Vi beskriver heri et assay ved kobling DNA methyltransferase adenin identifikation (DamID) til high throughput sekventering (DamID-seq). Denne forbedrede fremgangsmåde giver en højere opløsning og en større dynamikområde og muliggør analyse DamID-seq data i forbindelse med andre højt gennemløb sekventering data såsom chip-seq, RNA-seq, etc.
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.
DNA methyltransferase adenin identifikation (DamID) 1,2 er en metode til påvisning af protein-DNA-interaktioner in vivo og er en alternativ tilgang til kromatin immunofældning (chip) 3. Den benytter en relativt lav mængde af celler og kræver ikke kemisk tværbinding af protein med DNA eller et meget specifikt antistof. Sidstnævnte er særligt nyttigt, når målproteinet er løst eller indirekte er forbundet med DNA. DamID held har været anvendt til kortlægning af bindingssteder i en række proteiner, herunder nuklear kappeproteiner 4-10, chromatin-associerede proteiner 11-13, kromatin-modificerende enzymer 14, transkriptionsfaktorer og cofaktorer 15-18 og RNAi machineries 19. Metoden er anvendelig i flere organismer, herunder S. cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. thaliana 21,22 samt muse- og humane cellelinjer 6,8,10,23,24.
Udviklingen af DamID assay var baseret på den specifikke påvisning af adenin-methylerede DNA-fragmenter i eukaryote celler, som mangler endogene adenin methylering 2. Et udtrykt fusionsprotein, der består af DNA-bindende protein af interesse og E. coli-DNA adenin methyltransferase (Dam), kan methylere adeninbasen i GATC-sekvenser, som er i fysisk nærhed (mest markant inden for 1 kb og op til ca. 5 kb) til bindingsstederne af proteinet i genomet 2. De modificerede DNA-fragmenter kan amplificeres specifikt og hybridiseret til microarrays at detektere genomiske bindingssteder af proteinet af interesse 1,25,26. Denne originale DamID metode blev begrænset af tilgængeligheden af mikroarrays og tætheden af forudbestemte sonder. Vi har derfor integreret high throughput sekventeringtil DamID 10 og betegnes metoden som DamID-seq. Det store antal kort læser genereret fra DamID-seq muliggør nøjagtig lokalisering af protein-DNA-interaktioner genom-dækkende. Vi fandt, at DamID-seq forudsat en højere opløsning og et større dynamikområde end DamID af microarray til at studere genom-nukleare lamina (NL) foreninger 10. Denne forbedrede metode gør det muligt sondering NL foreninger inden for gen-strukturer 10 og letter sammenligninger med andre højt gennemløb sekventering data, såsom chip-seq og RNA-seq.
Den DamID-seq protokol beskrevet her blev oprindeligt udviklet til kortlægning genom-NL foreninger 10. Vi genererede et fusionsprotein ved tethering mus eller human Lamin B1 til E. coli DNA-adenin methyltransferase og testet protokollen i 3T3 muse embryonale fibroblaster, C2C12 musen myoblaster 10 og IMR90 humane føtale lungefibroblaster (data ikke offentliggjort). I denne protokol, starter vi med constructing vektorer og ekspression Dam-bundne fusionsproteiner ved lentiviral infektion i pattedyrceller 24. Dernæst beskriver vi de detaljerede protokoller fra forstærke adenin-methylerede DNA-fragmenter og forberede sekventering biblioteker, der skal være gældende i andre organismer.
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 …
The authors have nothing to disclose.
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).
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 |