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

Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A - Nat. Struct. Mol. Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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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Identification of amino acid mutations essential for viability under normal growth conditions. (a) Viability of histone alanine mutants. Strains expressing histones containing each single alanine mutation in the presence of wild-type histones (either HTA1 and HTB1, or HHT2 and HHF2) were plated with an initial OD600 of 0.5, followed by a four-fold serial dilution onto SC-Trp in the absence or presence of 5-FOA. * indicates that these mutants started to form revertants after 5 days of incubation at 30 °C; however, the appearance of the colonies of these mutants was repeatedly delayed and the number of colonies was extremely low. (b) Mapping of lethal residues on the nucleosome crystal structure as determined previously60. The locations of lethal residues are shown in red and numbers indicate the corresponding histone mutants shown in Figure 2a. Representation of nucleosome was generated using PyMOL. (c,d) Mapping of lethal residues in the surface of the yeast nucleosome on the crystal structure of the nucleosomes as determined previously (PDB 1ID3)60. H2A, H2B, H3 and H4 lethal residues are shown in orange, pink, blue and green, respectively. Red circles show H2A and H2B lethal residues. Representations of nucleosomes were generated using PyMOL (http://pymol.sourceforge.net/).
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Figure 3: Identification of amino acid mutations essential for viability under normal growth conditions. (a) Viability of histone alanine mutants. Strains expressing histones containing each single alanine mutation in the presence of wild-type histones (either HTA1 and HTB1, or HHT2 and HHF2) were plated with an initial OD600 of 0.5, followed by a four-fold serial dilution onto SC-Trp in the absence or presence of 5-FOA. * indicates that these mutants started to form revertants after 5 days of incubation at 30 °C; however, the appearance of the colonies of these mutants was repeatedly delayed and the number of colonies was extremely low. (b) Mapping of lethal residues on the nucleosome crystal structure as determined previously60. The locations of lethal residues are shown in red and numbers indicate the corresponding histone mutants shown in Figure 2a. Representation of nucleosome was generated using PyMOL. (c,d) Mapping of lethal residues in the surface of the yeast nucleosome on the crystal structure of the nucleosomes as determined previously (PDB 1ID3)60. H2A, H2B, H3 and H4 lethal residues are shown in orange, pink, blue and green, respectively. Red circles show H2A and H2B lethal residues. Representations of nucleosomes were generated using PyMOL (http://pymol.sourceforge.net/).

Mentions: Histone residues within nucleosomes are highly evolutionarily conserved. As a matter of fact, the amino acid residues in histones H2A and H2B are more than 70% conserved from yeast to humans, and in histones H3 and H4 more than 90% are conserved. When we decided to generate a complete alanine scanning collection of the histones, we considered its high evolutionary conservation and predicted that many of the mutants would be lethal. To our surprise, point mutations of less than 5% of the total residues of the four histones were lethal. Our comprehensive histone alanine scanning analysis identified only 18 residues within all of the core histones that were required for viability under normal growth conditions (Fig. 3a). All of the essential residues are found within the globular domains of histones (Fig. 3b). A few of these residues, marked by asterisk in Figure 3, are either slow growers or generate revertants as judged by growth after 5 d of slow or no growth. Although most of the post-translational modifications occur in histone tail domains, tail-less H3-H4, H2A and H2B histones are still capable of nucleosomal assembly in vitro. The histone H4 N-terminal tail is required for chromatin folding; however, it is dispensable for growth38. We have found that there are nine residues in histone H3 that are essential for viability: Tyr41, Leu48, Ile51, Gln55, Glu97, His113, Arg116, Thr118 and Asp123 (Fig. 3a). Five residues in histone H4 are also required for viability: Arg39, Arg40, Arg45, Tyr72 and Leu90. We have found that histone H2A and H2B together have only four residues required for yeast viability under normal growth conditions: three in H2A (Tyr58, Glu62 and Asp91) and one in H2B (Leu109) (Fig. 3a).


