[Methods in Molecular Biology] Chromatin Immunoprecipitation Assays Volume 567 || DamID: A Methylation-Based Chromatin Profiling Approach
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DamID: A Methylation-Based Chromatin Profiling Approach
Mona Abed, Dorit Kenyagin-Karsenti, Olga Boico, and Amir Orian
Gene expression is a dynamic process and is tightly connected to changes in chromatin structure and nuclearorganization (Schneider, R. and Grosschedl, R., 2007, Genes Dev. 21, 30273043; Kosak, S. T. and Groudine,M., 2004, Genes Dev. 18, 13711384). Our ability to understand the intimate interactions between proteinsand the rapidly changing chromatin environment requires methods that will be able to provide accurate,sensitive, and unbiased mapping of these interactions in vivo (van Steensel, B., 2005, Nat. Genet. 37 Suppl,S1824). One such tool is DamID chromatin profiling, a methylation-based tagging method used to identifythe direct genomic loci bound by sequence-specific transcription factors, co-factors as well as chromatin- andnuclear-associated proteins genome wide (van Steensel, B. and Henikoff, S., 2000, Nat. Biotechnol. 18,424428; van Steensel, Delrow, and Henikoff, 2001, Nat. Genet. 27, 304308). Combined with otherfunctional genomic methods and bioinformatics analysis (such as expression profiles and 5C analysis), DamIDemerges as a powerful tool for analysis of chromatin structure and function in eukaryotes. DamID allows thedetection of the direct genomic targets of any given factor independent of antibodies and without the need forDNA cross-linking. It is highly valuable for mapping proteins that associate with the genome indirectly or loosely(e.g., co-factors). DamID is based on the ability to fuse a bacterial Dam-methylase to a protein of interest andsubsequently mark the factors genomic binding site by adenine methylation. This marking is simple, highlyspecific, sensitive, inert, and can be done in both cell culture and living organisms. Below is a short description ofthe method, followed by a step-by-step protocol for performing DamID in Drosophila cells and embryos. Due tospace limitations, the reader is referred to recent reviews that compare the method with other profilingtechniques such as ChIP-chip as well as protocols for performing DamID in mammalian cells (NSouthall, T.D. and Brand, A. H., 2007, Nat. Struct. Mol. Biol. 14, 869871; Orian, A., 2006, Curr. Opin. Genet. Dev. 16,157164; Vogel, M. J., Peric-Hupkes, D. and van Steensel, B. 2007, Nat. Protoc. 2, 14671478).
Key words: DamID, gene regulation, chromatin, transcription, nuclear organization, genomics,Drosophila.
To monitor dynamic changes in chromatin and nuclear organiza-tion (1, 2), we describe below a step-by-step protocol for perform-ing DamID chromatin profiling.
Philippe Collas (ed.), Chromatin Immunoprecipitation Assays, Methods in Molecular Biology 567,DOI 10.1007/978-1-60327-414-2_11, Humana Press, a part of Springer Science+Business Media, LLC 2009
To perform a DamID profiling experiment, a bacterialDNA adenine methylase (DAM) is fused to the protein ofinterest (Fig. 11.1). Trace amounts of the chimeric proteinare expressed in cells or as a transgene in animals. DNAbinding of the chimeric protein results in local methylationin the vicinity of binding sites on adenine nucleotides withinthe Dam recognition sequence (GAmTC). Subsequently,GAmTC methylated DNA fragments are isolated using DpnIdigest, which cleaves specifically GAmTC. Considering thatGATC sequences are frequently present in the genome (onaverage every 0.22.5 kb), the fragments isolated containregions near by or within genes in addition to the bindingsite itself (Fig. 11.1). To account for accessibility and non-specific Dam binding, a DamID experiment is performed as acomparison between the relative binding of Protein X-Damchimeric protein to that of a free Dam protein. Isolated 0.22.5 kb DpnI genomic fragments from Dam-Only (reference)and Dam-X-Fusion (experimental) are directly labeled withCy3 and Cy5 dyes and hybridized directly to a cDNA/ESTor genomic tiling microarray (36). The Dam methylation ineukaryotes is transcriptionally as well as developmentally inert,and therefore is ideal for network analysis in vivo. IndeedDamID was used to map the binding site of sequence-specifictranscription factor networks, and to monitor co-factorsrecruitment (712). It is powerful for studying heterochroma-tin-associated proteins as well proteins required for nuclearorganization and dynamics (1318). DamID can also be usedto evaluate recruitment to a single gene of interest using aSouthern blot approach (4, 19, 20). DamID is not limited toDrosophila and has been used to map proteins in Arabidopsisthaliana and mammalian genomes (2123). In this chapter wedescribe a simple procedure to perform DamID using Drosophila
Fig. 11.1. The DamID method. Binding of the Dam-Fusion proteins to its cognate bindingsites for example CACGTG (dashed box) results in flanking DAM methylation (blackcircle). Subsequently, the methylated flanked fragment is isolated from the genomic DNAusing DpnI digest. Chromatin is represented as gray circles.
