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. 2005 Jun 6;33(10):3211-23.
doi: 10.1093/nar/gki635. Print 2005.

Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase

Affiliations

Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase

Pierre-Olivier Estève et al. Nucleic Acids Res. .

Abstract

Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 and the second between amino acids 394 and 401 of murine G9a. Our data show that both long and short G9a isoforms were capable of entering the nucleus to methylate chromatin. Full-length or N-terminus-deleted G9a isoforms were also catalytically active enzymes that methylated recombinant H3 or synthetic peptides representing the N-terminus tail of H3. In vitro methylation reactions using N-terminus tail peptides resulted in tri-methylation of K9 that remained processive, even in G9a enzymes that lacked an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di- and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa cells resulted in reduction of global H3-K9 di- and tri-methylation. A recombinant deletion mutant enzyme fused with maltose-binding protein (MBP-G9aDelta634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9aDelta634 for various substrates was approximately 3-fold less compared with G9aFL, while their Michaelis constants (K(m)) for recombinant H3 were similar. The K(AdoMet)m for MBP-G9aDelta634 was approximately 2.3-2.65 microM with various substrates. Catalytic efficiencies (kcat/K(m)) for both MBP-G9aDelta634 and G9aFL were similar, suggesting that the N-terminus is not essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic region, resulted in catalytically impaired enzymes, thereby confirming the involvement of the C-terminus of G9a in catalysis. Thus, distinct domains modulate nuclear targeting and catalytic functions of G9a.

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Figures

Figure 1
Figure 1
Dissection of nuclear localization signals of murine G9a. (A) A schematic diagram representing different GFP fusion proteins used to determine NLS. GFP is fused at the amino terminus of each fragment. Poly E (En), ANK repeats, preSET and SET domains are indicated. Amino acid residues are indicated below each construct. Nomenclature for the constructs is indicated on the left. The effects of deletions are summarized at the right, Nu (nuclear), Cy (cytoplasmic) and H (heterochromatic). (B) Western blot analysis of representative constructs from transfected cell extracts. Constructs are indicated on top and fusion proteins are marked by an arrow. Anti-GFP monoclonal antibody was used to probe the blot. (C) Cytochemical detection of GFP fusion proteins. Deletion constructs are indicated on the left. Nuclear staining is shown in purple, GFP-G9a fusions are in green and merge of both purple and green as light blue. Hoechst stain was used for nuclear staining. (D) Mapping and identification of NLS of G9a. Both NLS1 and NLS2 are shown in red. The translated protein sequence was based on the following GenBank accession numbers—murine G9a (mG9a): AB077210.gb_ro, human long isoform (hG9a) and short isoform G9a (shortG9a) are NM_006709 and BC020970.gb_pr, respectively.
Figure 2
Figure 2
Detailed mapping of the second NLS in G9a. (A) Amino acids sequence comparison of the putative second NLS between mouse and human G9a. Lys and Arg are shown in red. Lys- and Arg-rich blocks are shown as I and II. (B) Mutational analysis of the second NLS in G9a. Upper panel shows different constructs used for transfection along with nomenclature at the left and localization results after transfection at the right. Nu is nuclear and Cy is cytoplasmic localization of the fusion proteins. Bottom panel shows cells visualized for GFP fusion proteins in green, nucleus in purple and merge as whitish blue in the bottom row. Transfected constructs are indicated on top of the panel.
Figure 3
Figure 3
Catalytically active recombinant G9a and its amino terminus deletion mutants (A) Purified G9a full-length (G9aFL) along with amino terminus deletion mutants MBP-G9aΔ634 and MBP-G9aΔ775 resolved on SDS–PAGE. Molecular masses of the markers are indicated on the left. (B) Amino terminus deletion mutants and G9aFL are capable of histone methylation. Equimolar amounts of enzymes were used in this assay. The background value is deducted from the experimental result. (C) Representative initial substrate velocity curve for MBP-G9aΔ634 versus substrate concentration. MBP-G9aΔ634 activity with recombinant full-length histone H3 substrate is shown. Methylation reactions were performed with substrate concentrations of 0.03, 0.033, 0.0375, 0.05, 0.06, 0.075, 0.1, 0.15, 0.3, 0.44 and 0.55 μM and fixed enzyme and AdoMet concentrations of 25 nM and 5 μM, respectively. Product formation is plotted versus substrate concentration and nonlinear regression was performed for the determination of Km values. The inset shows the Lineweaver–Burke plot of the substrate velocity. Vmax and Km were calculated from the substrate velocity plot. (D) Purified wild-type H3 and point mutants shown in a Coomassie-stained denaturing gel on top panel. Histone methylation catalyzed by full-length baculovirus expressed G9a. The substrates were purified histones with different point mutations as indicated at the bottom. Background values were deducted and each bar represents two duplicates. (E) A similar assay as in (C), but the enzyme is MBP-G9aΔ634.
Figure 4
Figure 4
Mass spectroscopic determination of methylation reaction progression by recombinant MBP-G9aΔ634 (panels A–F). Spectra were taken at time (A) 0, (B) 15 min, (C) 30 min, (D) 120 min, (E) 8 h and (F) 15 h. Percentage intensity is shown at the left side and mass (m/z) on the bottom. Single methyl group as mono, double methyl groups as di and triple methyl groups as tri indicated on top of each peak with respective molecular masses of 2052 (mono), 2066 (di) and 2080 (tri) daltons. (G) Control experiment with a heat-inactivated enzyme is shown. (H) Western blot analysis of methylation status of lysine 9 histone H3 coexpressed in the presence of MBP-G9aΔ634 in E.coli. H3 alone or H3 plus MBP-G9aΔ634 as indicated on top were expressed. Top two panels show Coomassie staining of the co-eluted MBP-G9aΔ634 and H3 as well as the gradient of H3 loading. The bottom panels are western blot analysis of H3 as indicated under each panel for specific antibody. (I) siRNA-mediated knockdown of G9a resulted in the reduction of global di- and tri-methylated H3-K9 but not di-methylated H3-K27. siRNAs used for knockdown are indicated on top and antibodies for each panel on the bottom. Molecular mass is indicated as kDa. Densitometric scan of methylated histones is shown on the right.
Figure 5
Figure 5
Mutation of the conserved amino acids in the SET domain abolishes catalytic activity of G9a. (A) Sequence comparison between the Lys9 trimethylase DIM-5 of N.crassa and mouse G9a as indicated. Amino acids used for point mutation analysis are underlined. Cys residues shown in gray participate in metal binding in DIM-5 and are conserved in mouse G9a. The histone methyltransferase signature motif NHXCDPN is shaded. (B) Purified mutants resolved on SDS–PAGE and stained are shown. Mutants are similar to MBP-G9aΔ634, except mutation of indicated amino acids is indicated on top. (C) Methylation of wild-type H3 peptide by purified recombinant wt (MBP-G9aΔ634) and mutants. Enzymes used for methylation are shown at the bottom. (D) Tritiated AdoMet binding by recombinant purified mutant G9a as compared with wild-type (MBP-G9aΔ634) enzyme with the same set of point mutation as that of (C). All experiments were performed in duplicate and the background values were deducted from experimental values.

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