Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chromatin proteins captured by ChIP–mass spectrometry are linked to dosage compensation in Drosophila

Nature Structural & Molecular Biology volume 20, pages 202–209 (2013)Cite this article

Subjects

Abstract

X-chromosome dosage compensation by the MSL (male-specific lethal) complex is required in Drosophila melanogaster to increase gene expression from the single male X to equal that of both female X chromosomes. Instead of focusing solely on protein complexes released from DNA, here we used chromatin-interacting protein MS (ChIP-MS) to identify MSL interactions on cross-linked chromatin. We identified MSL-enriched histone modifications, including histone H4 Lys16 acetylation and histone H3 Lys36 methylation, and CG4747, a putative Lys36-trimethylated histone H3 (H3K36me3)-binding protein. CG4747 is associated with the bodies of active genes, coincident with H3K36me3, and is mislocalized in the Set2 mutant lacking H3K36me3. CG4747 loss of function in vivo results in partial mislocalization of the MSL complex to autosomes, and RNA interference experiments confirm that CG4747 and Set2 function together to facilitate targeting of the MSL complex to active genes, validating the ChIP-MS approach.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Use of ChIP-MS to identify proteins interacting with MSL3-HTB.
Figure 2: MS identification of histones recovered from MSL3-TAP purification.
Figure 3: CG4747 colocalization with H3K36me3 is dependent on Set2.
Figure 4: CG4747 colocalizes with H3K36me3 and MSL3 on the X chromosome.
Figure 5: MSL-complex localization is disrupted in CG4747 loss-of-function males.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Lucchesi, J.C., Kelly, W.G. & Panning, B. Chromatin remodeling in dosage compensation. Annu. Rev. Genet. 39, 615–651 (2005).

    Article  CAS  Google Scholar 

  2. Conrad, T. & Akhtar, A. Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription. Nat. Rev. Genet. 13, 123–134 (2011).

    Article  Google Scholar 

  3. Alekseyenko, A.A., Larschan, E., Lai, W.R., Park, P.J. & Kuroda, M.I. High-resolution ChIP-chip analysis reveals that the Drosophila MSL complex selectively identifies active genes on the male X chromosome. Genes Dev. 20, 848–857 (2006).

    Article  CAS  Google Scholar 

  4. Gilfillan, G.D. et al. Chromosome-wide gene-specific targeting of the Drosophila dosage compensation complex. Genes Dev. 20, 858–870 (2006).

    Article  CAS  Google Scholar 

  5. Gelbart, M.E. & Kuroda, M.I. Drosophila dosage compensation: a complex voyage to the X chromosome. Development 136, 1399–1410 (2009).

    Article  CAS  Google Scholar 

  6. Hilfiker, A., Hilfiker-Kleiner, D., Pannuti, A. & Lucchesi, J.C. mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J. 16, 2054–2060 (1997).

    Article  CAS  Google Scholar 

  7. Jin, Y., Wang, Y., Johansen, J. & Johansen, K.M. JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associates with the male specific lethal (MSL) dosage compensation complex. J. Cell Biol. 149, 1005–1010 (2000).

    Article  CAS  Google Scholar 

  8. Mendjan, S. et al. Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Mol. Cell 21, 811–823 (2006).

    Article  CAS  Google Scholar 

  9. Morales, V. et al. Functional integration of the histone acetyltransferase MOF into the dosage compensation complex. EMBO J. 23, 2258–2268 (2004).

    Article  CAS  Google Scholar 

  10. Smith, E.R. et al. The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol. Cell. Biol. 20, 312–318 (2000).

    Article  CAS  Google Scholar 

  11. Lambert, J.-P., Mitchell, L., Rudner, A., Baetz, K. & Figeys, D. A novel proteomics approach for the discovery of chromatin-associated protein networks. Mol. Cell. Proteomics 8, 870–882 (2009).

