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Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1.

Williamson K, Schneider V, Jordan RA, Mueller JE, Henderson Pozzi M, Bryk M - PLoS ONE (2013)

Bottom Line: The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1.Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity.Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

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Quantitative Western blots measuring steady-state levels of K4-methylated histone H3 in cells expressing wild-type and mutant alleles of SET1.Representative Western blotting experiments are shown for cells carrying wild-type SET1 at its endogenous location, set1Δ and set1 amino acid substitution alleles. Dilutions of protein extracts (µg loaded indicated below the lower panel) from wild-type (WT), set1Δ, and Set1 mutant cells were analyzed by Western blotting with specific antibodies to measure the in vivo steady-state levels of K4-monomethylated H3 (α-mono), K4-dimethylated H3 (α-di), and K4-trimethylated H3 (α-tri). The level of histone H3 (α-H3) or Pgk1 (α-Pgk1) protein was used to normalize the amount of protein loaded in each lane. The average level of normalized K4-methylated H3 detected in Set1 mutant extracts relative to wild-type extracts is shown below each blot (n = 2). See Table 1 for the normalized average +/− range for each mutant.
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pone-0057974-g003: Quantitative Western blots measuring steady-state levels of K4-methylated histone H3 in cells expressing wild-type and mutant alleles of SET1.Representative Western blotting experiments are shown for cells carrying wild-type SET1 at its endogenous location, set1Δ and set1 amino acid substitution alleles. Dilutions of protein extracts (µg loaded indicated below the lower panel) from wild-type (WT), set1Δ, and Set1 mutant cells were analyzed by Western blotting with specific antibodies to measure the in vivo steady-state levels of K4-monomethylated H3 (α-mono), K4-dimethylated H3 (α-di), and K4-trimethylated H3 (α-tri). The level of histone H3 (α-H3) or Pgk1 (α-Pgk1) protein was used to normalize the amount of protein loaded in each lane. The average level of normalized K4-methylated H3 detected in Set1 mutant extracts relative to wild-type extracts is shown below each blot (n = 2). See Table 1 for the normalized average +/− range for each mutant.

Mentions: Average levels +/− range of H3K4me1, H3K4me2 and H3K4me3 measured in whole cell extracts from Set1 mutants by quantitative western blotting (n = 2); values are normalized to the levels measured in whole cell extracts from a wild type Set1+ strain. No value for +/− range is given if the two measurements were identical. See Figure 3 and text for details.


Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1.

Williamson K, Schneider V, Jordan RA, Mueller JE, Henderson Pozzi M, Bryk M - PLoS ONE (2013)

Quantitative Western blots measuring steady-state levels of K4-methylated histone H3 in cells expressing wild-type and mutant alleles of SET1.Representative Western blotting experiments are shown for cells carrying wild-type SET1 at its endogenous location, set1Δ and set1 amino acid substitution alleles. Dilutions of protein extracts (µg loaded indicated below the lower panel) from wild-type (WT), set1Δ, and Set1 mutant cells were analyzed by Western blotting with specific antibodies to measure the in vivo steady-state levels of K4-monomethylated H3 (α-mono), K4-dimethylated H3 (α-di), and K4-trimethylated H3 (α-tri). The level of histone H3 (α-H3) or Pgk1 (α-Pgk1) protein was used to normalize the amount of protein loaded in each lane. The average level of normalized K4-methylated H3 detected in Set1 mutant extracts relative to wild-type extracts is shown below each blot (n = 2). See Table 1 for the normalized average +/− range for each mutant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585878&req=5

pone-0057974-g003: Quantitative Western blots measuring steady-state levels of K4-methylated histone H3 in cells expressing wild-type and mutant alleles of SET1.Representative Western blotting experiments are shown for cells carrying wild-type SET1 at its endogenous location, set1Δ and set1 amino acid substitution alleles. Dilutions of protein extracts (µg loaded indicated below the lower panel) from wild-type (WT), set1Δ, and Set1 mutant cells were analyzed by Western blotting with specific antibodies to measure the in vivo steady-state levels of K4-monomethylated H3 (α-mono), K4-dimethylated H3 (α-di), and K4-trimethylated H3 (α-tri). The level of histone H3 (α-H3) or Pgk1 (α-Pgk1) protein was used to normalize the amount of protein loaded in each lane. The average level of normalized K4-methylated H3 detected in Set1 mutant extracts relative to wild-type extracts is shown below each blot (n = 2). See Table 1 for the normalized average +/− range for each mutant.
Mentions: Average levels +/− range of H3K4me1, H3K4me2 and H3K4me3 measured in whole cell extracts from Set1 mutants by quantitative western blotting (n = 2); values are normalized to the levels measured in whole cell extracts from a wild type Set1+ strain. No value for +/− range is given if the two measurements were identical. See Figure 3 and text for details.

Bottom Line: The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1.Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity.Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.

Show MeSH
Related in: MedlinePlus