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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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GPS analyses in defining amino acid residues of histones H2A and H2B required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H2A mutants, d-f from H2B mutants) were subjected to SDS-PAGE and western blot analysis, and tested for the presence of dimethylated lysine 4 of histone H3 (H3K4Me2) and trimethylated lysine 4 of histone H3 (H3K4Me3) as indicated under panel. As the loading control, an antibody to histone H3 was also used. Red arrows indicate the possible hits, and blue asterisks (*) indicate the positions of empty wells (lethal residues), green arrows (C8) indicate the positions where the wild-type was used in place of a lethal residue-bearing strain as a positive control for growth in these regions. For the key to the organization of the strains within each plate, please see Figure 2. Positions I, II, III and IV indicate histone H2A Glu65, Leu66, Asn69 and Asp73, respectively. Positions V, VI and VII indicate histone H2B Lys123, Arg119 and His112, respectively. P2 indicates plate 2.
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Figure 5: GPS analyses in defining amino acid residues of histones H2A and H2B required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H2A mutants, d-f from H2B mutants) were subjected to SDS-PAGE and western blot analysis, and tested for the presence of dimethylated lysine 4 of histone H3 (H3K4Me2) and trimethylated lysine 4 of histone H3 (H3K4Me3) as indicated under panel. As the loading control, an antibody to histone H3 was also used. Red arrows indicate the possible hits, and blue asterisks (*) indicate the positions of empty wells (lethal residues), green arrows (C8) indicate the positions where the wild-type was used in place of a lethal residue-bearing strain as a positive control for growth in these regions. For the key to the organization of the strains within each plate, please see Figure 2. Positions I, II, III and IV indicate histone H2A Glu65, Leu66, Asn69 and Asp73, respectively. Positions V, VI and VII indicate histone H2B Lys123, Arg119 and His112, respectively. P2 indicates plate 2.

Mentions: In addition to cis-regulatory residues identified within histone H3, we have also identified several trans-regulatory residues that specifically regulate H3K4 di- and trimethylation (Figs. 5 and 6). These include four residues within histone H2A—Glu65, Leu66, Asn69 and Asp73 (indicated on Fig. 5a,b as I, II, III and IV, respectively)—and three residues within histone H2B—Lys123, Arg119 and His112 (indicated on Fig. 5e,f as V, VI and VII, respectively). When we localized these residues on the crystal structure of the nucleosomes, they formed a patch near Lys123 of H2B, the site of monoubiquitination required for H3K4 methylation by COMPASS (Fig. 6c). We tested whether the mutation of residues within this patch could alter H2B monoubiquitination levels and found that only mutations in H2BH112 and H2AL66 result in a defect in H2B monoubiquitination (Fig. 6d). The other residues within the patch, H2AN69, H2AD73 and H2AE65, regulate COMPASS’s activity independently of H2B monoubiquitination. We recently found that the Cps35 subunit of COMPASS interacts with chromatin in a monoubiquitination-dependent manner53 and that Cps35 is required for H3K4 methylation by COMPASS. Indeed, Cps35 loss phenocopies either Rad6 and/or H2BK123R mutations in regard to H3K4 di- and trimethylation. Conceivably, the residues within this patch that still have high levels of H2B monoubiquitination could be a part of a binding surface for proteins that recognize monoubiquitinated H2B in its nucleosomal context, or other factors, such as the Paf1 complex, that are also required for the association of COMPASS with transcribing polymerase.


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)

GPS analyses in defining amino acid residues of histones H2A and H2B required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H2A mutants, d-f from H2B mutants) were subjected to SDS-PAGE and western blot analysis, and tested for the presence of dimethylated lysine 4 of histone H3 (H3K4Me2) and trimethylated lysine 4 of histone H3 (H3K4Me3) as indicated under panel. As the loading control, an antibody to histone H3 was also used. Red arrows indicate the possible hits, and blue asterisks (*) indicate the positions of empty wells (lethal residues), green arrows (C8) indicate the positions where the wild-type was used in place of a lethal residue-bearing strain as a positive control for growth in these regions. For the key to the organization of the strains within each plate, please see Figure 2. Positions I, II, III and IV indicate histone H2A Glu65, Leu66, Asn69 and Asp73, respectively. Positions V, VI and VII indicate histone H2B Lys123, Arg119 and His112, respectively. P2 indicates plate 2.
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Related In: Results  -  Collection

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Figure 5: GPS analyses in defining amino acid residues of histones H2A and H2B required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H2A mutants, d-f from H2B mutants) were subjected to SDS-PAGE and western blot analysis, and tested for the presence of dimethylated lysine 4 of histone H3 (H3K4Me2) and trimethylated lysine 4 of histone H3 (H3K4Me3) as indicated under panel. As the loading control, an antibody to histone H3 was also used. Red arrows indicate the possible hits, and blue asterisks (*) indicate the positions of empty wells (lethal residues), green arrows (C8) indicate the positions where the wild-type was used in place of a lethal residue-bearing strain as a positive control for growth in these regions. For the key to the organization of the strains within each plate, please see Figure 2. Positions I, II, III and IV indicate histone H2A Glu65, Leu66, Asn69 and Asp73, respectively. Positions V, VI and VII indicate histone H2B Lys123, Arg119 and His112, respectively. P2 indicates plate 2.
Mentions: In addition to cis-regulatory residues identified within histone H3, we have also identified several trans-regulatory residues that specifically regulate H3K4 di- and trimethylation (Figs. 5 and 6). These include four residues within histone H2A—Glu65, Leu66, Asn69 and Asp73 (indicated on Fig. 5a,b as I, II, III and IV, respectively)—and three residues within histone H2B—Lys123, Arg119 and His112 (indicated on Fig. 5e,f as V, VI and VII, respectively). When we localized these residues on the crystal structure of the nucleosomes, they formed a patch near Lys123 of H2B, the site of monoubiquitination required for H3K4 methylation by COMPASS (Fig. 6c). We tested whether the mutation of residues within this patch could alter H2B monoubiquitination levels and found that only mutations in H2BH112 and H2AL66 result in a defect in H2B monoubiquitination (Fig. 6d). The other residues within the patch, H2AN69, H2AD73 and H2AE65, regulate COMPASS’s activity independently of H2B monoubiquitination. We recently found that the Cps35 subunit of COMPASS interacts with chromatin in a monoubiquitination-dependent manner53 and that Cps35 is required for H3K4 methylation by COMPASS. Indeed, Cps35 loss phenocopies either Rad6 and/or H2BK123R mutations in regard to H3K4 di- and trimethylation. Conceivably, the residues within this patch that still have high levels of H2B monoubiquitination could be a part of a binding surface for proteins that recognize monoubiquitinated H2B in its nucleosomal context, or other factors, such as the Paf1 complex, that are also required for the association of COMPASS with transcribing polymerase.

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