Dynamic Histone H1 Isotype 4 Methylation and Demethylation by Histone Lysine Methyltransferase G9a/KMT1C and the Jumonji Domain-containing JMJD2/KDM4 Proteins

Abstract

The linker histone H1 generally participates in the establishment of chromatin structure. However, of the seven somatic H1 isotypes in humans some are also implicated in the regulation of local gene expression. Histone H1 isotype 4 (H1.4) represses transcription, and its lysine residue 26 (Lys(26)) was found to be important in this aspect. H1.4K26 is known to be methylated and acetylated in vivo, but the enzymes responsible for these post-translational modifications and the regulatory cues that promote H1.4 residence on chromatin are poorly characterized. Here we report that the euchromatic histone lysine methyltransferase G9a/KMT1C mediates H1.4K26 mono- and dimethylation in vitro and in vivo and thereby provides a recognition surface for the chromatin-binding proteins HP1 and L3MBTL1. Moreover, we show evidence that G9a promotes H1 deposition and is required for retention of H1 on chromatin. We also identify members of the JMJD2/KDM4 subfamily of jumonji-C type histone demethylases as being responsible for the removal of H1.4K26 methylation.

FIGURE 1.

Purification of H1.4K26 HKMT activities from HeLa nuclear pellet. A, HKMT assays carried out with increasing amounts of nuclear extract (left) or nuclear pellet (right) using recombinant wild type histone H1.4 or a version with a Lys²⁶ to Ala point mutation as substrate. B, purification strategy to isolate H1.4K26-specific HKMT activities. C, Mono Q peak fraction of H1.4K26 HKMT activity was analyzed by SDS-PAGE and silver staining (left). Specificity is illustrated by HKMT assay using wild type or K26A mutant H1.4 as substrate (top). The fraction was subjected to mass spectrometric analysis and identified HKMTs were confirmed by immunoblotting (right). D, H1.4K26 HKMT activity peak from the Mono Q step was pooled and subjected to a Superose 6 gel filtration column. Fractions were either analyzed by HKMT assay or resolved by SDS-PAGE and analyzed by silver staining or immunoblotting. HKMT assays were performed using wild type or K26A mutant H1.4 as substrate. E, immunoprecipitation experiment from nuclear pellet fraction using IgG, anti-G9a, and anti-EZH2 antibodies. Precipitated material was analyzed by HKMT assays or immunoblotting.

FIGURE 2.

Recombinant G9a methylated H1.4 at Lys²⁶in vitro. A, recombinant full-length G9a derived from a baculoviral expression system was used to methylate increasing amounts of recombinant wild type or K26A mutant HA-tagged H1.4 using ³H-labeled AdoMet as methyl donor. Shown is the fluorography after an exposure time of 12 h (top panel) and the Coomassie Blue-stained membrane to visualize the amount of H1.4 and H1.4K26A proteins (bottom panel). B, as in A but using unlabeled AdoMet as methyl donor. Detection of methyl groups incorporated at H1.4K26 was carried out by Western blot using a specific anti-H1.4K26me2 antibody. An anti-HA antibody was used to determine the total H1.4 levels. C, as in A but using synthetic peptides corresponding to the sequence shown for H1.4 or H3 (top) as substrate. HKMT assay incubation time was limited to 30 min. D, recombinant wild type H1.4 was incubated with G9a in the presence or absence of unlabeled AdoMet as methyl donor. The reactions were resolved by SDS-PAGE, stained with Coomassie Brillant Blue, and the H1.4 bands then cut from the gel and subjected to mass spectrometry. Ion chromatograms of ²⁶KKSAGAAR peptides are shown with different modifications generated from LC-MS/MS data of propionylated partial tryptic digest of histone H1.4. Only the sample incubated with G9a and AdoMet led to the detection of monomethylated ([M + 2H]²⁺ = 578.1) and dimethylated Lys²⁶ ([M + 2H]²⁺ = 557.2) (spectra shown in black). Only unmodified H1.4K26 peptides were detected in the absence of AdoMet (spectra shown in red). In the absence of G9a only unmodified ([M + 2H]²⁺ = 570.8) Lys²⁶ peptides were detected. E, the table summarizes other potential H1.4 target sites and the percentage of methylation compared with an unmethylated H1.4 control.

FIGURE 3.

