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Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase

Nature Biotechnology volume 18, pages 424–428 (2000)Cite this article

Abstract

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.

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Figure 1: Targeting of Dam to a specific sequence.
Figure 2: In situ detection of targeted methylation by Dam-HP1.
Figure 3: HP1-targeted methylation of specific genomic loci.

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References

  1. Simpson, R.T. In vivo methods to analyze chromatin structure. Curr. Opin. Genet. Dev. 9, 225–229 ( 1999).

    Article  CAS  Google Scholar 

  2. Solomon, M.J., Larsen, P.L. & Varshavsky, A. Mapping protein–DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947 (1988).

    Article  CAS  Google Scholar 

  3. Orlando, V., Strutt, H. & Paro, R. Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205– 214 (1997).

    Article  CAS  Google Scholar 

  4. Law, A., Hirayoshi, K., O'Brien, T. & Lis, J.T. Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila. Nucleic Acids Res. 26, 919–924 (1998).

    Article  CAS  Google Scholar 

  5. Lee, J.S., Lee, C.H. & Chung, J.H. Studying the recruitment of Sp1 to the beta-globin promoter with an in vivo method: protein position identification with nuclease tail (PIN*POINT). Proc. Natl. Acad. Sci. USA 95, 969–974 (1998).

    Article  CAS  Google Scholar 

  6. Xu, G.L. & Bestor, T.H. Cytosine methylation targetted to pre-determined sequences. Nat. Genet. 17, 376–378 (1997).

    Article  CAS  Google Scholar 

  7. Gottschling, D.E. Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc. Natl. Acad. Sci. USA 89, 4062–4065 ( 1992).

    Article  CAS  Google Scholar 

  8. Singh, J. & Klar, A.J. Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast. Genes Dev. 6, 186–196 (1992).

    Article  CAS  Google Scholar 

  9. Kladde, M.P. & Simpson, R.T. Positioned nucleosomes inhibit Dam methylation in vivo. Proc. Natl. Acad. Sci. USA 91, 1361–1365 (1994).

    Article  CAS  Google Scholar 

  10. Wines, D.R., Talbert, P.B., Clark, D.V. & Henikoff S. Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo. Chromosoma 104, 332– 340 (1996).

    Article  CAS  Google Scholar 

  11. Lyko, F. et al. Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila. Nat. Genet. 23, 363–366 (1999).

    Article  CAS  Google Scholar 

  12. Fischer, J.A., Giniger, E., Maniatis, T. & Ptashne, M. GAL4 activates transcription in Drosophila. Nature 332, 853–856 (1988).

    Article  CAS  Google Scholar 

  13. Rørth, P. A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. USA 93, 12418–12422 (1996).

    Article  Google Scholar 

  14. Lie, Y.S. & Petropoulos, C.J. Advances in quantitative PCR technology: 5′ nuclease assays. Curr. Opin. Biotechnol. 9, 43–48 (1998).

    Article  CAS  Google Scholar 

  15. Caizzi, R., Caggese, C. & Pimpinelli, S. Bari-1, a new transposon-like family in Drosophila melanogaster with a unique heterochromatic organization. Genetics 133, 335–345 ( 1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Frommer, G., Schuh, R. & Jackle, H. Localized expression of a novel micropia-like element in the blastoderm of Drosophila melanogaster is dependent on the anterior morphogen bicoid. Chromosoma 103, 82– 89 (1994).

    Article  CAS  Google Scholar 

  17. Goldstein, A. Biostatistics. An introductory text. (MacMillan Co., New York, NY; 1964).

    Google Scholar 

  18. James, T.C., et al. Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J. Cell. Biol. 50, 170–180 ( 1989).

    CAS  PubMed  Google Scholar 

  19. Platero, J.S., Hartnett, T. & Eissenberg, J.C. Functional analysis of the chromo domain of HP1. EMBO J. 14, 3977–3986 ( 1995).

    Article  CAS  Google Scholar 

  20. Bringmann, P. & Lührmann, R. Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6. FEBS Lett. 213 , 309–315 (1987).

    Article  CAS  Google Scholar 

  21. Boivin, A. & Dura, J.-M. In vivo chromatin accessibility correlates with gene silencing in Drosophila. Genetics 150, 1539–1549 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Fitch, D.H., Strausbaugh, L.D. & Barrett, V. On the origins of tandemly repeated genes: does histone gene copy number in Drosophila reflect chromosomal location? Chromosoma 99, 118–124 ( 1990).

    Article  CAS  Google Scholar 

  23. Hilliker, A.J., Appels, R. & Schalet, A. The genetic analysis of D. melanogaster heterochromatin . Cell 21, 607–619 (1980).

    Article  CAS  Google Scholar 

  24. Scheer, U. & Hock, R. Structure and function of the nucleolus . Curr. Opin. Cell. Biol. 11, 385– 390 (1999).

    Article  CAS  Google Scholar 

  25. Barras, F. & Marinus, M.G. The great GATC: DNA methylation in E. coli. Trends Genet. 5, 139– 143 (1989).

    Article  CAS  Google Scholar 

  26. Pease, A.C. et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA 91, 5022–5026 (1994).

    Article  CAS  Google Scholar 

  27. Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 ( 1995).

    Article  CAS  Google Scholar 

  28. Henikoff, S., Ahmad, K., Platero, J.S. & van Steensel, B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc. Natl. Acad. Sci. USA 97, 716–721 (2000).

    Article  CAS  Google Scholar 

  29. van Steensel, B. et al. Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus. J. Cell. Sci. 108, 3003–3011 (1995).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr. R. Lührmann for providing anti-m6A-antibody; S. Elgin for anti-HP1 antibody; Joel Eissenberg and Susan Parkhurst for plasmids; Keith Kerkof for TaqMan PCR advice; Kami Ahmad for help with microinjection and fly genetics and for suggesting the DamID name; Jorja Henikoff for help with statistical analysis; Peter Kim and Judith O'Brien for technical assistance; and members of our lab for unlimited enthusiasm and helpful suggestions.

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  1. Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, 1100 Fairview Ave N, Seattle , 98109, WA

    Bas van Steensel & Steven Henikoff

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  1. Bas van Steensel
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Correspondence to Bas van Steensel.

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Steensel, B., Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase. Nat Biotechnol 18, 424–428 (2000). https://doi.org/10.1038/74487

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