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Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters

Nature Structural & Molecular Biology volume 13, pages 256–263 (2006)Cite this article

Abstract

Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.

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Figure 1: System for in vivo kinetic analysis of PHO5 chromatin remodeling.
Figure 2: MTase accessibility is highest at HSR2-proximal nucleosomes in the population of activated PHO5 promoters.
Figure 3: Nucleosomes adjacent to HSR2 show higher rates of increasing MTase accessibility in the population of activated PHO5 promoters.
Figure 4: Variable numbers of nucleosomes are disrupted at individual PHO5 promoters.
Figure 5: M.HhaI methylates protein-free DNA randomly.
Figure 6: Contiguous nucleosomes are disrupted at the induced PHO5 promoter.

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Acknowledgements

We thank S. Conard for excellent technical assistance, R. Roberts (NEB) for the M.HhaI gene, R. Morse (Wadsworth Center) for providing the plasmid expressing LexA-ER-VP16, S. Hanes (Wadsworth Center) for the plasmid containing the minimal GAL1 promoter with lexO sites, D. Pettigrew for generous advice and discussion and M. Bryk, S. Dent, W. Hörz and J. Mueller for helpful discussions and critical reading of an earlier version of this manuscript. The work was supported by a grant from the National Cancer Institute of the US National Institutes of Health to M.P.K. and in part by an Advanced Research Program award from the Texas Higher Education Coordinating Board.

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  1. Walter J Jessen

    Present address: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 7024, Cincinnati, Ohio, 45229-3039, USA

  2. Walter J Jessen and Scott A Hoose: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas, 77843-2128, USA

    Jessica A Kilgore & Michael P Kladde

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Correspondence to Michael P Kladde.

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Supplementary information

Supplementary Fig. 1

Chromatin structure and induction kinetics of PHO5 and PHO5HhaI promoters are indistinguishable. (PDF 354 kb)

Supplementary Fig. 2

Increased rate of disruption of UAS-adjacent nucleosome in an extended activation time course. (PDF 243 kb)

Supplementary Fig. 3

Pho4-dependent increases in M.HhaI accessibility in populations. (PDF 380 kb)

Supplementary Fig. 4

Associated non-histone factors do not protect against methylation. (PDF 270 kb)

Supplementary Fig. 5

M.HhaI lacks site preference for protein-free PHO5HhaI DNA. (PDF 191 kb)

Supplementary Table 1

Statistical significances of increased M.HhaI accessibility. (PDF 67 kb)

Supplementary Table 2

Additional oligonucleotides used in this study. (PDF 75 kb)

Supplementary Methods

Micrococcal nuclease analysis. (PDF 72 kb)

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Jessen, W., Hoose, S., Kilgore, J. et al. Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters. Nat Struct Mol Biol 13, 256–263 (2006). https://doi.org/10.1038/nsmb1062

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