Chromatin Immunoprecipitation를 통한 Transcription factor binding site 분석 분자유전학 연구실 남제현

목차 실험 목적 실험 배경 및 원리 실험 개요 실험 재료 실험 방법

실험 목적 Chromatin Immunoprecipitation; ChIP -To determine whether a protein binds a particular DNA site in a living cell

실험 배경 및 원리 Transcription Factor -Protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to mRNA

Gene Regulation in Eukaryotes 실험 배경 및 원리 Gene Regulation in Eukaryotes Special transcription factor

Tissue specific transcription factor 실험 배경 및 원리 Tissue specific transcription factor Recombinant DNA techniques have been used to alter the orientation and location of DNA control elements to study the effect of the change on the level of transcription. The core promoter alone (just upstream of gene) allows a basal level of transcription to occur. When the core promoter is removed from the gene, no transcription occurs. An enhancer alone cannot substitute for the promoter region, but combining an enhancer with a core promoter results in a significantly higher level of transcription than occurs with the promoter alone. This increase in transcription is observed when the enhancer is 1) moved farther upstream,  2)  inverted in orientation or 3) moved to the 3 prime side of the structural gene. A Model for Enhancer Action In this model, an enhancer located at a great distance along the DNA from the protein-coding gene it regulates is brought close to the core promoter by a looping of the DNA. The influence of an enhancer on the promoter is mediated by regulatory transcription factors called activators. 1) The activator proteins bind to the enhancer elements, forming an enhanceosome. 2) Bending of the DNA brings the enhanceosome closer to the core promoter. The general transcription factor TFIID is in the promoter's vicinity. For the purpose of this figure, two of the protein subunits of TFIID, which will function as coactivators in step 3, are distinguished from the rest of the factor. 3) The DNA-bound activators interact with specific coactivators that are part of TFIID. This interaction facilitates the correct positioning of TFIID on the promoter. 4) The other general transcription factors and RNA polymerase join the complex, and transcription is initiated. The gene for the protein albumin, like other genes, is associated with an array of regulatory DNA elements; here we show only two control elements, as well as the core promoter. Cells of all tissues contain RNA polymerase and the general transcription factors, but the set of regulatory transcription factors available varies with the cell type. Liver cells contain a set of regulatory transcription factors that includes the factors for recognizing all the albumin gene control elements. When these factors bind to the DNA, they facilitate transcription of the albumin gene at a high level. Brain cells, however, have a different set of regulatory transcription factors, which does not include all the ones for the albumin gene. Consequently, in brain cells, the transcription complex can assemble at the promoter, but not very efficiently. The result is that brain cells transcribe the albumin gene only at a low level.

실험 배경 및 원리 What is Chromatin? <The function of chromatin> Package DNA to enable it to fit in the cell, strengthen DNA to assist with mitosis and meiosis, and serve as a mechanism to control gene expression, DNA repair, and DNA replication. Histone proteins play an important role in the regulation of these processes. (acetylation, methylation, phosphorylation and etc..) The function of chromatin is to package DNA to enable it to fit in the cell, strengthen DNA to assist with mitosis and meiosis, and serve as a mechanism to control gene expression, DNA repair, and DNA replication. Histone proteins play an important role in the regulation of these processes. A large number of residues found on the histones can be covalently modified with chemical groups by processes such as acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation and deimination . Furthermore, cis-trans proline isomerisation while not strictly a modification results in a conformational change These modifications function either by disrupting chromatin contacts or by affecting the recruitment of non-histone proteins to chromatin. A number of enzymes have been identified which catalyze the addition of these modifications to histone proteins.

Chromatin Immunoprecipitation (ChIP) 실험 배경 및 원리 Chromatin Immunoprecipitation (ChIP) The principle of ChIP : The selective enrichment of a chromatin fraction containing a special protein of interest. Antibodies that recognize a protein or protein modification of interest can be used to determine the relative abundance of that antigen. Basically, if you’re looking to observe your protein of interest and its interactions with the genome in its ‘natural’ state ChIP is a great choice.

ChIP is extremely versatile 실험 개요 ChIP is extremely versatile Chip-seq Chip-on-chip

<Overview of ChIP assay> 실험 개요 <Overview of ChIP assay> step1. Fixation을 통해 DNA-protein crosslink를 얻어내고 Sonication 하여 DNA를 일정한 크기로 끊어준다. step2. Immunoprecipitation을 통해 target protein을 침전시켜 얻는다. step3. Purification된 DNA를 qPCR하여 transcription factor가 target gene에 binding 하는지 알아본다.