    Article  CAS  Google Scholar 

  12. Lambert, J.-P. et al. Defining the budding yeast chromatin-associated interactome. Mol. Syst. Biol. 6, 448 (2010).

    Article  CAS  Google Scholar 

  13. Smart, S.K., Mackintosh, S.G., Edmondson, R.D., Taverna, S.D. & Tackett, A.J. Mapping the local protein interactome of the NuA3 histone acetyltransferase. Protein Sci. 18, 1987–1997 (2009).

    Article  CAS  Google Scholar 

  14. Guerrero, C., Tagwerker, C., Kaiser, P. & Huang, L. An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol. Cell. Proteomics 5, 366–378 (2006).

    Article  CAS  Google Scholar 

  15. Tagwerker, C. et al. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. Mol. Cell. Proteomics 5, 737–748 (2006).

    Article  CAS  Google Scholar 

  16. Tardiff, D.F., Abruzzi, K.C. & Rosbash, M. Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking. Proc. Natl. Acad. Sci. USA 104, 19948–19953 (2007).

    Article  CAS  Google Scholar 

  17. Déjardin, J. & Kingston, R.E. Purification of proteins associated with specific genomic loci. Cell 136, 175–186 (2009).

    Article  Google Scholar 

  18. Larschan, E. et al. Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation. PLoS Genet. 8, e1002830 (2012).

    Article  CAS  Google Scholar 

  19. Cronan, J.E. Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J. Biol. Chem. 265, 10327–10333 (1990).

    CAS  PubMed  Google Scholar 

  20. Copps, K. et al. Complex formation by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly. EMBO J. 17, 5409–5417 (1998).

    Article  CAS  Google Scholar 

  21. Hamada, F.N., Park, P.J., Gordadze, P.R. & Kuroda, M.I. Global regulation of X chromosomal genes by the MSL complex in Drosophila melanogaster. Genes Dev. 19, 2289–2294 (2005).

    Article  CAS  Google Scholar 

  22. Straub, T., Gilfillan, G.D., Maier, V.K. & Becker, P.B. The Drosophila MSL complex activates the transcription of target genes. Genes Dev. 19, 2284–2288 (2005).

    Article  CAS  Google Scholar 

  23. Kharchenko, P.V. et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471, 480–485 (2011).

    Article  CAS  Google Scholar 

  24. Kho, Y. et al. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. USA 101, 12479–12484 (2004).

    Article  CAS  Google Scholar 

  25. Turner, B.M., Birley, A.J. & Lavender, J. Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375–384 (1992).

    Article  CAS  Google Scholar 

  26. Larschan, E. et al. MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism. Mol. Cell 28, 121–133 (2007).

    Article  CAS  Google Scholar 

  27. Sural, T.H. et al. The MSL3 chromodomain directs a key targeting step for dosage compensation of the Drosophila melanogaster X chromosome. Nat. Struct. Mol. Biol. 15, 1318–1325 (2008).

    Article  CAS  Google Scholar 

  28. Bell, O. et al. Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster. Mol. Cell Biol. 28, 3401–3409 (2008).

    Article  CAS  Google Scholar 

  29. Plazas-Mayorca, M.D. et al. One-pot shotgun quantitative mass spectrometry characterization of histones. J. Proteome Res. 8, 5367–5374 (2009).

    Article  CAS  Google Scholar 

  30. Akhtar, A. & Becker, P.B. Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol. Cell 5, 367–375 (2000).

    Article  CAS  Google Scholar 

  31. Smith, E.R., Allis, C.D. & Lucchesi, J.C. Linking global histone acetylation to the transcription enhancement of X-chromosomal genes in Drosophila males. J. Biol. Chem. 276, 31483–31486 (2001).

    Article  CAS  Google Scholar 

  32. Kind, J. et al. Genome-wide analysis reveals MOF as a key regulator of dosage compensation and gene expression in Drosophila. Cell 133, 813–828 (2008).

