DamID-SEQ : 아데닌 - 메틸화 된 DNA 단편의 높은 처리량 시퀀싱에 의해 단백질 DNA 상호 작용의 게놈 넓은 매핑
Summary
우리는 높은 처리량 시퀀싱 (DamID-SEQ)에 DNA 아데닌의 메틸화 식별 (DamID)을 결합하여 여기에 분석을 설명합니다. 이 개선 된 방법은 높은 해상도와 넓은 동적 범위를 제공하며, 허용 등과 칩 서열, RNA-SEQ 같은 다른 높은 처리량 시퀀싱 데이터와 함께 DamID-서열 데이터 분석
Abstract
The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.
Introduction
아데닌 DNA 메틸화 식별 (DamID) -1,2-는 생체 내에서 DNA 단백질 상호 작용을 검출하는 방법이며 염색질 면역 (칩) 3로 대체 방법이다. 그것은 세포의 상대적으로 적은 양을 사용하고, DNA 또는 고도로 특이 항체와 단백질의 화학적 가교 결합을 필요로하지 않는다. 표적 단백질은 DNA 느슨하게 또는 간접적으로 연결된 경우 후자는 특히 유용하다. DamID 성공적 핵막 단백질 4-10, 11-13 염색질 관련 단백질, 염색질 변형 효소 14, 전사 인자 및 15-18 공동 인자와의 RNAi 기계 (19)를 포함하는 단백질의 다양한 결합 부위를 매핑하는데 사용되어왔다. 이 방법은 S. 등 다양한 유기체에서 적용 할 수있다 cerevisiae의 13, S. pombe 7, C. 엘레 9,17, D. melanogaster의 5,11,18,20, A. (21, 22)뿐만 아니라 마우스와 인간의 세포 라인 6,8,10,23,24 장대.
DamID 분석법의 개발은 내인성 아데닌 2 메틸화 결여 진핵 세포에서 아데닌 메틸화 DNA 단편의 특정 검출에 기초 하였다. 관심 E.의 DNA 결합 단백질로 이루어진 융합 단백질을 발현, 콜라이 DNA 메틸화 된 아데닌 (DAM)은 공간적 인접 (가장 크게 1킬로바이트 내의 최대 약 5킬로바이트까지)이다 게놈 2 단백질의 결합 부위에 GATC 서열의 아데닌 염기를 메틸화 할 수있다. 개질 된 DNA 단편은 특별히 관심 1,25,26의 단백질의 유전자 결합 부위를 검출하는 마이크로 어레이 혼성화 및 증폭 될 수있다. 이것은 원래 DamID 방법은 마이크로 어레이의 프로브 가용성 및 소정의 밀도에 의해 제한되었다. 따라서 우리는 높은 처리량 시퀀싱을 통합 한DamID 10이고 DamID-SEQ와 같은 방법을 지정. 짧은의 많은 수의 DamID-SEQ에서 생성 된 읽기 게놈 전체 단백질 DNA 상호 작용의 정확한 위치 파악을 할 수 있습니다. 우리는 DamID-서열이 게놈 핵 라미 (NL) 협회 (10) 유학을위한 마이크로 어레이에 의해 DamID보다 높은 해상도와 넓은 동적 범위를 한 것으로 나타났습니다. 이 개선 된 방법은 유전자 구조 (10) 내에 연관 NL 프로빙 수 있으며 이러한 칩 서열 및 RNA-SEQ 다른 높은 처리량 시퀀싱 데이터와의 비교를 용이하게한다.
여기에 설명 된 DamID-SEQ 프로토콜은 초기에 매핑 게놈 NL 협회 (10)을 위해 개발되었다. 우리는 E.에 마우스 또는 인간 라민의 B1을 테 더링하여 융합 단백질을 생성 대장균의 DNA 메틸화 된 아데닌과 C2C12 마우스 (데이터가 공개되지 않음) (10)과 IMR90 인간의 태아 폐 섬유 아 세포를 근육 모세포, 3T3 마우스 배아 섬유 아세포에서 프로토콜을 테스트했다. 이 프로토콜에서는, 우리는 C로 시작벡터 및 포유류 세포 onstructing 24 렌티 바이러스 감염에 의한 댐 연결형 융합 단백질을 발현. 다음으로, 우리는 아데닌 메틸화 DNA 단편을 증폭하고 다른 생물에 적용되어야 서열 라이브러리의 제조 상세한 프로토콜을 설명한다.
Protocol
Representative Results
Discussion
Whether Dam-tagged proteins retain the functions of endogenous proteins should be examined before a DamID-seq experiment. The subcellular localization of Dam-tagged nuclear envelope proteins should always be determined and compared with that of the endogenous proteins. For studying transcription factors, it is suggested to examine whether the Dam-fusion protein can rescue the functions of the endogenous protein in regulating gene expression. This functional test can be performed in organisms in which knockout mutants of …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Bas van Steensel for providing the DamID mammalian expression vectors. We thank Yale Center for Genome Analysis and the Genomics Core in Yale Stem Cell Center for advice on preparing NGS libraries and implementing high throughput DNA sequencing. This work was supported by the startup funding from Yale School of Medicine, a Scientist Development Grant from American Heart Association (12SDG11630031) and a Seed Grant from Connecticut Innovations, Inc. (13-SCA-YALE-15).
