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| Status |
Public on Aug 02, 2007 |
| Title |
Genome-wide Replication-independent H3 Exchange Occurs Predominantly at Promoters and Implicates H3K56ac and Asf1 |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Genome binding/occupancy profiling by genome tiling array
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| Summary |
In yeast, histone H3/H4 exchange independent of replication is poorly understood. Here, we analyzed the deposition of histone H3 molecules, synthesized during G1, using a high-density microarray histone exchange assay. While we found that H3 exchange in coding regions requires high levels of transcription, promoters exchange H3 molecules in absence of transcription. In inactive promoters, H3 is deposited predominantly in well-positioned nucleosomes surrounding nucleosome free regions, indicating that some nucleosomes in promoters are dynamic. This could facilitate induction of repressed genes. Importantly, we show that histone H3 K56 acetylation, a replication-associated mark, is also present in replication-independent newly assembled nucleosomes and correlates perfectly with the deposition of new H3. Finally, we found that transcription-dependent incorporation of H3 at promoters is highly dependent on Asf1. Taken together our data underline the dynamic nature of replication-independent nucleosome assembly/disassembly, specify a link to transcription and implicate Asf1 and H3 K56 acetylation.
Keywords: ChIP-chip
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| Overall design |
To analyze histone H3 dynamics we used an experimental system based on the method developed to study chromatin reassembly at the PHO5 promoter upon repression (Schermer et al., 2005) and coupled it to high-resolution microarrays. In this system there are two different sources of histone H3 in the cell, the endogenous histone tagged with the Myc epitope and a galactose-inducible form fused to the Flag tag coexpressed with histone H4. In order to eliminate the contribution of DNA replication-dependant histone deposition, exponentially growing cells containing the double tag system are blocked in G1 with alpha-factor. After incubation with alpha-factor, cells are either fixed or induced to express Flag-H3 prior to formaldehyde treatment. Next, the level of Myc-H3, Flag-H3, and RNAP II are assayed either by standard ChIP-QPCR or by ChIP-chip using tiling path DNA microarrays (Guillemette et al., 2005; Pokholok et al., 2005).
The raw data were corrected (foreground-background) then normalized using the limma's loess function (Yang et al., 2002) in BioConductor (from the ArrayPipe Analysis Pipeline (Hokamp et al., 2004)) and replicates were combined using a weighted average method as described previously (Ren et al., 2000, Pokholok et al., 2005).
Histone H3 exchange in WT and in the asf1Δ mutant, RNAPII occupancy, and H3 K56 acetylation experiments were done in duplicate, the Asf1-dependence of H3 exchange was assayed in triplicate, and the MNase ChIP-chip experiment once. The combined datasets are available in the supplemental files of the original publication.
REFERENCES
Guillemette, B., Bataille, A. R., Gevry, N., Adam, M., Blanchette, M., Robert, F., and Gaudreau, L. (2005). Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol 3, e384.
Hokamp, K., Roche, F. M., Acab, M., Rousseau, M. E., Kuo, B., Goode, D., Aeschliman, D., Bryan, J., Babiuk, L. A., Hancock, R. E., and Brinkman, F. S. (2004). ArrayPipe: flexible processing pipeline for microarray data. Nucleic Acids Res 32, W457-459.
Pokholok, D. K., Harbison, C. T., Levine, S., Cole, M., Hannett, N. M., Lee, T. I., Bell, G. W., Walker, K., Rolfe, P. A., Herbolsheimer, E., et al. (2005). Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517-527.
Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306-2309.
Schermer, U. J., Korber, P., and Horz, W. (2005). Histones are incorporated in trans during reassembly of the yeast PHO5 promoter. Mol Cell 19, 279-285.
Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.
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| Contributor(s) |
Rufiange* A, Jacques* PE, Bhat W, Robert F, Nourani A |
| Citation(s) |
17679090 |
| Submission date |
Jun 26, 2007 |
| Last update date |
Feb 15, 2018 |
| Contact name |
François Robert |
| E-mail(s) |
Francois.Robert@ircm.qc.ca
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| URL |
http://www.ircm.qc.ca/microsites/francoisrobert/en/index.html
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| Organization name |
Institut de recherches cliniques de Montréal (IRCM)
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| Department |
Chromatin and Genomic Expression
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| Street address |
110, avenue des Pins Ouest
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| City |
Montréal |
| State/province |
Québec |
| ZIP/Postal code |
H2W 1R7 |
| Country |
Canada |
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| Platforms (3) |
| GPL3737 |
Agilent-012713 Yeast Whole Genome ChIP-on-chip Microarray (G4486A) |
| GPL4130 |
Agilent-014741 Yeast Whole Genome ChIP-on-Chip Microarray 244K (G4491A) |
| GPL4131 |
Agilent-014810 Yeast Whole Genome ChIP-on-Chip Microarray 4x44K (G4493A) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA101263 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE8299_RAW.tar |
997.6 Mb |
(http)(custom) |
TAR (of GPR, TIFF) |
| Processed data included within Sample table |
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