Symmetrical modification within a nucleosome is not required globally for histone lysine methylation

Abstract

Two copies of each core histone exist in every nucleosome; however, it is not known whether both histones within a nucleosome are required to be symmetrically methylated at the same lysine residues. We report that for most lysine methylation states, wild-type histones paired with mutant, unmethylatable histones in mononucleosomes have comparable methylation levels to bulk histones. Our results indicate that symmetrical histone methylation is not required on a global scale. However, wild-type H4 histones paired with unmethylatable H4K20R histones showed reduced levels of H4K20me2 and H4K20me3, suggesting that some fractions of these modifications might exist symmetrically, and enzymes mediating these modifications might, to some extent, favour nucleosome substrates with premethylated H4K20.

Figure 1

Experimental scheme. HeLa cells labelled with K8 ([¹³C₆, ¹⁵N₂] heavy isotope-labelled L-lysine) were cultured in K8 medium for 1 month. If any methylation is required to exist in a symmetrical manner, unmethylatable histones should have a negative impact on the methylation status of its partner histone (Scenario A). Asterisk: The expected K8/K0 ratio for scenario A is approximately 10, because the labelling efficiency of the HeLa cells used in this study is approximately 90% (supplementary Fig S4 online). PAGE, polyacrylamide gel electrophoresis.

Figure 2

Symmetrical methylation is not required for H3K9. (A) Table summarizing the quantified peptides of methylated H3K9. (B) Mass spectra for the quantified peptides, including the backbone peptide and peptides from H3K9me1, H3K9me2 and H3K9me3. AMU, atomic mass unit; pro, propionylation.

Figure 3

Symmetrical methylation is not required for H3K27. (A) Table summarizing the quantified peptides of methylated H3K27. (B) Mass spectra for the quantified peptides, including the backbone peptide and peptides from H3K27me1, H3K27me2 and H3K27me3. AMU, atomic mass unit; pro, propionylation.

Figure 4

Symmetrical methylation is not required for H3K79. (A) Table summarizing the quantified peptides of methylated H3K79. (B) Mass spectra for the quantified peptides including the backbone peptide and peptides from H3K79me1, H3K79me2 and H3K79me3. AMU, atomic mass unit; oxi, oxidation.

Figure 5

Symmetrical methylation is not required for H4K20. (A) Table summarizing the quantified peptides of methylated H3K79. (B) Mass spectra for the quantified peptides including the backbone peptide and peptides from H4K20me1 and H4K20me2. (C) Western blot analysis quantifying relative abundance of H4K20me3. AMU, atomic mass unit; pro, propionylation.