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Automethylation of G9a and its implication in wider substrate specificity and HP1 binding.

Chin HG, Estève PO, Pradhan M, Benner J, Patnaik D, Carey MF, Pradhan S - Nucleic Acids Res. (2007)

Bottom Line: Automethylation of G9a did not impair or activate the enzymatic activity in vitro.In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1alpha and HP1gamma isoforms whereas the G9a mutant with K239A displayed poor co-localization.Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, 240 County Road, Ipswich, MA 01938-2723, USA.

ABSTRACT
Methylation of lysine residues on histones participates in transcriptional gene regulation. Lysine 9 methylation of histone H3 is a transcriptional repression signal, mediated by a family of SET domain containing AdoMet-dependent enzymes. G9a methyltransferase is a euchromatic histone H3 lysine 9 methyltransferase. Here, G9a is shown to methylate other cellular proteins, apart from histone H3, including automethylation of K239 residue. Automethylation of G9a did not impair or activate the enzymatic activity in vitro. The automethylation motif of G9a flanking target K239 (ARKT) has similarity with histone H3 lysine 9 regions (ARKS), and is identical to amino acids residues in EuHMT (ARKT) and mAM (ARKT). Under steady-state kinetic assay conditions, full-length G9a methylates peptides representing ARKS/T motif of H3, G9a, mAM and EuHMT efficiently. Automethylation of G9a at ARKT motif creates a binding site for HP1 class of protein and mutation of lysine in the motif impairs this binding. In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1alpha and HP1gamma isoforms whereas the G9a mutant with K239A displayed poor co-localization. Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein-protein interactions.

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Analysis of the post-translational modifications of G9a by mass spectroscopy. Three post-translationally modified peptides were observed from digestion and nano-ESI-MS/MS analysis. MS/MS Ion Trap spectra: The major ions are labeled with the corresponding m/z value, a or b or y ion fragments and neutral loss species where they could be assigned. The observed b and y ions are indicated by \ and / and where both were present. Any position that gave rise to a neutral loss species is indicated in bold. (A) Phosphoserine-containing peptide. The MS/MS spectrum of an ion with m/z value of 672.9, corresponding to a G9a tryptic peptide that eluted at 36.4 min. M S M/TG/AG/K Sp/P/P/S VQ/SL/A/M/R where Sp = phosphoserine (B) Dimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 842.8, corresponding to a G9a tryptic peptide that eluted at 31.2 min. IVLGH\ATKdS/FPS/S/P/S/K where Kd = dimethyllysine. (C) Trimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 976.8, corresponding to a G9a tryptic peptide that eluted at 31.1 min. Kt T M S K P S NG/Q/P P/I P E/K R/P P E/V Q/H F R where Kt = Trimethyllysine.
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Figure 3: Analysis of the post-translational modifications of G9a by mass spectroscopy. Three post-translationally modified peptides were observed from digestion and nano-ESI-MS/MS analysis. MS/MS Ion Trap spectra: The major ions are labeled with the corresponding m/z value, a or b or y ion fragments and neutral loss species where they could be assigned. The observed b and y ions are indicated by \ and / and where both were present. Any position that gave rise to a neutral loss species is indicated in bold. (A) Phosphoserine-containing peptide. The MS/MS spectrum of an ion with m/z value of 672.9, corresponding to a G9a tryptic peptide that eluted at 36.4 min. M S M/TG/AG/K Sp/P/P/S VQ/SL/A/M/R where Sp = phosphoserine (B) Dimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 842.8, corresponding to a G9a tryptic peptide that eluted at 31.2 min. IVLGH\ATKdS/FPS/S/P/S/K where Kd = dimethyllysine. (C) Trimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 976.8, corresponding to a G9a tryptic peptide that eluted at 31.1 min. Kt T M S K P S NG/Q/P P/I P E/K R/P P E/V Q/H F R where Kt = Trimethyllysine.

Mentions: To establish the authenticity of the methylated lysine residue of G9a, the tryptic fragments of the purified G9a were subjected to mass-spectroscopy analysis. Mascot, Spectrum Mill and X! Tandem (29) analysis identified the major protein present in the sample as the murine G9a methyltransferase. All three programs identified a serine (residue S194) as being in both a phosphorylated and unmodified state in multiple peptides. Figure 3A shows a spectrum obtained from a phosphorylated peptide. Mascot, X! Tandem and Spectrum Mill also identified a dimethyllysine at residue 167 (K167me2) only in the modified form (Figure 3B). Mascot and Spectrum Mill also identified a trimethyllysine at residue 239 (K239me3) only in the modified form (Figure 3C). The spectra of the peptide containing a mass 42 modification has a characteristic loss of mass 59 known to be associated with trimethyllysine (30) hence distinguishing it from an acetylation.Figure 3.


Automethylation of G9a and its implication in wider substrate specificity and HP1 binding.

