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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.
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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