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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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Automethylated G9a binds to HP1 family of proteins. (A) GST pull-down assay with G9afl (automethylated) or G9amut containing K239A (unmethylated) enzyme. The top panel shows ponceau stain of the western blotted membrane demonstrating similar loading of GST-HP1α, GST-HP1β and GST-HP1γ. The blot was probed with anti-G9a as indicated. The molecular weight markers are on the left. Immunofluorescence detection of co-localization events are as follows (B) GFP-G9a and anti-HP1α; (C) GFP-G9amut and anti-HP1α; (D) GFP-G9a and anti-HP1γ; (E) GFP-G9amut and anti-HP1γ. Both HP1α and HP1γ are shown in red.
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Figure 6: Automethylated G9a binds to HP1 family of proteins. (A) GST pull-down assay with G9afl (automethylated) or G9amut containing K239A (unmethylated) enzyme. The top panel shows ponceau stain of the western blotted membrane demonstrating similar loading of GST-HP1α, GST-HP1β and GST-HP1γ. The blot was probed with anti-G9a as indicated. The molecular weight markers are on the left. Immunofluorescence detection of co-localization events are as follows (B) GFP-G9a and anti-HP1α; (C) GFP-G9amut and anti-HP1α; (D) GFP-G9a and anti-HP1γ; (E) GFP-G9amut and anti-HP1γ. Both HP1α and HP1γ are shown in red.

Mentions: Since G9a automethylates K239, it creates a motif very similar to methylated H3 tail (G9a: ARKme3T; H3: ARKme3S). The similarity of these amino acids residues between G9a and H3 led us to investigate if G9aK239me3 would act like H3K9me3 and bind to HP1. To examine the HP1 interaction with G9a, we performed GST pull-down assay with immobilized GST-HP1α, GST-HP1β and GST-HP1γ. A fixed amount of G9a wild type or G9amut was added to the beads and incubated for a fixed time followed by three washes to remove unbound enzymes. The bound proteins were separated on a SDS/PAGE and western blotted with anti-G9a antibody. Indeed, all the three isoforms of HP1 were able to bind strongly to the wild-type G9a that predominately contained K239me3, as compared to the G9amut (Figure 6A). This suggests that the K239 trimethylation creates an anchoring site for HP1α, HP1β and HP1γ, similar to H3K9me3-HP1 binding.


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)

Automethylated G9a binds to HP1 family of proteins. (A) GST pull-down assay with G9afl (automethylated) or G9amut containing K239A (unmethylated) enzyme. The top panel shows ponceau stain of the western blotted membrane demonstrating similar loading of GST-HP1α, GST-HP1β and GST-HP1γ. The blot was probed with anti-G9a as indicated. The molecular weight markers are on the left. Immunofluorescence detection of co-localization events are as follows (B) GFP-G9a and anti-HP1α; (C) GFP-G9amut and anti-HP1α; (D) GFP-G9a and anti-HP1γ; (E) GFP-G9amut and anti-HP1γ. Both HP1α and HP1γ are shown in red.
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Figure 6: Automethylated G9a binds to HP1 family of proteins. (A) GST pull-down assay with G9afl (automethylated) or G9amut containing K239A (unmethylated) enzyme. The top panel shows ponceau stain of the western blotted membrane demonstrating similar loading of GST-HP1α, GST-HP1β and GST-HP1γ. The blot was probed with anti-G9a as indicated. The molecular weight markers are on the left. Immunofluorescence detection of co-localization events are as follows (B) GFP-G9a and anti-HP1α; (C) GFP-G9amut and anti-HP1α; (D) GFP-G9a and anti-HP1γ; (E) GFP-G9amut and anti-HP1γ. Both HP1α and HP1γ are shown in red.
Mentions: Since G9a automethylates K239, it creates a motif very similar to methylated H3 tail (G9a: ARKme3T; H3: ARKme3S). The similarity of these amino acids residues between G9a and H3 led us to investigate if G9aK239me3 would act like H3K9me3 and bind to HP1. To examine the HP1 interaction with G9a, we performed GST pull-down assay with immobilized GST-HP1α, GST-HP1β and GST-HP1γ. A fixed amount of G9a wild type or G9amut was added to the beads and incubated for a fixed time followed by three washes to remove unbound enzymes. The bound proteins were separated on a SDS/PAGE and western blotted with anti-G9a antibody. Indeed, all the three isoforms of HP1 were able to bind strongly to the wild-type G9a that predominately contained K239me3, as compared to the G9amut (Figure 6A). This suggests that the K239 trimethylation creates an anchoring site for HP1α, HP1β and HP1γ, similar to H3K9me3-HP1 binding.

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