-DamID siguientes: Genoma de toda la cartografía de la proteína-ADN Interacciones de alto rendimiento de secuenciación de fragmentos de ADN adenina-metilado
Summary
Se describe en este documento un ensayo mediante el acoplamiento de identificación de ADN metiltransferasa adenina (DamID) a alto rendimiento de secuenciación (DamID-ss). Este método mejorado proporciona una mayor resolución y un rango dinámico más amplio, y permite el análisis de datos DamID-Seq en conjunción con otros datos de secuenciación de alto rendimiento tales como chip-ss, RNA-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 metiltransferasa identificación adenina (DamID) 1,2 es un método para detectar la proteína-DNA in vivo y es un enfoque alternativo a la cromatina immunoprecipitation (CHIP) 3. Se utiliza una cantidad relativamente baja de células y no requiere la reticulación química de la proteína con el ADN o un anticuerpo altamente específico. Este último es particularmente útil cuando la proteína diana está vagamente o indirectamente asociada con el ADN. DamID ha sido utilizado con éxito para mapear los sitios de unión de una variedad de proteínas, incluyendo proteínas de la envoltura nuclear, la cromatina asociada 4-10 11-13 proteínas, enzimas modificadoras de la cromatina 14, factores de transcripción y co-factores 15-18 y maquinarias RNAi 19. El método es aplicable en múltiples organismos incluyendo S. cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. thaliana 21,22, así como de ratón y células humanas líneas 6,8,10,23,24.
El desarrollo del ensayo DamID se basó en la detección específica de fragmentos de ADN adenina-metilado en células eucariotas que carecen de adenina endógena metilación 2. Una proteína de fusión expresada, que consiste en la proteína de unión a ADN de interés y E. ADN metiltransferasa coli adenina (Dam), puede metilar la base adenina en las secuencias GATC que están en proximidad espacial (más significativamente dentro de 1 kb y hasta aproximadamente 5 kb) a los sitios de unión de la proteína en el genoma 2. Los fragmentos de ADN modificados se pueden amplificar específicamente y se hibridaron a microarrays para detectar los sitios de unión genómicas de la proteína de interés 1,25,26. Este método original DamID estaba limitada por la disponibilidad de microarrays y la densidad de sondas predeterminados. Por ello, hemos integrado secuenciación de alto rendimiento ena DamID 10 y designado el método DamID-ss. El gran número de cortos lee genera a partir DamID-ss permite la localización precisa de la proteína-DNA del genoma de ancho. Encontramos que DamID-ss proporciona una mayor resolución y un rango dinámico más amplio que DamID de microarrays para el estudio de la lámina genoma nuclear (NL) 10 asociaciones. Este método mejorado permite sondear asociaciones NL dentro de las estructuras de genes 10 y facilita las comparaciones con otros datos de secuenciación de alto rendimiento, tales como chip-ss y ARN-ss.
El protocolo DamID-ss aquí descrito fue desarrollado inicialmente para el mapeo de asociaciones del genoma NL 10. Hemos generado una proteína de fusión mediante la inmovilización del ratón o Lamin B1 humano a E. coli DNA metiltransferasa adenina y se prueba el protocolo en 3T3 fibroblastos embrionarios de ratón, ratón C2C12 mioblastos 10 y IMR90 fibroblastos de pulmón fetal humano (datos no publicados). En este protocolo, empezamos con constructing vectores y expresar proteínas de fusión Dam-atados por la infección lentiviral en células de mamíferos 24. A continuación, se describen los protocolos detallados de amplificación de fragmentos de ADN adenina-metilado y la preparación de bibliotecas de secuenciación que deberían ser aplicables en otros organismos.
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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