156 Abed et al.
Kc167 cells and Dam-transgenic Drosophila melanogaster embryosusing a sucrose gradient (Fig. 11.2). We also included protocolsfor constructing Dam-fusions proteins, transfection of Drosophilacells, and isolation of genomic DNA from large quantities ofDrosophila embryos. While we have tried to be as conclusive aspossible, an excellent DamID source can be found at: http://research.nki.nl/Vansteensellab/, which contains technical infor-mation, published DamID data sets, and answers to frequentlyasked questions.
All materials should be of high molecular and analytic grade.
1. pNDamMyc and pCMycDam expression vectors. Vectors canbe obtained from the Van Steensel laboratory (for academicand non-profit use). A complete list of vectors; theirsequences, maps and cloning strategies are available for down-load from the Van Steensel lab (see above link).
Fig. 11.2. Design and flow-chart for a DamID experiment.
DamID: A Methylation-Based Chromatin Profiling Approach 157
2. Full-length cDNA encoding the protein of interest.
of Kc Cells
1. HyQ-SFX-Insect MP (#SH30350.03, HyClone) supplemen-ted with 20 mM L-glutamine.
2. 100 20 mm2 tissue culture plates (Falcon).3. 0.4 cm gap electroporation cuvettes (Bio-Rad).
4. Dam expression vectors (pNDamMyc (see Note 1), a vectorencoding the Dam-fusion protein of interest) and a heatshock (hs)-Casper GFP vector (transfection control). All con-structs should be prepared with a high-quality Plasmid MaxiKit (such as #12163, Qiagen) or by CsCl2 purification.
5. Bio-Rad Gene Pulser II/Capacitance Extender II Electro-phoresis System (Bio-Rad), or a similar cell electroporator.
6. Tissue culture grade sterile tips and pasture pipettes, as well as15 and 50 mL plastic tubes.
of Genomic DNA
from Transfected Kc
Cells for DamID
1. T10E10 buffer: 10 mM Tris-HCl, pH 7.5, 10 mM EDTA.
2. T10E0.1 buffer: 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA.
3. TENS buffer: 10 mM Tris-HCl, pH 7.5, 10 mM EDTA,100 mM NaCl, 0.5% SDS. Store solutions 13 at roomtemperature (RT).
4. TENS/K solution: 200 mg/mL proteinase K (#03-115-887,Roche Diagnostics) in TENS. Prepare freshly before use andkeep at room temperature.
5. Buffer-saturated phenol:chloroform:isoamylalcohol (25:24:1)saturated with 10 mM Tris-HCl pH 8.0, 1 mM EDTA.
6. 3 M Na-Acetate (NaAc), pH 5.2.
7. DNase-free RNaseA (10 mg/mL).
of Genomic DNA
from Fly Embryos
for DamID Labeling
1. Yeast paste. Dissolve baking yeast in water to form paste. Keepat room temperature or 4C. Prepare freshly every 2 days.
2. Household bleach.
3. 1 M Tris-base, pH 9.0.
4. 0.5 M EDTA.
5. 5 M NaCl.
6. 50% sucrose, filtered.
7. 20% SDS.