    Article  CAS  Google Scholar 

  33. Gelbart, M.E., Larschan, E., Peng, S., Park, P.J. & Kuroda, M.I. Drosophila MSL complex globally acetylates H4K16 on the male X chromosome for dosage compensation. Nat. Struct. Mol. Biol. 16, 825–832 (2009).

    Article  CAS  Google Scholar 

  34. Pokholok, D.K. et al. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517–527 (2005).

    Article  CAS  Google Scholar 

  35. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  Google Scholar 

  36. Nguyen, A.T. & Zhang, Y. The diverse functions of Dot1 and H3K79 methylation. Genes Dev. 25, 1345–1358 (2011).

    Article  CAS  Google Scholar 

  37. Vermeulen, M. et al. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell 142, 967–980 (2010).

    Article  CAS  Google Scholar 

  38. Carrozza, M.J. et al. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 123, 581–592 (2005).

    Article  CAS  Google Scholar 

  39. Huh, J.-W. et al. Multivalent di-nucleosome recognition enables the Rpd3S histone deacetylase complex to tolerate decreased H3K36 methylation levels. EMBO J. 31, 3564–3574 (2012).

    Article  CAS  Google Scholar 

  40. Ge, H., Si, Y. & Wolffe, A.P. A novel transcriptional coactivator, p52, functionally interacts with the essential splicing factor ASF/SF2. Mol. Cell 2, 751–759 (1998).

    Article  CAS  Google Scholar 

  41. Pradeepa, M.M., Sutherland, H.G., Ule, J., Grimes, G.R. & Bickmore, W.A. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLoS Genet. 8, e1002717 (2012).

    Article  CAS  Google Scholar 

  42. Maurer-Stroh, S. et al. The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28, 69–74 (2003).

    Article  CAS  Google Scholar 

  43. Vezzoli, A. et al. Molecular basis of histone H3K36me3 recognition by the PWWP domain of Brpf1. Nat. Struct. Mol. Biol. 17, 617–619 (2010).

    Article  CAS  Google Scholar 

  44. Dhayalan, A. et al. The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation. J. Biol. Chem. 285, 26114–26120 (2010).

    Article  CAS  Google Scholar 

  45. Eissenberg, J.C. & Shilatifard, A. Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev. Biol. 339, 240–249 (2010).

    Article  CAS  Google Scholar 

  46. Mohan, M. et al. The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31, 4310–4318 (2011).

    Article  CAS  Google Scholar 

  47. Cheng, H., He, X. & Moore, C. The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3. Mol. Cell. Biol. 24, 2932–2943 (2004).

    Article  CAS  Google Scholar 

  48. Regnard, C. et al. Global analysis of the relationship between JIL-1 kinase and transcription. PLoS Genet. 7, e1001327 (2011).

    Article  CAS  Google Scholar 

  49. Qiu, C., Sawada, K., Zhang, X. & Cheng, X. The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds. Nat. Struct. Biol. 9, 217–224 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Handler, D. et al. A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J. 30, 3977–3993 (2011).

    Article  CAS  Google Scholar 

  51. Ni, J.-Q. et al. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat. Methods 8, 405–407 (2011).

    Article  CAS  Google Scholar 

  52. Mito, Y., Henikoff, J.G. & Henikoff, S. Genome-scale profiling of histone H3.3 replacement patterns. Nat. Genet. 37, 1090–1097 (2005).

    Article  CAS  Google Scholar 

  53. Moore, S.A., Ferhatoglu, Y., Jia, Y., Al-Jiab, R.A. & Scott, M.J. Structural and biochemical studies on the chromo-barrel domain of male specific lethal 3 (MSL3) reveal a binding preference for mono or dimethyl lysine 20 on histone H4. J. Biol. Chem. 285, 40879–40890 (2010).