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

Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A - Nat. Struct. Mol. Biol. (2008)

Identification of amino acid mutations essential for viability under normal growth conditions. (a) Viability of histone alanine mutants. Strains expressing histones containing each single alanine mutation in the presence of wild-type histones (either HTA1 and HTB1, or HHT2 and HHF2) were plated with an initial OD600 of 0.5, followed by a four-fold serial dilution onto SC-Trp in the absence or presence of 5-FOA. * indicates that these mutants started to form revertants after 5 days of incubation at 30 °C; however, the appearance of the colonies of these mutants was repeatedly delayed and the number of colonies was extremely low. (b) Mapping of lethal residues on the nucleosome crystal structure as determined previously60. The locations of lethal residues are shown in red and numbers indicate the corresponding histone mutants shown in Figure 2a. Representation of nucleosome was generated using PyMOL. (c,d) Mapping of lethal residues in the surface of the yeast nucleosome on the crystal structure of the nucleosomes as determined previously (PDB 1ID3)60. H2A, H2B, H3 and H4 lethal residues are shown in orange, pink, blue and green, respectively. Red circles show H2A and H2B lethal residues. Representations of nucleosomes were generated using PyMOL (http://pymol.sourceforge.net/).
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Related In: Results  -  Collection

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Figure 3: Identification of amino acid mutations essential for viability under normal growth conditions. (a) Viability of histone alanine mutants. Strains expressing histones containing each single alanine mutation in the presence of wild-type histones (either HTA1 and HTB1, or HHT2 and HHF2) were plated with an initial OD600 of 0.5, followed by a four-fold serial dilution onto SC-Trp in the absence or presence of 5-FOA. * indicates that these mutants started to form revertants after 5 days of incubation at 30 °C; however, the appearance of the colonies of these mutants was repeatedly delayed and the number of colonies was extremely low. (b) Mapping of lethal residues on the nucleosome crystal structure as determined previously60. The locations of lethal residues are shown in red and numbers indicate the corresponding histone mutants shown in Figure 2a. Representation of nucleosome was generated using PyMOL. (c,d) Mapping of lethal residues in the surface of the yeast nucleosome on the crystal structure of the nucleosomes as determined previously (PDB 1ID3)60. H2A, H2B, H3 and H4 lethal residues are shown in orange, pink, blue and green, respectively. Red circles show H2A and H2B lethal residues. Representations of nucleosomes were generated using PyMOL (http://pymol.sourceforge.net/).
Mentions: Histone residues within nucleosomes are highly evolutionarily conserved. As a matter of fact, the amino acid residues in histones H2A and H2B are more than 70% conserved from yeast to humans, and in histones H3 and H4 more than 90% are conserved. When we decided to generate a complete alanine scanning collection of the histones, we considered its high evolutionary conservation and predicted that many of the mutants would be lethal. To our surprise, point mutations of less than 5% of the total residues of the four histones were lethal. Our comprehensive histone alanine scanning analysis identified only 18 residues within all of the core histones that were required for viability under normal growth conditions (Fig. 3a). All of the essential residues are found within the globular domains of histones (Fig. 3b). A few of these residues, marked by asterisk in Figure 3, are either slow growers or generate revertants as judged by growth after 5 d of slow or no growth. Although most of the post-translational modifications occur in histone tail domains, tail-less H3-H4, H2A and H2B histones are still capable of nucleosomal assembly in vitro. The histone H4 N-terminal tail is required for chromatin folding; however, it is dispensable for growth38. We have found that there are nine residues in histone H3 that are essential for viability: Tyr41, Leu48, Ile51, Gln55, Glu97, His113, Arg116, Thr118 and Asp123 (Fig. 3a). Five residues in histone H4 are also required for viability: Arg39, Arg40, Arg45, Tyr72 and Leu90. We have found that histone H2A and H2B together have only four residues required for yeast viability under normal growth conditions: three in H2A (Tyr58, Glu62 and Asp91) and one in H2B (Leu109) (Fig. 3a).

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus