DamID-seq: L'échelle du génome Cartographie des protéine-ADN Interactions par séquençage à haut débit de fragments d'ADN adénine-méthylé
Summary
Nous décrivons ici un essai en couplant l'identification ADN adénine méthyltransférase (DamID) pour le séquençage à haut débit (DamID-seq). Cette méthode améliorée offre une résolution plus élevée et une plage dynamique plus large, et permet l'analyse des données DamID-Seq en conjonction avec d'autres données de séquençage à haut débit tels que ChIP-seq, ARN-Seq, etc.
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
ADN identification adénine méthyltransférase (DamID) 1,2 est une méthode pour détecter les interactions protéine-ADN in vivo et est une approche alternative à immunoprécipitation de la chromatine (ChIP) 3. Il utilise une quantité relativement faible de cellules et ne nécessite pas de reticulation chimique de l'ADN ou de protéines avec un anticorps hautement spécifique. Ce dernier est particulièrement utile lorsque la protéine cible est associée de façon lâche ou indirectement avec de l'ADN. DamID a été utilisé avec succès pour cartographier les sites de liaison d'une variété de protéines y compris des protéines d'enveloppe nucléaire 4-10, 11-13 protéines associées à la chromatine, les enzymes modifiant la chromatine, 14 facteurs de transcription et des co-facteurs d'ARNi 15-18 et 19 machines. Le procédé est applicable dans de nombreux organismes, y compris S. 13 cerevisiae, S. 7 pombe, C. 9,17 elegans, D. melanogaster 5,11,18,20, A. thaliana 21,22 ainsi que la souris et cellules humaines lignes 6,8,10,23,24.
Le développement de l'essai DamID était basée sur la détection spécifique de fragments d'ADN adénine-méthylé dans des cellules eucaryotes qui ne disposent pas la méthylation 2 adénine endogène. Une protéine de fusion exprimée, consistant en la protéine de liaison à l'ADN d'intérêt et E. adénine méthyltransférase coli DNA (barrage), peut méthyler la base adénine dans des séquences GATC qui sont à proximité spatiale (la plus significative au sein de 1 kb et jusqu'à environ 5 ko) pour les sites de liaison de la protéine dans le génome 2. Les fragments d'ADN peuvent être modifiées spécifiquement amplifiés et hybrides à des puces pour détecter les sites de fixation génomiques de la protéine d'intérêt 1,25,26. Cette méthode originale a été DamID limitée par la disponibilité de puces et la densité de sondes prédéterminées. Nous avons donc intégré séquençage à haut débit dansà DamID 10 et désigné la méthode que DamID-seq. Le grand nombre de courte lectures généré à partir DamID-seq permet une localisation précise des interactions protéine-ADN pangénomique. Nous avons constaté que DamID-seq fourni une résolution plus élevée et une plage dynamique plus large que DamID par microarray pour l'étude du génome nucléaire lamina (NL) 10 associations. Cette méthode permet une meilleure sonder les associations NL au sein des structures de gènes 10 et facilite les comparaisons avec d'autres données de séquençage à haut débit, tels que ChIP-seq et de l'ARN-Seq.
Le protocole DamID-seq décrit ici a été initialement développé pour la cartographie du génome NL 10 associations. Nous avons généré une protéine de fusion de souris ou attacher Lamin B1 humaine à E. coli ADN adénine méthyltransférase et testé le protocole 3T3 souris fibroblastes embryonnaires, C2C12 myoblastes de souris 10 et IRM90 fibroblastes pulmonaires de foetus humain (données non publiées). Dans ce protocole, nous commençons avec constructing vecteurs et exprimant Dam-captifs protéines de fusion par l'infection lentiviral dans des cellules mammifères 24. Ensuite, nous décrivons les protocoles détaillés de l'amplification de fragments d'ADN adénine-méthylé et de préparer des bibliothèques de séquençage qui devraient être applicables dans d'autres organismes.
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 …
Divulgations
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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