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  • Protocol Extension
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Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

Nature Protocols volume 11, pages 1586–1598 (2016)Cite this article

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Abstract

This protocol is an extension to: Nat. Protoc. 2, 1467–1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007

The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure—from collecting tissue samples to generating sequencing libraries—can be accomplished within 5 d.

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Figure 1: TaDa can profile the interaction of any protein with DNA or chromatin in a cell-type-specific manner.
Figure 2: Flowchart for processing of TaDa material, with protocol steps listed.
Figure 3: Sample TapeStation plots showing the expected fragment-size distribution following TaDa.
Figure 4: Example log2 ratio profile of RNA Pol II binding in Drosophila neural stem cells.

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References

  1. Southall, T.D. et al. Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells. Dev. Cell. 26, 101–112 (2013).

    Article  CAS  Google Scholar 

  2. van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18, 424–428 (2000).

    Article  CAS  Google Scholar 

  3. Vogel, M.J., Peric-Hupkes, D. & van Steensel, B. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat. Protoc. 2, 1467–1478 (2007).

    Article  CAS  Google Scholar 

  4. Brand, A.H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  Google Scholar 

  5. Luukkonen, B.G., Tan, W. & Schwartz, S. Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance. J. Virol. 69, 4086–4094 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Van Blokland, H.J.M., Hoeksema, F., Siep, M., Otte, A.P. & Verhees, J.A. Methods to create a stringent selection system for mammalian cell lines. Cytotechnology 63, 371–384 (2011).

    Article  CAS  Google Scholar 

  7. Marshall, O.J. & Brand, A.H. damidseq_pipeline: an automated pipeline for processing DamID sequencing datasets. Bioinformatics 31, 3371–3373 (2015).

    Article  Google Scholar 

  8. Filion, G.J. et al. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143, 212–224 (2010).

    Article  CAS  Google Scholar 

  9. Woolcock, K.J., Gaidatzis, D., Punga, T. & Bühler, M. Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe. Nat. Struct. Mol. Biol. 18, 94–99 (2011).

    Article  CAS  Google Scholar 

  10. Germann, S., Juul-Jensen, T., Letarnec, B. & Gaudin, V. DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci. Plant J. 48, 153–163 (2006).

    Article  CAS  Google Scholar 

  11. Schuster, E. et al. DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO. Mol. Syst. Biol. 6, 399 (2010).

    Article  Google Scholar 

  12. Guelen, L. et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453, 948–951 (2008).

    Article  CAS  Google Scholar 

  13. Vogel, M. et al. Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res. 16, 1493–1504 (2006).

    Article  CAS  Google Scholar 

  14. Bryant, Z. et al. Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis. Proc. Natl. Acad. Sci. USA 96, 5559–5564 (1999).

    Article  CAS  Google Scholar 

  15. Bonn, S. et al. Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat. Genet. 44, 148–156 (2012).

    Article  CAS  Google Scholar 

  16. Bonn, S. et al. Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP. Nat. Protoc. 7, 978–994 (2012).

    Article  CAS  Google Scholar 

  17. Deal, R.B. & Henikoff, S. A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell 18, 1030–1040 (2010).

    Article  CAS  Google Scholar 

  18. Henry, G.L., Davis, F.P., Picard, S. & Eddy, S.R. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691–9704 (2012).

    Article  CAS  Google Scholar 

  19. Steiner, F.A., Talbert, P.B., Kasinathan, S., Deal, R.B. & Henikoff, S. Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res. 22, 766–777 (2012).

    Article  CAS  Google Scholar 

  20. Schauer, T. et al. CAST-ChIP maps cell-type-specific chromatin states in the Drosophila central nervous system. Cell Rep. 5, 271–282 (2013).

    Article  CAS  Google Scholar 

  21. Otsuki, L., Cheetham, S.W. & Brand, A.H. Freedom of expression: cell-type-specific gene profiling. Wiley Interdiscip. Rev. Dev. Biol. 3, 429–443 (2014).

    Article  CAS  Google Scholar 

  22. Roy, P.J., Stuart, J.M., Lund, J. & Kim, S.K. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature 418, 975–979 (2002).

    Article  CAS  Google Scholar 

  23. Thomas, A. et al. A versatile method for cell-specific profiling of translated mRNAs in Drosophila. PLoS ONE 7, e40276 (2012).

    Article  CAS  Google Scholar 

  24. Miller, M.R., Robinson, K.J., Cleary, M.D. & Doe, C.Q. TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat. Methods 6, 439–441 (2009).

    Article  CAS  Google Scholar 

  25. Modrich, P. & Lahue, R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101–133 (1996).

    Article  CAS  Google Scholar 

  26. Baena-Lopez, L.A., Alexandre, C., Mitchell, A., Pasakarnis, L. & Vincent, J.-P. Accelerated homologous recombination and subsequent genome modification in Drosophila. Development 140, 4818–4825 (2013).

    Article  CAS  Google Scholar 

  27. Venken, K.J.T. et al. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nat. Methods 8, 737–743 (2011).

    Article  CAS  Google Scholar 

  28. Luan, H., Peabody, N.C., Vinson, C. & White, B.H. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron 52, 425–436 (2006).

    Article  CAS  Google Scholar 

  29. Matsumoto, K., Toh-e, A. & Oshima, Y. Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4. J. Bacteriol. 134, 446–457 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. McGuire, S.E., Le, P.T., Osborn, A.J., Matsumoto, K. & Davis, R.L. Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765–1768 (2003).

    Article  CAS  Google Scholar 

  31. Osterwalder, T., Yoon, K.S., White, B.H. & Keshishian, H.A Conditional tissue-specific transgene expression system using inducible GAL4. Proc. Natl. Acad. Sci. USA 98, 12596–12601 (2001).

    Article  CAS  Google Scholar 

  32. Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by a Wellcome Trust Senior Investigator Award (103792), Wellcome Trust Programme Grant (092545) and BBSRC Project Grant (BB/L00786X/1) to A.H.B. A.H.B. acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492).

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Author notes

  1. Owen J Marshall & Tony D Southall

    Present address: Present addresses: Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia (O.J.M.), Department of Life Sciences, Imperial College London, London, UK (T.D.S.).,

  2. Owen J Marshall and Tony D Southall: These authors contributed equally to this work.

Authors and Affiliations

  1. The Gurdon Institute, University of Cambridge, Cambridge, UK

    Owen J Marshall, Tony D Southall, Seth W Cheetham & Andrea H Brand

  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Owen J Marshall, Tony D Southall, Seth W Cheetham & Andrea H Brand

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Contributions

O.J.M., T.D.S., S.W.C. and A.H.B. designed the research and analyzed the data; T.D.S., S.W.C. and O.J.M. developed the protocols; and O.J.M., T.D.S., S.W.C. and A.H.B. wrote the paper.

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Correspondence to Andrea H Brand.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Details of the pUAST-attB-LT3-NDam vector used for cloning Dam-fusion proteins for TaDa

(A) Schematic vector map illustrating the key features of the vector. (B) Sequence details of the linker and MCS regions of the vector; translated codons in frame with Dam are shaded. Vector map created using SnapGene software.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 389 kb)

Supplementary Table 1

Sequencing adaptor oligos (DST purity) (XLSX 10 kb)

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Marshall, O., Southall, T., Cheetham, S. et al. Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nat Protoc 11, 1586–1598 (2016). https://doi.org/10.1038/nprot.2016.084

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