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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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Multiple substrate specificity of recombinant G9a. HCT116 cell extracts were incubated with recombinant G9a, heat killed G9a and tritiated AdoMet as indicated with a plus symbol. The radiolabeled protein mixture was separated on denaturing PAGE, stained and fluorographed. The Coomassie-stained gel is shown at the left panel and the radioactive proteins are seen as specific dark bands at the right panel. The apparent position of G9a and H3 are shown at the right. The molecular weight markers are on the left.
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Figure 1: Multiple substrate specificity of recombinant G9a. HCT116 cell extracts were incubated with recombinant G9a, heat killed G9a and tritiated AdoMet as indicated with a plus symbol. The radiolabeled protein mixture was separated on denaturing PAGE, stained and fluorographed. The Coomassie-stained gel is shown at the left panel and the radioactive proteins are seen as specific dark bands at the right panel. The apparent position of G9a and H3 are shown at the right. The molecular weight markers are on the left.

Mentions: Previously, we have studied the substrate specificity and steady-state kinetic properties of the murine G9a in detail. The enzyme is capable of mono-, di- and trimethylation of the target lysine residues on the amino terminal tail of histone H3 (22). Subsequent in vitro biochemical studies suggested that the substrate recognition and sequence specificity of G9a involve seven amino acids (TARKSTG) of the histone H3 tail and that K9 methylation is impaired by S10 or T11 phosphorylation (28). In the mammalian proteome similar motifs are expected to be found in protein other than histone H3. To determine if the G9a methyltransferase is capable of methylating other cellular proteins, we incubated purified recombinant G9a enzyme with HCT116 plus tritiated AdoMet co-factor. The radioactive protein samples were separated on a SDS gel and fluorographed to visualize G9a-mediated protein methylation. Surprisingly, several prominent radioactive protein bands were visible in autoradiography after overnight exposure of the film (Figure 1). The apparent molecular weights of these bands were between 7 and 175 kDa (Figure 1, right panel, lane 1). Histone H3 molecules were radioactively labeled and visible at 17 kDa region as expected. Experiments with heat killed recombinant G9a in the reaction mixture or without additional G9a enzyme did not display strong radioactive bands (Figure 1, right panel, lanes 2 and 3). The minor bands observed in the presence of heat killed G9a and extract alone suggests the cell extracts contain other protein methyltransferases. Several signals on the autoradiograph are specific to recombinant G9a-mediated tritiated methyl group incorporation. Similar autoradiography profile was observed with HCT116 DNMT1 , mouse 3T3 and Hela cell extracts (data not shown), suggesting G9a is capable of methylating proteins other than histone H3.Figure 1.


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)

Multiple substrate specificity of recombinant G9a. HCT116 cell extracts were incubated with recombinant G9a, heat killed G9a and tritiated AdoMet as indicated with a plus symbol. The radiolabeled protein mixture was separated on denaturing PAGE, stained and fluorographed. The Coomassie-stained gel is shown at the left panel and the radioactive proteins are seen as specific dark bands at the right panel. The apparent position of G9a and H3 are shown at the right. The molecular weight markers are on the left.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175347&req=5

Figure 1: Multiple substrate specificity of recombinant G9a. HCT116 cell extracts were incubated with recombinant G9a, heat killed G9a and tritiated AdoMet as indicated with a plus symbol. The radiolabeled protein mixture was separated on denaturing PAGE, stained and fluorographed. The Coomassie-stained gel is shown at the left panel and the radioactive proteins are seen as specific dark bands at the right panel. The apparent position of G9a and H3 are shown at the right. The molecular weight markers are on the left.
Mentions: Previously, we have studied the substrate specificity and steady-state kinetic properties of the murine G9a in detail. The enzyme is capable of mono-, di- and trimethylation of the target lysine residues on the amino terminal tail of histone H3 (22). Subsequent in vitro biochemical studies suggested that the substrate recognition and sequence specificity of G9a involve seven amino acids (TARKSTG) of the histone H3 tail and that K9 methylation is impaired by S10 or T11 phosphorylation (28). In the mammalian proteome similar motifs are expected to be found in protein other than histone H3. To determine if the G9a methyltransferase is capable of methylating other cellular proteins, we incubated purified recombinant G9a enzyme with HCT116 plus tritiated AdoMet co-factor. The radioactive protein samples were separated on a SDS gel and fluorographed to visualize G9a-mediated protein methylation. Surprisingly, several prominent radioactive protein bands were visible in autoradiography after overnight exposure of the film (Figure 1). The apparent molecular weights of these bands were between 7 and 175 kDa (Figure 1, right panel, lane 1). Histone H3 molecules were radioactively labeled and visible at 17 kDa region as expected. Experiments with heat killed recombinant G9a in the reaction mixture or without additional G9a enzyme did not display strong radioactive bands (Figure 1, right panel, lanes 2 and 3). The minor bands observed in the presence of heat killed G9a and extract alone suggests the cell extracts contain other protein methyltransferases. Several signals on the autoradiograph are specific to recombinant G9a-mediated tritiated methyl group incorporation. Similar autoradiography profile was observed with HCT116 DNMT1 , mouse 3T3 and Hela cell extracts (data not shown), suggesting G9a is capable of methylating proteins other than histone H3.Figure 1.

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