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. 2010 Mar 24;3(1):7.
doi: 10.1186/1756-8935-3-7.

Histone H1 Variant-Specific Lysine Methylation by G9a/KMT1C and Glp1/KMT1D

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Free PMC article

Histone H1 Variant-Specific Lysine Methylation by G9a/KMT1C and Glp1/KMT1D

Thomas Weiss et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Background: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown.

Results: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle.

Conclusions: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.

Figures

Figure 1
Figure 1
G9a and its interaction partner Glp1 methylate H1. (a) Candidate methylation assay on core nucleosomes and H1. Indicated HKMTs fused to GST (see also Methods) were used for methylation assays to identify enzymes methylating H1. Core nucleosomes (purified from HEK293) were mixed with H1 as substrates. All enzymes were tested in (lane 1) Tris, (lane 2) PBS, (lane 3) MAB and (lane 4) R methylation buffers. Incorporation of the methyl group from the donor adenosyl-L-methionine S-[methyl-3H] was detected by autoradiography (for a specificity control see Additional file 1). (b) Recombinantly expressed SET domains of Glp1 (610-917) and G9a (682-1000) and recombinant H1.2FL and H1.2CT were used for methylation assays. G9a and Glp1 methylate the H1.2 C-terminus (CT). Ponceau staining of the membrane (Pon, upper panels) and autoradiography (Rad, lower panel) are shown.
Figure 2
Figure 2
G9a and Glp1 methylate H1.2K187. (a) Amino acid sequence of the C-terminal tail of H1.2. Lysines as potential methylation sites are highlighted in grey. (b) Cluster mutation approach to map methylation sites in the C-terminal part with its 40 potential sites. Cluster definitions and their substituting amino acid sequence are depicted. All lysines in the C-terminal tail of H1.2 were classified into 10 clusters (grey rectangles). Cluster 12 is a fusion of clusters 1 and 2. (Upper) Wild-type H1.2 sequence; (lower) the substituting amino acids. (c) The methylated site is between amino acids 187 and 196. Methylation assay of the cluster mutation constructs with immunoprecipitated Glp1. Used constructs are schematically indicated on top. Grey rectangles, mutated clusters; white rectangles, wild-type clusters. In addition, all constructs carry a lysine to arginine mutation introducing an arginase C cutting site. (Upper panel) Ponceau (Pon) staining as a loading control. The different migration levels of the constructs are due to the substitutions and replacements of different numbers of uncharged and positively charged lysines by uncharged amino acids. Note a strong reduction in methylation in lane 16, corresponding to a cluster 8 mutation (mutated cluster containing K187 is indicated by asterix). (d) Methylation assay with wild-type H1.2CT, H1.2CT K172R, H1.2CT K187R, H1.2CT K172R and K187R and wild-type H1.2CT (2× z-tag) as substrates. Mutation of K187 results in a strong reduction of methylation by immunoprecipitated Glp1. (Upper panel) Pon; (lower panel) autoradiography (Rad). Wild-type H1.2CT (2× z-tag) was added to the methylation assays as an internal control of methyltransferase activity (lanes 5 to 8). (e) Methylation assays on peptides. Peptides containing unmodified K187, monomethylated K187 and dimethylated K187 and immunoprecipitated G9a were used for methylation assay. Rad is shown. Note that unmodified and monomethylated peptide were methylated to similar extents, whereas the dimethylated peptide was not methylated.
Figure 3
Figure 3
G9a and Glp1 methylate H1.2K187 in vivo. (a) Reverse transcription PCR analysis of siRNA-transfected cells harvested at indicated time points. HEK293 cells were either transfected with a control siRNA (negative control; NK) or simultaneously with siRNAs against G9a and Glp1 (double knockdown; dkn). mRNA levels were normalised to β-actin expression. The mRNA levels of G9a and Glp1 in the control cells were set as 1 and levels in the double knockdown cells calculated as the ratio of this level (light grey, G9a; dark grey, Glp1). (b) MS analysis of H1.2 after knockdown. Full MS result showing the quantitative comparison of H1 peptides from control and G9a/Glp1 double knockdown samples. We observed an approximate twofold decrease in a peptide (414.750 m/z) from the G9a/Glp1 dkn sample compared with the control. (c) This peptide at 414.750 m/z was sequenced by MS/MS experiments and determined to be the peptide containing the K187 monomethylation mark. The dimethylation of H1.2K187 might have escaped our mass-spectrometric analysis by being below of our current detection threshold.
Figure 4
Figure 4
Variant-specific methylation of H1 by G9a/Glp1. (a) Alignment of H1 variants H1.2, H1.3, H1.4, H1.5 and H1.0. Amino acids identical in at least three variants are highlighted in blue. The regions around H1.4K26 and H1.2K187 are boxed. (b) H1.4K26 and H1.2K187 are G9a and Glp1 targets. Methylation assay on indicated H1.2 and H1.4 mutants using (left) immunoprecipitated Glp1 and (right) G9a. Note that the high expression of H1 led to partial N-terminal degradation of some constructs in bacteria. (Upper panels) Ponceau (Pon) staining: *degradation fragments. (Lower panels) Autoradiography (Rad). (c) H1.2, H1.3, H1.4, H1.5 and H1.0 were methylated by G9a and Glp1. Methylation assays on recombinant human H1 variants using immunoprecipitated G9a and Glp1. (Upper panels) Ponceau (Pon) staining; (lower panels) autoradiography (Rad). (d) Methylation assays on human recombinant H1 followed by chymotrypsin digestion, which separates the N-terminus and core from the C-terminus. Only the N-terminus and core of H1.4 is a major G9a/Glp1 target whereas H1.2, H1.3, H1.5 and H1.0 are methylated by G9a/Glp1 preferentially in the C-terminal part. Autoradiography (Rad) is shown (loading control in Additional file 3).
Figure 5
Figure 5
H1.2K187 methylation is cell cycle-independent and HP1 binds only to methylated H1.4K26. (a) H1.2K187me is cell cycle-independent. Quantitative MS analysis of H1 extracted from unsynchronized HEK293 cells and synchronized cells in G1 and M phase. A selected ion monitoring experiment was used on the ion 414.745 m/z (corresponding to H1K187 methylation) to monitor the levels of methylation. Normalisation was done relative to unsynchronized cells. (b) Recombinant HP1 binds to H1.4K26me2 but not to H1.2K187me2. Peptide affinity purification of recombinant HP1β and γ (GST fusion protein). The upper panels show the anti GST WB of the elution and lower panels the Coomassie staining of eluted fraction. (c) Endogenous HP1 binds to H1.4K26me2 but not H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with HP1α/β specific antibody. (d) Glp1 does not bind to H1.4K26me2 or H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with Glp1 specific antibody. (e) JMJD2D demethylates H1.4K26me2 only. Methylation assay with G9a and Glp1 after demethylation of peptides by JMJD2D (Rad).

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