G9a methylates H1.4K26 in vivo. A, 293T cells were transiently transfected with GAL4, GAL4-PR-SET7, or GAL4-G9a. Whole cell extract was prepared from transfected cells or acid extraction was performed to isolate core and linker histones and both fractions were analyzed by Western blot using antibodies indicated on the right. Anti-GAL4 was used for the top panel. B, acid extraction of histones was performed from wild type (TT2) or G9a^–/– (2210) embryonic stem cells and purified histones were analyzed by Western blot using antibodies indicated on the right. C, 293T cells were treated for 48 h with DMSO, compound BIX-01294 (a specific G9a inhibitor; final concentration 9 μm), or trichostatin A (final concentration 300 nm). High salt nuclear extracts were prepared from treated cells and analyzed by Western blot using the antibodies indicated on the right.

FIGURE 4.

G9a methylates H1.4K26 on a specific genomic location and promotes H1 recruitment. A, illustration of the transgene system that allows tetracycline-induced expression of GAL4-G9a and targeting to the luciferase promoter containing GAL4 DNA binding sites. Notably, luciferase is constitutively expressed in the default state (–Tet). B, induction of GAL4-G9a effectively represses luciferase expression as demonstrated by luciferase assays of two independent clones. The experiments were carried out in duplicate and the result of one representative experiment is shown. C, whole cell extracts were prepared from two independent clones that were grown in the absence or presence of tetracycline for 24 h. Extracts were analyzed by Western blot using the antibodies indicated on the right. D, ChIP experiments from cells grown in the absence or presence of tetracycline for 24 h. Antibodies used for the ChIPs are indicated on the right. One representative of two independent experiments is shown. E, ChIPs with anti-H1.4K26me2 antibodies either untreated or preincubated with synthetic peptides corresponding to various histone sequences and modification states. Quantitative real time PCR analysis for each sample was carried out in triplicate. p values are indicated and were calculated by paired t-tests. Competition with H3K9me2 and H1.4K26me2 peptides did not result in statistically relevant (p > 0.05) reduction of PCR product. F, quantitative real time RT-PCR measuring MAGE-A1 and -A3 transcript levels from HeLa cells treated for 72 h with DMSO or decitabine. The GAPDH transcript level was used for normalization. Three biological replicates were used and all PCR were performed in triplicate. G, ChIP experiments carried out from 293 cells analyzing the MAGE-A1 promoter in the absence or presence of decitabine. ChIP at the GAPDH gene served as a negative control. One representative of three independent experiments is shown.

FIGURE 5.

G9a-methylated H1.4 is recognized by L3MBTL1 and HP1γ in vitro. A, GST pull-down experiment with GST or GST fused to the three MBT domains of L3MBTL1 (GST-3MBT) and recombinant HA-tagged H1.4 either unmethylated or methylated by G9a. Notably, a point mutation in the second MBT domain (P2a) abolished binding but mutations in the first and third MBT domains (P1a and P3a) retained binding to G9a-methylated H1.4. Methylated H1.4 was visualized by ³H-fluorography. B, GST pull-down experiment with GST or GST-HP1γ and recombinant HA-tagged H1.4 either unmethylated or methylated by G9a. Methylated H1.4 was visualized by Western blot using anti-H1.4K26me2 antibodies.

FIGURE 6.

Members of the JMJD2 subfamily of the JmjC domain-containing proteins demethylate H1.4K26 in vitro and in vivo. A, identification of mouse Jmjd2d H1.4K26 demethylase activity by MALDI-TOF mass spectrometry. Each panel contains a spectrum for H3K4me3, H3K9me3, H3K27me3, H4K20me3, H1.4K26me3, H1.4K26me2, and H1.4K26me1 peptides incubated with or without mouse Jmjd2d protein. The masses corresponding to unmodified (0), mono- (1), di- (2), or trimethylated (3) peptides are indicated in dotted lines. The appearance of a peak corresponding to the demethylated peptide is marked with an arrowhead. The shift corresponds to a loss of 14, 28, or 42 daltons due to the removal of methyl group(s). B, Jmjd2a, Jmjd2b, and Jmjd2c were also identified as H1K26me3 demethylases. Addition of the iron chelator deferoxamine at the beginning of the reaction suppressed the formation of a demethylated peptide peak. C, NIH3T3 cells stably expressing mouse Jmjd2b display a reduction in H1.4K26me2/3 and H3K9me3. Total H1 and H3 levels served as a loading control. Acid-extracted histones (1 or 2 μg) were analyzed as indicated by triangles on the top. D, ectopic expression of human JMJD2D wild type but not its catalytic point mutant (H192A) in 293T cells causes global reduction in H1.4K26me2/3 and H3K9me2. β-Actin, total H1, and total H3 served as loading controls.