Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini

Abstract

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or "bivalent domains," in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1-50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.

Fig. 1.

Analysis of somatic chromatin suggests long-range connectivity among modifications on histone H3. (A) Schematic of PTMs observed on the Glu-C generated N-terminal peptide of histone H3 [for the entire sequence of Tetrahymena H3.2, see supporting information (SI) Fig. 4]. Conventional mass spectrometry protocols often require digestion into much smaller pieces such that connectivity between modifications spaced by many amino acids as may occur in bivalent domains (open arrows on K4 and K27) is poorly understood. (B) Highly purified Tetrahymena MIC and MAC histones were resolved by SDS/PAGE and detected by Western blot analysis with antibodies recognizing monomethylated, dimethylated, or trimethylated species of H3K4. (C) Histones were purified and resolved as in B; however, Western blot analysis was performed with antibodies recognizing monomethylated, dimethylated, or trimethylated species of H3K27. A MIC-specific H3 isoform that is proteolytically processed endogenously is indicated by ∗. (D) MAC histones were acid-extracted and resolved by acid-urea gel to separate positively and negatively charged histone H3 species into six rungs representing H3 isoforms that range from relatively hypoacetylated (0) to hyperacetylated (5). Equivalent Ponceau staining was used as a loading control.

Fig. 2.

Schematic of the mass spectrometry approach. (A) PolyCAT A separation of intact histone H3. (B) FTMS mass measurements of the N-terminal H3 Glu-C peptide (H3_1–50). Shown are the +9 charge states of the species present in fraction 30 of group 5 (I) and the +10 charge states of fraction 67 of group 0 (II). (C) The resulting ETD/PTR zoom scan spectrum of the indicated species from groups 5 (I) and 0 (II) are shown. Singly charged c-type (containing the N terminus) and z-type fragment ions (containing the C terminus) are labeled in the spectra. Doubly charged fragment ions of type c and z are indicated by ∗. (D) Selected m/z range of the corresponding spectra displayed in C. Note the double ion series in CII. Two c₄ fragment ions are observed corresponding to two H3 species; one is unmodified and one is monomethylated at K4. This ion series is traceable up to the +2 fragment ion series as shown by the two c₈⁺² and c₂₄⁺² fragment ions. The H3 species in this spectrum that is not methylated at K4 affords a methylation on a different residue to satisfy the FTMS measurements. The second H3 species is methylated at K27 as two z₂₄⁺² fragment ions corresponding to K27 and differing by nominal 14 Da are observed. The spectra in CII and DII correspond to two species, one monomethylated on K4 and one monomethylated on K27. (E) Sequence of H3_1–50. Modified residues are indicated by blue flags for acetylation (Ac) and by yellow circles for methylation (M). Peptide backbone cleavages observed in the spectra are indicated. EI shows a species trimethylated on K4 and acetylated on K9, K14, K18, K23, and K27, as interpreted from CI and DI. EII shows two species, one monomethylated on K4 and one monomethylated on K27, as interpreted from CII and DII.

Fig. 3.

Summary of H3 species identified in the mass spectrometry analysis. Shown are the H3_1–50 modifications states identified in this study, categorized by fraction number. Species identified within the same spectrum are indicated with a left bracket, and the relative abundance of each species within this spectrum is indicated by the number of + symbols. The species with the most + symbols is most abundant, and the species with the least number of + symbols is of lowest abundance. Where no + symbol is noted next to a fraction number, only one species per spectrum was identified. The numbers on the right indicate grouping by acetylation occupancy.