The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules

Abstract

Histone modifications have important roles in transcriptional control, mitosis and heterochromatin formation. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by mono- and dimethylating histone H3 at Lys9 (H3K9). Here we demonstrate that the ankyrin repeat domains of G9a and GLP bind with strong preference to N-terminal H3 peptides containing mono- or dimethyl K9. X-ray crystallography revealed the basis for recognition of the methylated lysine by a partial hydrophobic cage with three tryptophans and one acidic residue. Substitution of key residues in the cage eliminated the H3 tail interaction. Hence, G9a and GLP contain a new type of methyllysine binding module (the ankyrin repeat domains) and are the first examples of protein (histone) methyltransferases harboring in a single polypeptide the activities that generate and read the same epigenetic mark.

Figure 1

G9a and G9a-like protein (GLP) bind to mono- and dimethylated histone H3. (a,b) Peptide pull-down assays with peptides that were unmodified (U) or mono-, di- or trimethylated (1, 2 or 3) at K4 or K9 (H3 1–21) or at K27 (H3 21–44) using G9a full-length (FL, residues 1–1263) or fragments containing the SET domain (residues 936–1263 of G9a) or the ankyrin repeats (ANK, residues 730–965 of G9a and 734–968 of GLP). Proteins were detected by SDS-PAGE and autoradiography. 1/10, 10% input. Shorter exposures of H3K9me1 and H3K9me2 binding are shown below each panel. (c,d) Binding of G9a and GLP ankyrin repeats with N-terminal fluoresceinated H3 peptides (residues 1–15), as determined by fluorescence polarization.

Figure 2

Structural basis for ankyrin repeat recognition of dimethylated histone H3 lysine 9 (H3K9me2). (a) The helix-turn-helix-β-turn structure of G9a-like protein (GLP) ankyrin repeats. The H3 peptide (gray) binds between the fourth and fifth repeats. For convenience, G9a residue numbers are shown (Supplementary Fig. 2). (b) H3K9me2 binds in a partial hydrophobic cage. Peptide binding is further specified by the interactions with H3 Ser10, Thr11, Gly12 and Gly13. (c) Peptide pull-down assays with H3 peptides (residues 1–21) using G9a wild-type (WT) or mutant ankyrin repeats. H3 peptides were either unmodified (U), or mono-, di- or trimethylated (1, 2 and 3). 1/10, 10% input. (d) Structural comparison of GLP ankyrin repeats with (green) and without (blue) bound peptide. The Trp839 indole ring rotated along the side chain torsion angles _χ1 (∼20°) and _χ2 (∼80°). In addition, the carboxyl group of Glu870 moved approximately 2.5 Å toward Ser10. (e) Dimethyllysine from the crystal structure of GLP ankyrin repeats is satisfactorily accommodated in the cage. Spheres represent approximate van der Waals radii of atoms. (f) Trimethyllysine is excluded from the GLP ankyrin repeat cage. Modeling a trimethyllysine in the cage causes steric clashes. The conformation with the fewest clashes is shown. (g) Binding of GLP ankyrin repeats with wild-type (WT) or mutated H3 peptides (residues 1–15) containing K9me2, as determined by fluorescence polarization.

Figure 3

Analysis of G9a ankyrin mutants that lack H3K9me1 and H3K9me2 peptide binding. (a) Expression and methyltransferase activity of full-length G9a proteins. Immunoprecipitated (IP) proteins, derived from transfected Cos cells, were analyzed for either methyltransferase activity using core histones as a substrate (above) or immunoblotted with anti-Flag antibody (below). Wild-type G9a (WT), indicated point mutants or Flag vector control (vector) are shown. (b,c) Coimmunoprecipitation (Co-IP) of wild-type G9a (WT) or the indicated mutant Flag-G9a full-length (1–1263) or Flag-G9a ankyrin region (730–965) with HA-GRIP1 (5–479). Above, IP and immunoblot of G9a with Flag antibodies. Middle, immunoblot of input GRIP1 with HA antibodies. Below, Co-IP of GRIP1, immunoblotted with HA antibodies. IgG heavy (IgH) and light (IgL) chains are indicated. (d,e) Coimmunoprecipitation of WT or mutant full-length Flag-G9a with HA-GFI1 delta zinc finger mutant (ΔZF). Cos7 cells were cotransfected with plasmids encoding Flag-G9a and HA-GFI1 ΔZF. Above, anti-Flag blots of IP proteins. Below, anti-HA blots of Co-IP proteins. Mock, no G9a; 1/10, 10% input; IgG, IP with normal mouse IgG; Flag, IP with anti-Flag.