(using centrifuge 240g, 5min) (Using sonicator: 10sec, 10times) 실험 개요 1.Fixation (cross-linking) MYC NPM1 Promoter 2. Cell collection (using centrifuge 240g, 5min) 3. Cell lysis (Lysis Buffer 200uL) 4.Sonication (Using sonicator: 10sec, 10times) MYC Promoter

(Using protein agarose A/G beads) 실험 개요 ※Preclear (Using protein agarose A/G beads) MYC protein Promoter Spin down and throw beads away Get supernatant MYC protein MYC MYC MYC MYC MYC protein

실험 개요 5.Immunoprecipitation (Put MYC antibody) Promoter (Put protein agarose A/G beads) MYC MYC MYC MYC MYC MYC MYC (Get Protein-Antibody-Beads complex)

(Using NaCl and Proteinase K) (Using PCR purification kit) 실험 개요 6. Reverse cross-link (Using NaCl and Proteinase K) MYC Promoter MYC NPM1 Promoter 7. Purification (Using PCR purification kit) 8. Real-time PCR

실험 방법 Transfac Before ChIP assay Program을 이용한 transcription factor 검색(Target prediction) Transcription Factor Binding Prediction software Put NPM1 Promoter sequence Find transcription factor Transfac aacCATGTGgtc

실험 방법 1일차. Live cell을 Cross-linking한 후, lysis 된 샘플을 Sonication하고, sonication된 DNA단편의 크기(size)를 DNA gel electrophoresis를 통해 확인한다. 1000bp 1000bp 500bp 1kbp부근에서 detection 됨 500bp 부근에서 detection 됨

실험 방법 2일차. Immunoprecipitation을 통해 target protein을 침전시킨다. 이후 reverse cross-linking을 통해 DNA만 얻어낸다. 그 이후 qPCR을 진행하여 NPM1 gene을 증폭시킨다. NPM1

실험 방법 3일차.Purification 된 DNA sample을 이용하여 Real-time PCR을 진행한다. -Percent Input 또는 Fold enrichment calculation을 통해 transcription factor가 target gene에 binding하는지 알아본다. Fold Enrichment NPM1

실험 계획(1주차) - 주어진 cell을 이용하여 Fixation부터 sonication 단계까지 진행한다. (Immunoprecipitation 전 단계까지 진행) -DNA gel electrophoresis(전기영동)을 이용하여 sonication 된 DNA의 크기를 알아본다.

실험 재료 <실험 1주차 실험재료> 1x10^7 cells 1% 포름알데하이드 272uL for fixation. Glycine(stock 1.25M) 1.15mL for stop fixation. Centrifuge PBS 1mL for wash Cell lysis buffer (200ul) Nuclei lysis buffer(500ul) IP dilution buffer(2.5ml) Sonicator DNA dye와 DNA gel electrophoresis 기계 Protein A/G agarose Beads

1% Formaldehyde를 이용한 Cross linking 및 cell lysis 과정 실험 방법 1% Formaldehyde를 이용한 Cross linking 및 cell lysis 과정 1% formaldehyde 272uL를 1x10^7(1천만개)의 cell이 담겨있는 tube에 넣고 10분간 상온에서 기다린다. Tube에 Glycine(1.25M)을 1.15mL 넣고 5분간 기다린다. Centrifuge를 이용하여 240g에서 5분간 돌린 후 Pellet만 남긴 후, PBS를 이용하여 Cell을 washing해준다. NPM1

1% Formaldehyde를 이용한 Cross linking 및 cell lysis 과정 실험 방법 1% Formaldehyde를 이용한 Cross linking 및 cell lysis 과정 4. Centrifuge를 이용하여 Pellet만 남긴 후, Cell lysis buffer를 200ul 넣어주고 pellet을 풀어준다. 5. 얼음에 5분간 박아둔다. 6.Centrifuge를 이용해 (2600rpm)에서 3분간 돌려준다. 7.Pellet만 남기고 상층액은 버려준 후, Nuclei lysis buffer 500ul를 이용하여 pellet을 풀어준 후 10분간 얼음에 박아둔다. NPM1

실험 방법 Sonicator를 이용한 sonication 과정 8. IP dilution buffer를 이용해 total volume을 3mL로 맞춰준다. 9. Sonicator를 이용하여 (Power는 level 8로 고정) sonication을 진행한다. (10sec sonication, 50sec stop)총 6회 10. Centrifuge를 이용하여 13,000rpm에서 10분간 돌려준다.

