DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序
Summary
我们描述这里的一个检测通过结合DNA腺嘌呤甲基识别（DamID），以高通量测序（DamID-SEQ）。这种改进的方法提供了更高的分辨率和更宽的动态范围，并允许在分析与其他高通量测序数据如芯片起，RNA测序等一起DamID-SEQ数据
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
脱氧核糖核酸腺嘌呤甲基识别（DamID）1,2是这样一种方法来检测体内蛋白-DNA相互作用，是一种可供选择的方法来染色质免疫沉淀（ChIP）3。它采用的是相对低的细胞的量，并且不需要化学交联蛋白与DNA或高度特异性的抗体。后者是特别有用当靶蛋白是松散或间接地与DNA相关联。 DamID已成功地用于绘制的多种蛋白质，包括核包膜蛋白4-10，相关联的染色质蛋白11-13染色质修饰酶14，转录因子和辅因子15-18和RNAi机械19的结合位点。该方法适用于多种生物，包括S.酵母 13，S。酵母 7，C。线虫 9,17，D。黑 5,11,18，20，A。拟南芥 21,22以及小鼠和人的细胞系6,8,10,23,24。
所述DamID测定的发展是基于在缺乏内源性腺嘌呤甲基化2的真核细胞腺嘌呤甲基化的DNA片段的特异性检测。表达的融合蛋白，包括利息和 E的DNA结合蛋白的大肠杆菌DNA腺嘌呤甲基（水坝），可以甲基化腺嘌呤碱中GATC序列是在空间接近性（最显著内1 kb和多达大约5kb的）的蛋白质的基因组中的2的结合位点。修饰的DNA片段可以特异性地扩增和杂交到微阵列检测感兴趣1,25,26的蛋白质的基因组结合位点。这个原始DamID方法是由微阵列的可用性和预定探针的密度的限制。因此，我们集成了高通量测序到DamID 10和指定的方法DamID-起。读取从DamID-SEQ产生大量的短使蛋白质-DNA相互作用的全基因组的精确定位。我们发现，DamID-SEQ提供比DamID更高的分辨率和更宽的动态范围芯片用于研究基因组核纤层（NL）协会10。这种改进的方法允许基因结构 10内探测NL协会和便于与其它高通量测序数据，如芯片起和RNA-SEQ比较。
这里描述的DamID-SEQ协议最初开发用于映射基因组-NL协会10。我们通过圈养小鼠或人类核纤层蛋白B1至E.产生的融合蛋白大肠杆菌DNA甲基腺嘌呤和测试协议中3T3小鼠胚胎成纤维细胞，C2C12小鼠成肌细胞10和IMR90人胚肺成纤维细胞（数据未公布）。在这个协议中，我们开始用Constructing载体和表达坝-拴系融合蛋白由慢病毒感染的哺乳动物细胞24。接着，我们描述放大腺嘌呤甲基化的DNA片段，并准备测序文库，应该适用于其它生物体的详细方案。
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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