein 이 어느 유전자 부위에 결합하는지를 알아보는 방법Principle

Protein 과 specific DNA region 을 알아보기 위해 보고자 하는 protein Ab 로 immunoprecifitation 실시 후 primer 를 이용 , PCR 을 수행하는 방법


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


Method

Figure. Yeast two hybrid system

포름알데하이드로 단백질 (DNA 에 결합된 단백질 ) 과 DNA 를 결합시킵니다 .

Sonication 시켜서 세포와 DNA 를 잘게 부순다 .

Immunoprecipitation 과정 수행

DNA 를 분리 정제

보고자 하는 유전자 의 프로모터 주변에서 primer 를 디자인 하여 PCR 을 수행 .

만약 어떤 전사인자가 A 라고 하는 유전자의 프로모터에 결합한다면 위의 과정을 거치면 대부분의 DNA 는 제거되고 A 유 전 자 프 로 모 터 부 위 는 남 게 됨 , 그 러 면 A 유 전 자 프로모터에 결합하는 primer 를 이용 , PCR 을 수행하면 밴드를 얻게되고 . 그래서 밴드가 진하게 나오면 결합하는 것이고 그렇지 않으면 결합하지 않는다고 봄

- Purification of nuclei from 0-16 hrs embryos- UV cross-linking

- in vivo IP with anti-EN Ab

- Sonication (0.1 - 3 Kb)

-Addition of linkersPCR amplification

PCR amplification.HindIII digestion and cloning in Ks+

603 individual clones isolated and sequenced

Chromatin ImmunoPrecipitation (ChIP)

315 independent clones

- in vitro IP with anti-EN Ab


Snail promoter 지역에는 MTA3 와 MBD3 분자가 Association 되어 있음 .

MTA3 와 MBD3 분자는 Snail promoterStart 지점의 500bp 정도 떨어진upstream 부분에서 강하게 Binding 한다 .

Example

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)


Part A. Optimization of DNA ShearingEstablish 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.

protocol


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.


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 # 20-153), 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.

protocol


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):


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.

protocol


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.


ChIP on ChipChromatin immunopreciptation on a microarray

Chip

http://www.chiponchip.org/

Fluorescently label

DNA from binding site

Protein – protein interaction

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