Identification of novel histone post-translational modifications by peptide mass fingerprinting

Abstract

The extent and pattern of histone post-translational modifications is a key determinant dictating the structure of chromatin. We employed mass spectrometry to map the post-translational modifications present on mammalian core histones. Using accurate peptide mass fingerprinting on proteolytic digests of purified histones, we identified more than 20 novel sites of histone modification. One newly identified site of methylation, histone H4 lysine 59, maps to the surface of the nucleosome in close proximity to the site of the only identified histone core modification, histone H3 lysine 79. Consistent with the role of histone H3 lysine 79 methylation in the formation of silent chromatin structure, histone H4 lysine 59 is essential for transcriptional silencing at the yeast silent mating loci and telomeres.

Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1

“…Only a slight decrement in affinity was observed, compared to the wild-type peptides (H3 and H4 scrambled in Table 1). However, when we tested additional methylated lysine residues (H4K59, H4K79 and H3K79) (Zhang et al, 2003), which are not embedded in basic sequence environments (pI o10), we found no binding of 3xMBT (Table 1, H3-and H4-C-terminal domain peptides). Thus, the binding of 3xMBT to mono-and dimethylated lysines is only seen using 'basic' peptides (pI>11, Table 1).…”

Widespread regulation of gene expression in the Drosophila genome by the histone acetyltransferase dTip60

“…The majority of these modifications are positioned at the N-terminal tails of histones, with the exception of ubiquitylation, which is found at the C-terminal part of H2A and H2B. However, it is becoming increasingly evident that histones can also carry posttranslational modifications in the core region (Zhang et al, 2003).…”

Chemical derivatization of histones for facilitated analysis by mass spectrometry

“…However, these techniques are time consuming and have certain limitations that do not enable complete characterization of the myriad of modifications present on histones. Mass spectrometry (MS) has complemented the biological work being conducted on histone PTMs, categorizing novel modifications on histones that were previously not detected by any other means [4][5][6][7][8] . Histone proteins pose a formidable challenge to the mass spectrometrist, as their sequences are decorated with an overwhelming number of arginine and lysine residues, especially on the N-termini where most PTMs are known to reside.…”

“…Enzymatic digestion with trypsin results in small peptides that are difficult to retain on RP-HPLC columns and analyzed by MS. On the other hand, proteolysis with enzymes that cleave after acidic residues (e.g., Glu-C) generates larger multiply charged peptides whose MS/MS spectra are difficult if not impossible to interpret. MS-related histone work has benefited from the use of limited trypsin or other enzyme digestion [4][5][6] ; however, these methods typically generate several truncated peptides containing the same modification sites, rendering quantification of histone PTMs cumbersome. More recently, the Arg-C digestion of histones has been performed to produce histone peptides that can be used for relative quantification purposes 8 .…”