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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 to defining amino acid residues of histone H3 and H4 required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H3 mutants, d-f from H4 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 below 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). For the key to the organization of the strains within each plate, see Figure 2. Positions I, II, III, IV and V indicate histone H3 Arg2, Lys4, Gln5, Thr3 and Lys14, respectively. P2 indicates plate 2.
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Figure 4: GPS analyses to defining amino acid residues of histone H3 and H4 required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H3 mutants, d-f from H4 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 below 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). For the key to the organization of the strains within each plate, see Figure 2. Positions I, II, III, IV and V indicate histone H3 Arg2, Lys4, Gln5, Thr3 and Lys14, respectively. P2 indicates plate 2.

Mentions: So far, we know that two identified residues (H2BK123 and H3R2) have important roles in proper methylation by COMPASS. Therefore, we wanted to determine whether any other residues within histones are required for proper H3K4 methylation. We performed a GPS analysis of the extracts from the entire histone-mutant collection. Our analysis has resulted in the identification of several previously uncharacterized key regulatory residues within the histones required for normal levels of H3K4 methylation (Figs. 4-6). The key for the position of the mutants is shown in Figure 2. First and foremost, we have identified a few consistently reproducible cis-regulatory residues within histone H3 regulating H3K4 trimethylation (Fig. 4). These include Arg2, Lys4, Gln5, Thr3 and Lys14 of H3, indicated on Figure 4a,b as residues I, II, III, IV and V, respectively. Of these, Lys4 mutated to alanine can no longer be methylated. Arg2 has already been shown to be required for substrate recognition by COMPASS33,34. Thr3 could be phosphorylated, and its phosphorylation could regulate COMPASS’s activity; however, it is also possible that H3T3A mutation may result in epitope masking and therefore loss of immunoreactivity by the H3K4 polyclonal antibodies. Further studies should clarify these possibilities. Mutations in H3Q5 also result in the loss of the mono-, di- and trimethylation of H3K4. As many of these residues lay near the H3K4 site, their mutation could result in a defect in epitope recognition by the antibodies or defect in the substrate recognition by Set1-COMPASS.


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 to defining amino acid residues of histone H3 and H4 required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H3 mutants, d-f from H4 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 below 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). For the key to the organization of the strains within each plate, see Figure 2. Positions I, II, III, IV and V indicate histone H3 Arg2, Lys4, Gln5, Thr3 and Lys14, respectively. P2 indicates plate 2.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: GPS analyses to defining amino acid residues of histone H3 and H4 required for proper H3K4 methylation. Cell extracts prepared from the entire histone-mutant collection (a-c from H3 mutants, d-f from H4 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 below 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). For the key to the organization of the strains within each plate, see Figure 2. Positions I, II, III, IV and V indicate histone H3 Arg2, Lys4, Gln5, Thr3 and Lys14, respectively. P2 indicates plate 2.
Mentions: So far, we know that two identified residues (H2BK123 and H3R2) have important roles in proper methylation by COMPASS. Therefore, we wanted to determine whether any other residues within histones are required for proper H3K4 methylation. We performed a GPS analysis of the extracts from the entire histone-mutant collection. Our analysis has resulted in the identification of several previously uncharacterized key regulatory residues within the histones required for normal levels of H3K4 methylation (Figs. 4-6). The key for the position of the mutants is shown in Figure 2. First and foremost, we have identified a few consistently reproducible cis-regulatory residues within histone H3 regulating H3K4 trimethylation (Fig. 4). These include Arg2, Lys4, Gln5, Thr3 and Lys14 of H3, indicated on Figure 4a,b as residues I, II, III, IV and V, respectively. Of these, Lys4 mutated to alanine can no longer be methylated. Arg2 has already been shown to be required for substrate recognition by COMPASS33,34. Thr3 could be phosphorylated, and its phosphorylation could regulate COMPASS’s activity; however, it is also possible that H3T3A mutation may result in epitope masking and therefore loss of immunoreactivity by the H3K4 polyclonal antibodies. Further studies should clarify these possibilities. Mutations in H3Q5 also result in the loss of the mono-, di- and trimethylation of H3K4. As many of these residues lay near the H3K4 site, their mutation could result in a defect in epitope recognition by the antibodies or defect in the substrate recognition by Set1-COMPASS.

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