8. Proteinase K, 20 mg/mL stock.
10. 3 M NaAc, pH 5.2.
11. DNase-free RNase A (10 mg/mL; #R5503, Sigma).
158 Abed et al.
12. Homogenizing buffer: 0.1 M Tris-HCl, pH 9.0, 0.1 MEDTA, 0.1 M NaCl, 5% sucrose. Store at 4C.
13. 3 mL glass homogenizer fitted with pestle A (tight).
14. Embryo collection sieves (#052-006, 230 260 mm2,Whatman Biometra)
15. 15 cm embryo collection plates (grape plates)
16. Population cage containing 100200 fly bottles.
2.5. DpnI Digestion
of Genomic DNA
1. DpnI (New England Biolabs).
2. Restriction buffer No. 4 (New England Biolabs; supplied withDpnI).
3. DNase-free RNase A (10 mg/mL; #R5503, Sigma).
2.6. Sucrose Gradient
1. 5% sucrose sol.: 5% sucrose, 10 mM Tris-HCl, pH 7.5,10 mM EDTA, 150 mM NaCl.
2. 30% sucrose sol: 30% sucrose, 10 mM Tris-HCl, pH 7.5,10 mM EDTA, 150 mM NaCl, a dash of Bromophenol-Bluecrystals to give the solution a bit of color. Filter each solutionthrough a 0.22 mm filter and keep sterile at 4C.
3. 3 M NaAc, pH 5.2
Tubes (14 89 mm2, #BC-344059, Beckman).5. Gradient mixer with a peristaltic pump.
6. Ultra centrifuge with a SW40-Ti swing-out rotor.
7. 1% agarose gel.
8. Wide-spectrum DNA ladder.
2.7. Labeling of DpnI
1. BioPrime DNA labeling kit (Invitrogen).
2. PCR grade dNTPs (#28-4065-51, Amersham).
3. 10X dNTP Genomic labeling mix: 1.2 mM each dATP,dGTP and dTTP, 0.6 mM dCTP, 10 mM Tris-HCl pH 8.0,1 mM EDTA.
4. Yeast tRNA (# 15401-011, Invitrogen); 5 mg/mL stock.
5. Cy3-dCTP (PA53021, Amersham); 1 mM stock.
6. Cy5-dCTP (PA55021, Amersham), 1 mM stock.
7. 25 mg competitor DNA, i.e., the plasmid encoding the Dam-fusion protein that was used to transfect the Kc cells.
8. Strataclean Resin (#400714, Stratagene).
9. Glycogen (Roche).
10. Poly [dA]-Poly [dT] 1 mg/mL stock (#P9764-25UN,Sigma).
11. Microcon YM-30 filters (#42410, Millipore).
DamID: A Methylation-Based Chromatin Profiling Approach 159
12. 20X SSC.
13. Hybridization oven set at 55C.14. 37C heating block or water bath.
of Dam Expression
1. Clone the gene of interest in frame into the multiple cloningsites (MCS) of both pNDamMyc and pCMycDam expressionvectors (see Notes 2, 3). The ORF of the gene of interest canbe cloned upstream of the Myc-tag (EQKLISEEDL, 9E10)in the pCMycDam vector. Similarly, the gene of interest canbe cloned downstream of the Myc tag in the pNDamMycvector using its MCS. In both cases the short Myc-tag servesas a linker between the protein of interest and the Dam, andcould be used for detection of the chimeric protein.
2. We recommend that the sequence, proper expression, andnuclear localization of the chimeric protein be verified priorto performing the DamID experiment.
of Kc Cells
1. One 90% confluent 100 20 mm dish (1 108 cells) isrequired per transfection. A 1:10 split of sub-confluent Kccells growing in SFX supplemented with L-glutamine willprovide this appropriate cell density after 48 h at 25C (seeNotes 4, 5). The protocol described below is for a single platetransfection. Note that five starting plates (five independenttransfections for each construct) are required for DamIDanalysis of a single protein.
2. Resuspend cells and pool in a 15 mL sterile tube. Spin at1,000g for 3 min, aspirate supernatant, and resuspend cellpellet in 0.81 mL SFX-glutamine.
3. Mix 10 mg of the expression vector with the cell suspensionand transfer to a 0.4 cm gap electroporation cuvette.
4. Electroporator setup: turn the capacitance rotary switch tohigh capacitance, set the voltage at 0.25 kV and highcapacitance at 1. A good electroporation should yield a timeconstant in the range of 1622.
5. In the hood, carefully remove the cell suspension from thecuvette while avoiding the upper layer of foam and cell debris.Split the cell suspension evenly (380400 mL) to two 100 20 mm dishes supplemented with 10 mL SFX-glutamine.
6. Grow cells at 25C for approximately 3648 h before continu-ing to the DNA purification and labeling stages (see Notes 6, 7).
160 Abed et al.
7. If the transfection is intended for analysis of the nuclearlocalization of the Dam-fusion protein by immunofluores-cence or for Western blot analysis, heat shock induction ofthe protein is required. Heat shock is carried out by incubat-ing the cells at 37C for 1 h and subsequently 6 h recoveryperiod at 25C (see Note 6).
of Genomic DNA
from Transfected Kc
Cells for DamID
1. Collect cells from 10 plates of transfected Kc cells into two 50mL tubes. Spin at 1,500g for 3 min in a tabletop centrifuge.
2. Remove the supernatant and pool the pellets in 7 mL ice-coldT10 E10 by gently pipetting up and down.
3. Squirt in 7.5 mL freshly prepared room temperature TENS/K. Gently invert the tube a few times to induce sufficientmixing.
4. Incubate the tube at 55C for 2 h in a hybridization oven withgentle shaking. Mix gently after 30 min and return to oven.
5. Add 15 mL buffer-saturated phenol:chloroform:isoamylalco-hol and mix gently by inverting the tube. Spin for 20 min at2,200g at RT.
6. Gently transfer the supernatant to a clean tube and add 15 mLisopropanol and 1.5 mL of 3 M NaAc, pH 5.2.
7. Mix gently until DNA forms a large spool. Carefully removethe DNA spool using a large pipetting tip and drain it gentlyon the side of the tube. Continue transferring the DNA anddraining it on the side of a set of clean Eppendorf tubes inorder to further assist the drying process.
8. Transfer DNA to a clean Eppendorf tube and add 0.3 mLof T10E10 and 20 mg DNase-free RNase. Incubate at37C for 30 min. Mix the DNA gently by pipetting upand down using a blue tip, which has been cut at the tip.Return the DNA to 37C and incubate overnight. Impor-tant: The DNA must be completely dissolved before thenext step.
9. Add 0.3 mL TENS/K and gently mix by pipetting up anddown with a blue tip. Incubate tube at 55C for 2 h.
10. Add 0.6 mL phenol:chloroform:isoamylalcohol, mix gently,and spin 15 min 10,000g in a tabletop centrifuge (seeNote 8).
11. Transfer the supernatant to a clean Eppendorf tube. Add60 mL 3 M NaAc, pH 5.2, and 0.6 mL isopropanol. Carefullymix by gently inverting the tube a few times.
12. Spool the DNA onto a yellow tip and briefly dip into anEppendorf tube with 70% ethanol in order to remove thesalt.
DamID: A Methylation-Based Chromatin Profiling Approach 161
13. Transfer DNA into a clean Eppendorf and dissolve it byincubating the DNA with 500 mL T10E0.1for several hoursat 37C. Pipette up and down a few times with a blue tip todissolve the DNA. At this stage the tubes can be incubatedovernight at 37C. Important: Only go to the DpnI diges-tion step (Section 3.5) if the DNA is completely dissolved insolution.
of Genomic DNA from
Fly Embryos for DamID
1. Set up a population cage with approximately 100 bottles offlies (see Notes 912).
2. Synchronize flies by changing the embryo collection platestwice over 1 h.
3. Collect embryos of the appropriate age (for example 46 h forearly developmental stages) by washing the embryos off thecollection plate with water and a paintbrush into an embryocollection sieve.
4. Wash the embryos thoroughly with water and dry off the sieveusing a p...