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A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation

Nature Structural & Molecular Biology volume 15, pages 881–888 (2008)Cite this article

Abstract

Methylation of histone 3 lysine 4 (H3K4) by yeast Set1-COMPASS requires prior monoubiquitination of histone H2B. To define whether other residues within the histones are also required for H3K4 methylation, we systematically generated a complete library of the alanine substitutions of all of the residues of the four core histones in Saccharomyces cerevisiae. From this study we discovered that 18 residues within the four histones are essential for viability on complete growth media. We also identified several cis-regulatory residues on the histone H3 N-terminal tail, including histone H3 lysine 14 (H3K14), which are required for normal levels of H3K4 trimethylation. Several previously uncharacterized trans-regulatory residues on histones H2A and H2B form a patch on nucleosomes and are required for methylation mediated by COMPASS. This library will be a valuable tool for defining the role of histone residues in processes requiring chromatin.

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Figure 1: Schematic representation of the experimental procedure.
Figure 2: Key to the organization of the SHIMA library.
Figure 3: Identification of amino acid mutations essential for viability under normal growth conditions.
Figure 4: GPS analyses to defining amino acid residues of histone H3 and H4 required for proper H3K4 methylation.
Figure 5: GPS analyses in defining amino acid residues of histones H2A and H2B required for proper H3K4 methylation.
Figure 6: Identification of a nucleosomal patch regulating the H3K4 methylation pattern.

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Acknowledgements

We thank L. Shilatifard for editorial assistance and E. Smith for critical reading of the manuscript. We are grateful for M. Ruhlman and N. Dillon for large-scale preparation of polyacrylamide gels, and C. Wimberly and H. Strobietto for large-scale plasmid preparation and DNA sequencing. We are also grateful to B. Li from the Workman laboratory at the Stowers Institute for providing YBL574 and Y131 strains and shuffling plasmids. This work was supported by the US National Institute of Health grants 2R01CA89455 and 2R01GM069905 to A.S.

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  1. Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, 64110, Missouri, USA

    Shima Nakanishi, Brian W Sanderson, Kym M Delventhal, William D Bradford, Karen Staehling-Hampton & Ali Shilatifard

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Contributions

S.N. and A.S. carried out the research and wrote the manuscript; B.W.S. and K.M.D. performed site-directed mutagenesis of histones and validation of the generated vectors; W.D.B. performed validation of the transformed yeast library; K.S.-H. and A.S. provided support and advice.

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Correspondence to Ali Shilatifard.

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Nakanishi, S., Sanderson, B., Delventhal, K. et al. A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation. Nat Struct Mol Biol 15, 881–888 (2008). https://doi.org/10.1038/nsmb.1454

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