Chin HG, Estève PO, Pradhan M, Benner J, Patnaik D, Carey MF, Pradhan S - Nucleic Acids Res. (2007)

Analysis of the post-translational modifications of G9a by mass spectroscopy. Three post-translationally modified peptides were observed from digestion and nano-ESI-MS/MS analysis. MS/MS Ion Trap spectra: The major ions are labeled with the corresponding m/z value, a or b or y ion fragments and neutral loss species where they could be assigned. The observed b and y ions are indicated by \ and / and where both were present. Any position that gave rise to a neutral loss species is indicated in bold. (A) Phosphoserine-containing peptide. The MS/MS spectrum of an ion with m/z value of 672.9, corresponding to a G9a tryptic peptide that eluted at 36.4 min. M S M/TG/AG/K Sp/P/P/S VQ/SL/A/M/R where Sp = phosphoserine (B) Dimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 842.8, corresponding to a G9a tryptic peptide that eluted at 31.2 min. IVLGH\ATKdS/FPS/S/P/S/K where Kd = dimethyllysine. (C) Trimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 976.8, corresponding to a G9a tryptic peptide that eluted at 31.1 min. Kt T M S K P S NG/Q/P P/I P E/K R/P P E/V Q/H F R where Kt = Trimethyllysine.
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Figure 3: Analysis of the post-translational modifications of G9a by mass spectroscopy. Three post-translationally modified peptides were observed from digestion and nano-ESI-MS/MS analysis. MS/MS Ion Trap spectra: The major ions are labeled with the corresponding m/z value, a or b or y ion fragments and neutral loss species where they could be assigned. The observed b and y ions are indicated by \ and / and where both were present. Any position that gave rise to a neutral loss species is indicated in bold. (A) Phosphoserine-containing peptide. The MS/MS spectrum of an ion with m/z value of 672.9, corresponding to a G9a tryptic peptide that eluted at 36.4 min. M S M/TG/AG/K Sp/P/P/S VQ/SL/A/M/R where Sp = phosphoserine (B) Dimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 842.8, corresponding to a G9a tryptic peptide that eluted at 31.2 min. IVLGH\ATKdS/FPS/S/P/S/K where Kd = dimethyllysine. (C) Trimethyllysine-containing peptide. The MS/MS spectrum of an ion with m/z value of 976.8, corresponding to a G9a tryptic peptide that eluted at 31.1 min. Kt T M S K P S NG/Q/P P/I P E/K R/P P E/V Q/H F R where Kt = Trimethyllysine.
Mentions: To establish the authenticity of the methylated lysine residue of G9a, the tryptic fragments of the purified G9a were subjected to mass-spectroscopy analysis. Mascot, Spectrum Mill and X! Tandem (29) analysis identified the major protein present in the sample as the murine G9a methyltransferase. All three programs identified a serine (residue S194) as being in both a phosphorylated and unmodified state in multiple peptides. Figure 3A shows a spectrum obtained from a phosphorylated peptide. Mascot, X! Tandem and Spectrum Mill also identified a dimethyllysine at residue 167 (K167me2) only in the modified form (Figure 3B). Mascot and Spectrum Mill also identified a trimethyllysine at residue 239 (K239me3) only in the modified form (Figure 3C). The spectra of the peptide containing a mass 42 modification has a characteristic loss of mass 59 known to be associated with trimethyllysine (30) hence distinguishing it from an acetylation.Figure 3.

Bottom Line: Automethylation of G9a did not impair or activate the enzymatic activity in vitro.In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1alpha and HP1gamma isoforms whereas the G9a mutant with K239A displayed poor co-localization.Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, 240 County Road, Ipswich, MA 01938-2723, USA.

ABSTRACT
Methylation of lysine residues on histones participates in transcriptional gene regulation. Lysine 9 methylation of histone H3 is a transcriptional repression signal, mediated by a family of SET domain containing AdoMet-dependent enzymes. G9a methyltransferase is a euchromatic histone H3 lysine 9 methyltransferase. Here, G9a is shown to methylate other cellular proteins, apart from histone H3, including automethylation of K239 residue. Automethylation of G9a did not impair or activate the enzymatic activity in vitro. The automethylation motif of G9a flanking target K239 (ARKT) has similarity with histone H3 lysine 9 regions (ARKS), and is identical to amino acids residues in EuHMT (ARKT) and mAM (ARKT). Under steady-state kinetic assay conditions, full-length G9a methylates peptides representing ARKS/T motif of H3, G9a, mAM and EuHMT efficiently. Automethylation of G9a at ARKT motif creates a binding site for HP1 class of protein and mutation of lysine in the motif impairs this binding. In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1alpha and HP1gamma isoforms whereas the G9a mutant with K239A displayed poor co-localization. Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein-protein interactions.

Show MeSH
Related in: MedlinePlus