Materials
| ViraPower Lentiviral Expression Systems | Life Technologies | K4950-00, K4960-00, K4970-00, K4975-00, K4980-00, K4985-00, K4990-00, K367-20, K370-20, and K371-20 | |
| Gateway BP Clonase II Enzyme Mix | Life Technologies | 11789-020 | |
| Gateway LR Clonase II Enzyme Mix | Life Technologies | 11791-020 | |
| DNeasy Blood & Tissue Kit (250) | QIAGEN | 69506 or 69504 | |
| Gateway pDONR 201 | Life Technologies | 11798-014 | |
| 293T cells | American Type Culture Collection | CRL-11268 | |
| Trypsin-EDTA (0.05%), phenol red | Life Technologies | 25300-054 | |
| DMEM, high glucose, pyruvate | Life Technologies | 11995-065 | |
| Fetal Bovine Serum | Sigma | F4135 | |
| Tris | brand not critical | ||
| EDTA | brand not critical | ||
| 200 Proof EtOH | brand not critical | ||
| Isopropanol | brand not critical | ||
| Sodium Acetate | brand not critical | ||
| DpnI | New England Biolabs | R0176 | supplied with buffer |
| DamID adaptors "AdRt" and "AdRb" | Integrated DNA Technologies | sequences available in ref. 24; no phosphorylation of the 5' or 3' end to prevent self-ligation. | |
| T4 DNA Ligase | Roche Life Science | 10481220001 | supplied with buffer |
| DpnII | New England Biolabs | R0543 | supplied with buffer |
| DamID PCR primer "AdR_PCR" | Integrated DNA Technologies | sequences available in ref. 24 | |
| Deoxynucleotide (dNTP) Solution Set | New England Biolabs | N0446 | 100 mM each of dATP, dCTP, dGTP and dTTP |
| Advantage 2 Polymerase Mix | Clontech | 639201 | supplied with buffer |
| 1Kb Plus DNA Ladder | Life Technologies | 10787018 | 1.0 µg/µl |
| QIAquick PCR Purification Kit | QIAGEN | 28104 or 28106 | |
| MinElute PCR Purification Kit | QIAGEN | 28004 or 28006 | for an elution volume of less than 30 µl |
| SPRI beads / Agencourt AMPure XP | Beckman Coulter | A63880 | apply extra mixing and more elution time if less than 40 µl elution buffer is used |
| Buffer EB | QIAGEN | 19086 | |
| NEBNext dsDNA Fragmentase | New England Biolabs | M0348 | supplied with buffer |
| T4 DNA Ligase Reaction Buffer | New England Biolabs | B0202 | |
| T4 DNA Polymerase | New England Biolabs | M0203 | |
| DNA Polymerase I, Large (Klenow) Fragment | New England Biolabs | M0210 | |
| T4 Polynuleotide Kinase | New England Biolabs | M0201 | |
| Klenow Fragment (3’ -> 5’ exo-) | New England Biolabs | M0212 | supplied with buffer |
| sequencing adaptors | Integrated DNA Technologies | sequences available in ref. 28 | |
| Quick Ligation Kit | New England Biolabs | M2200 | used in 11.2; supplied with Quick Ligation Reaction Buffer and Quick T4 DNA Ligase |
| sequencing primer 1 and 2 | Integrated DNA Technologies | sequences available in ref. 28 | |
| KAPA HiFi PCR Kit | Kapa Biosystems | KK2101 or KK2102 | supplied with KAPA HiFi DNA Polymerase, 5X KAPA HiFi Fidelity Buffer and 10mM dNTP mix |
| agarose | Sigma Aldrich | A4679 | |
| ethidium bromide | Sigma Aldrich | E1510-10ML | 10 mg/ml |
| QIAquick Gel Extraction Kit | QIAGEN | 28704 or 28706 | |
| iTaq Universal SYBR Green Supermix | Bio-Rad Laboratories | 1725121 or 1725122 | |
| Spectrophotometer | brand not critical | ||
| 0.45 um PVDF Filter | brand not critical | ||
| 25 ml Seringe | brand not critical | ||
| 10 cm Tissue Culture Plates | brand not critical | ||
| 6-well Tissue Culture Plates | brand not critical | ||
| S1000 Thermal Cycler | Bio-Rad Laboratories | ||
| C1000 Touch Thermal Cycler | Bio-Rad Laboratories | for qPCR | |
| Vortex Mixer | brand not critical | ||
| Dry Block Heater or Thermomixer | brand not critical | ||
| Microcentrifuge | brand not critical | ||
| Gel electrophoresis system with power supply | brand not critical | ||
| Magnet stand | for purification of DNA with SPRI beads; should hold 1.5-2 ml tubes; brand not critical | ||
| UV transilluminator | brand not critical | ||
| E-gel electrophoresis system | Life Technologies | G6400, G6500, G6512ST |
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