실험 방법 DNA gel electrophoresis를 통해 sonication 정도를 확인 Sonication을 진행한 sample에서 20uL를 따낸다. Loading dye 3uL 를 넣어준다. DNA agarose gel에 loading한다. DNA gel imager를 통해 band를 확인한다. 500bp 500bp 부근에서 detection 됨

실험 방법 Immunoprecipitation: Preclear 과정 Protein agarose A/G beads 15uL를 Sample 에 넣어준다. 1Hrs , 4℃ rotor 에 올려둔다. Centrifuge를 이용하여 beads 를 down시킨 후, 상층액을 얻어낸다. E-tube 에 각각 250uL씩 나눠준다. Normal IgG antibody와 MYC antibody를 각각 5uL씩 넣어준다.

1주차 결과 1조 2조 3조 4조 5조 6조 3000bps 1000bps

실험 계획(2주차) Sonication을 통해 얻은 Sample(1주차) 을 이용하여 Immunoprecipitation한다. Immunoprecipitation을 한 후, Nacl를 이용해 Reverse Crosslink 시킨다. Proteinase K를 이용하여 DNA에 붙어있는 Antibody를 제거한다.

실험 방법 IP sample 준비 10% Input 30uL Normal IgG 300uL MYC antibody 300uL

실험 방법 MYC antibody를 이용한 IP 에서 10% Input sample에서 NPM1 유전자 20개 30uL MYC antibody 300uL

Calculation by Band thickness 실험 방법 Calculation by Band thickness 1% 1% Input Band 의 두께 X % : = Myc Antibody Band 의 두께 0.4 두께 0.7 1% Input Normal IgG MYC antibody

실험 재료 <실험 2주차 실험재료> Protein A/G agarose Beads NaCl 5M IP wash buffer1 + IP wash buffer2 IP elution buffer Proteinase K (10mg/mL) PCR purification kit Realtime PCR mix

100*2^(Adjusted input –Ct(IP)) 실험 방법 Calculation by % Input STEP1 Adjusted input to 100% Raw Ct Ct value - 6.644 NPM1 Input (1%) 32.644 26 STEP2 Percent input % Input Average Ct 100*2^(Adjusted input –Ct(IP)) Adjusted input 26 IgG 34 0.39% Normal igG MYC antibody Myc antibody 29 12.5%

실험 방법 Immunoprecipitation and DNA reverse cross-linking Protein agarose A/G beads 10uL를 Sample 에 넣어준다. 1Hrs , 4℃ rotator 에 올려둔다. Centrifuge를 이용하여 beads 를 down시킨 후 Pellet을 얻어낸다. IP wash buffer1 300uL로 1회 washing한다. IP wash buffer2 300uL로 1회 washing 한다. IP elution buffer 100uL 를 넣은 후, rotator에 5분간 Incubation 한다. IP elution buffer 200uL+ NaCl 15uL를 추가로 넣어준다. 마지막으로 65 ℃ 에서 15분간 Incubation 한다.

실험 방법 Proteinase K 처리 및 DNA purification Sample에 Proteinase K 5uL를 넣어준다. 10min , 54℃ heat block에 넣어 둔다. PCR purification kit를 이용하여 DNA를 purification 한다. NPM1

실험 방법 PCR purification kit를 이용하여 DNA를 purification 한다. Proteinase K를 이용하여 reverse crosslink가 종료된 sample을 얻는다. DNA binding buffer 를 500uL따서, sample과 잘 섞어준 후 column tube로 옮겨준다. Centrifuge 를 이용하여 14000rpm, 30초간 돌려준다. Tube 밑으로 내려온 버퍼를 버려 준 후, DNA Wash Buffer 200ul 를 column에 넣어준다. Tube 밑으로 내려온 버퍼를 버려준 후, 5번 과정을 한번 더 시행한다. Column을 새로운 tube로 옮겨 준 후, DW 50uL를 넣어주고 1분간 기다린다. Centrifuge, 14000rpm, 1min 하여 tube 바닥에 얻어진 DNA sample을 새로운 tube로 옮겨준다.

실험 방법 Real time PCR을 통한 NPM1 expression 확인 준비된 Real-time PCR sample mix에 DNA sample 10uL를 넣어준다. Real time PCR machine을 이용해 real time PCR을 진행한다. 결과를 분석한다.

예비 보고서 Site-directed Mutagenesis의 실험목적, 원리, 방법 및 예시 Ex> PCR, transformation, mini-prep 과정을 꼭 서술 하셔야 됩니다.