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.

Genome-scale profiling of histone H3.3 replacement patterns

Nature Genetics volume 37, pages 1090–1097 (2005)Cite this article

Abstract

Histones of multicellular organisms are assembled into chromatin primarily during DNA replication. When chromatin assembly occurs at other times, the histone H3.3 variant replaces canonical H3. Here we introduce a new strategy for profiling epigenetic patterns on the basis of H3.3 replacement, using microarrays covering roughly one-third of the Drosophila melanogaster genome at 100-bp resolution. We identified patterns of H3.3 replacement over active genes and transposons. H3.3 replacement occurred prominently at sites of abundant RNA polymerase II and methylated H3 Lys4 throughout the genome and was enhanced on the dosage-compensated male X chromosome. Active genes were depleted of histones at promoters and were enriched in H3.3 from upstream to downstream of transcription units. We propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Replication-independent deposition corresponds to active modifications at genes.
Figure 2: H3.3 enrichment corresponds to H3K4me2 and RNA Pol II at genes.
Figure 3: Transposons have distinct chromatin patterns.
Figure 4: Changes in nucleosome and histone density profiles with increasing RNA Pol II density.
Figure 5: H3.3 shows enrichment patterns over genes and flanking sequences.
Figure 6: H3.3, RNA Pol II and H3K4me2 show similar enrichment patterns from upstream to downstream of transcription start sites.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Wolffe, A.P. Chromatin: Structure and Function (Academic, San Diego, 1992).

    Google Scholar 

  2. Ringrose, L. & Paro, R. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu. Rev. Genet. 38, 413–443 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Schubeler, D. et al. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18, 1263–1271 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bernstein, B.E. et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169–181 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Kurdistani, S.K., Tavazoie, S. & Grunstein, M. Mapping global histone acetylation patterns to gene expression. Cell 117, 721–733 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Ahmad, K. & Henikoff, S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Janicki, S.M. et al. From silencing to gene expression: Real-time analysis in single cells. Cell 116, 683–698 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schwartz, B.E. & Ahmad, K. Transcriptional activation triggers deposition and removal of the histone variant H3.3. Genes Dev. 19, 804–814 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chow, C.M. et al. Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division. EMBO Rep. 6, 354–360 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. McKittrick, E., Gafken, P.R., Ahmad, K. & Henikoff, S. Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc. Natl. Acad. Sci. USA 101, 1525–1530 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Waterborg, J.H. Sequence analysis of acetylation and methylation in two histone H3 variants of alfalfa. J. Biol. Chem. 265, 17157–17161 (1990).

    CAS  PubMed  Google Scholar 

  13. Johnson, L. et al. Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications. Nucleic Acids Res. 32, 6511–6518 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. de Boer, E. et al. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 100, 7480–7485 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. MacAlpine, D.M., Rodriguez, H.K. & Bell, S.P. Coordination of replication and transcription along a Drosophila chromosome. Genes Dev. 18, 3094–3105 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Martens, J.H. et al. The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J. 24, 800–812 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bernstein, B.E., Liu, C.L., Humphrey, E.L., Perlstein, E.O. & Schreiber, S.L. Global nucleosome occupancy in yeast. Genome Biol. 5, R62 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lee, C.K., Shibata, Y., Rao, B., Strahl, B.D. & Lieb, J.D. Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet. 36, 900–905 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Ahmad, K. & Henikoff, S. Centromeres are specialized replication domains in heterochromatin. J. Cell Biol. 153, 101–110 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Schwabish, M.A. & Struhl, K. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24, 10111–10117 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kristjuhan, A. & Svejstrup, J.Q. Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo . EMBO J. 23, 4243–4252 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Boeger, H., Griesenbeck, J., Strattan, J.S. & Kornberg, R.D. Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell 11, 1587–1598 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Reinke, H. & Horz, W. Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. Mol. Cell 11, 1599–1607 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Yuan, G.C. et al. Genome-scale identification of nucleosome positions in S. cerevisiae . Science 309, 626–630 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Workman, J.L. & Abmayr, S.M. Histone H3 variants and modifications on transcribed genes. Proc. Natl. Acad. Sci. USA 101, 1429–1430 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Henikoff, S., Furuyama, T. & Ahmad, A. Histone variants, nucleosome assembly and epigenetic inheritance. Trends Genet. 20, 320–326 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Henikoff, S. & Meneely, P. Unwinding dosage compensation. Cell 72, 1–2 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. 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  PubMed  Google Scholar 

  29. Straub, T., Dahlsveen, I.K. & Becker, P.B. Dosage compensation in flies: Mechanism, models, mystery. FEBS Lett. 579, 3258–3263 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. 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  PubMed  PubMed Central  Google Scholar 

  31. Roh, T.Y., Ngau, W.C., Cui, K., Landsman, D. & Zhao, K. High-resolution genome-wide mapping of histone modifications. Nat. Biotechnol. 22, 1013–1016 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. van Steensel, B. Mapping of genetic and epigenetic regulatory networks using microarrays. Nat. Genet. 37 Suppl, S18–S24 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Kirmizis, A. et al. Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev. 18, 1592–1605 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sarraf, S.A. & Stancheva, I. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol. Cell 15, 595–605 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Tyler, J.K. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555–560 (1999).

    Article  CAS  PubMed  Google Scholar 

  36. Thiriet, C. & Hayes, J.J. Replication-independent core histone dynamics at transcriptionally active loci in vivo . Genes Dev. 19, 677–682 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. & Fraser, P. Intergenic transcription and developmental remodeling of chromatin subdomains in the human β-globin locus. Mol. Cell 5, 377–386 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Lipshitz, H.D., Peattie, D.A. & Hogness, D.S. Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev. 1, 307–322 (1987).

    Article  CAS  PubMed  Google Scholar 

  39. Wyers, F. et al. Cryptic Pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121, 725–737 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Johnson, J.M., Edwards, S., Shoemaker, D. & Schadt, E.E. Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments. Trends Genet. 21, 93–102 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Hogga, I. & Karch, F. Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing. Development 129, 4915–4922 (2002).

    CAS  PubMed  Google Scholar 

  42. Rank, G., Prestel, M. & Paro, R. Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch. Mol. Cell. Biol. 22, 8026–8034 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schmitt, S., Prestel, M. & Paro, R. Intergenic transcription through a polycomb group response element counteracts silencing. Genes Dev. 19, 697–708 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bunch, T.A., Grinblat, Y. & Goldstein, L.S. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16, 1043–1061 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Beckett, D., Kovaleva, E. & Schatz, P.J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Iwaki, T., Figuera, M., Ploplis, V.A. & Castellino, F.J. Rapid selection of Drosophila S2 cells with the puromycin resistance gene. Biotechniques 35, 482–484, 486 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Echalier, G. Drosophila Cells in Culture (Academic, New York, 1997).

    Google Scholar 

  48. 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  PubMed  PubMed Central  Google Scholar 

  49. Blower, M.D., Sullivan, B.A. & Karpen, G.H. Conserved organization of centromeric chromatin in flies and humans. Dev. Cell 2, 319–330 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pollack, J.R. et al. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat. Genet. 23, 41–46 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Furuyama for providing the biotin pull-down system and for advice in generating lines; Y. Dalal for advice on chromatin procedures; T. Iwaki for the puromycin resistance gene; J. Delrow, M. Aronszajn, L. Chow and K. Munn for help and advice; D. Schübeler for sharing unpublished information; J. Lucchesi for discussions of dosage compensation; and members of our laboratory for discussions and comments on the manuscript.

Author information

Authors and Affiliations

  1. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, 98109, Washington, USA

    Yoshiko Mito, Jorja G Henikoff & Steven Henikoff

  2. Howard Hughes Medical Institute, USA

    Steven Henikoff

Authors
  1. Yoshiko Mito
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jorja G Henikoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steven Henikoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steven Henikoff.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Scatterplots comparing signal ratios of H3.3core or H3 to signal ratios of individual modifications based on cDNA array analysis. (PDF 428 kb)

Supplementary Fig. 2

Transposon profiles for H3.3/H3 datasets. (PDF 63 kb)

Supplementary Fig. 3

Diagram of the method used for averaging genes and removing overlaps. (PDF 64 kb)

Supplementary Fig. 4

Enhancement of H3.3 replacement at genes on the male chromosome in S2 cells. (PDF 61 kb)

Supplementary Fig. 5

Typical growth curve for a stably transformed cell line, showing timing of induction and harvesting for chromatin affinity purification. (PDF 56 kb)

Supplementary Fig. 6

Western blot analyses of histones in cells and after extraction from nuclei. (PDF 361 kb)

Supplementary Fig. 7

MNase cleavage and pull-down of nucleosomes used for profiling. (PDF 126 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mito, Y., Henikoff, J. & Henikoff, S. Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 37, 1090–1097 (2005). https://doi.org/10.1038/ng1637

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng1637

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