    Article  CAS  Google Scholar 

  54. Kim, D. et al. Corecognition of DNA and a methylated histone tail by the MSL3 chromodomain. Nat. Struct. Mol. Biol. 17, 1027–1029 (2010).

    Article  CAS  Google Scholar 

  55. Alekseyenko, A.A. et al. A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome. Cell 134, 599–609 (2008).

    Article  CAS  Google Scholar 

  56. Schotta, G. & Reuter, G. Controlled expression of tagged proteins in Drosophila using a new modular P-element vector system. Mol. Gen. Genet. 262, 916–920 (2000).

    Article  CAS  Google Scholar 

  57. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  Google Scholar 

  58. Kelley, R.L. et al. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98, 513–522 (1999).

    Article  CAS  Google Scholar 

  59. Saumweber, H., Symmons, P., Kabisch, R., Will, H. & Bonhoeffer, F. Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster: establishment of antibody producing cell lines and partial characterization of corresponding antigens. Chromosoma 80, 253–275 (1980).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Kaiser (University of California, Irvine, Irvine, California, USA) for the HTB tag and insightful discussions. We thank G. Schotta (Ludwig-Maximilians Universität, Munich, Germany) for the pGS-mw[+] vector. The anti-Z4 antibody was a generous gift from H. Saumweber (Humboldt University, Berlin, Germany). We thank E. Gerace from the Moazed lab (Harvard Medical School, Boston, Massachusetts, USA) for providing the protocol for coupling IgG to magnetic beads. We thank the Bloomington Stock Center and TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing fly stocks used in this study. We are grateful to M. Gelbart for support and expertise, E. Smith for technical assistance and A. Ciccia, B. Adamson and A. Plachetka for critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health (NIH) to M.I.K. (GM45744 and GM101958), P.V.K. (K25AG037596) and S.J.E. (GM44664). A.E.H.E. is supported by fellowships from The Jane Coffin Childs Foundation and The American Society for Radiation Oncology. B.A.G. is supported by a US National Science Foundation Early Faculty CAREER award and NIH award number DP2OD007447 from the Office of the Director.

Author information

Authors and Affiliations

  1. Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Charlotte I Wang, Artyom A Alekseyenko, Andrew EH Elia, Andrey A Gorchakov, Stephen J Elledge & Mitzi I Kuroda

  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Charlotte I Wang, Artyom A Alekseyenko, Andrew EH Elia, Andrey A Gorchakov, Stephen J Elledge & Mitzi I Kuroda

  3. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.,

    Gary LeRoy, Laura-Mae P Britton & Benjamin A Garcia

  4. Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Andrew EH Elia

  5. Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Andrew EH Elia & Stephen J Elledge

  6. Institute of Molecular and Cell Biology, Novosibirsk, Russia

    Andrey A Gorchakov

  7. Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA

    Peter V Kharchenko

  8. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Benjamin A Garcia

Authors
  1. Charlotte I Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Artyom A Alekseyenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gary LeRoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew EH Elia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrey A Gorchakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Laura-Mae P Britton
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephen J Elledge
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter V Kharchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Benjamin A Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mitzi I Kuroda
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.I.W., A.A.A. and A.A.G. performed ChIP-MS experiments; A.E.H.E. performed LC-MS/MS to identify MSL-interacting proteins; G.L. and L.-M.P.B. performed quantitative MS of histone PTM; C.I.W. performed all other experiments; P.V.K. performed all bioinformatics analyses; S.J.E., B.A.G. and M.I.K. supervised analyses; C.I.W. and M.I.K. prepared the manuscript in consultation with all coauthors.

Corresponding author

Correspondence to Mitzi I Kuroda.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1–3 and Supplementary Note (PDF 347 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, C., Alekseyenko, A., LeRoy, G. et al. Chromatin proteins captured by ChIP–mass spectrometry are linked to dosage compensation in Drosophila. Nat Struct Mol Biol 20, 202–209 (2013). https://doi.org/10.1038/nsmb.2477

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2477

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing