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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.

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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.

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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puromycin resistance gene; J. Delrow, M. Aronszajn, L. Chow and K. Munn for help and advice; D. Schübeler for sharing unpublished information; J. Lucchesi for discussions of dosage compensation; and members of our laboratory for discussions and comments on the manuscript.

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  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

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  1. Yoshiko Mito
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Correspondence to Steven Henikoff.

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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)

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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

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