Methylation of H2AR29 is a novel repressive PRMT6 target - PubMed
- ️Sat Jan 01 2011
Methylation of H2AR29 is a novel repressive PRMT6 target
Tanja Waldmann et al. Epigenetics Chromatin. 2011.
Abstract
Background: Covalent histone modifications are central to all DNA-dependent processes. Modifications of histones H3 and H4 are becoming well characterised, but knowledge of how H2A modifications regulate chromatin dynamics and gene expression is still very limited.
Results: To understand the function of H2A modifications, we performed a systematic analysis of the histone H2A methylation status. We identified and functionally characterised two new methylation sites in H2A: R11 (H2AR11) and R29 (H2AR29). Using an unbiased biochemical approach in combination with candidate assays we showed that protein arginine methyltransferase (PRMT) 1 and PRMT6 are unique in their ability to catalyse these modifications. Importantly we found that H2AR29me2 is specifically enriched at genes repressed by PRMT6, implicating H2AR29me2 in transcriptional repression.
Conclusions: Our data establishes R11 and R29 as new arginine methylation sites in H2A. We identified the specific modifying enzymes involved, and uncovered a novel functional role of H2AR29me2 in gene silencing in vivo. Thus this work reveals novel insights into the function of H2A methylation and in the mechanisms of PRMT6-mediated transcriptional repression.
Figures

H2A is methylated by protein arginine methyltransferase (PRMT)1 at novel sites. (A) Biochemical purification scheme (for details see Additional file 5, supplementary materials and methods) of H2A-specific histone methyltransferase from HeLa nuclear extract. (B) (Upper panel) Silver stain of heparin-Sepharose fractions 1 to 8. PRMT1 was identified by mass spectrometry (asterisk) (see Additional file 1, Figure S 1). Autoradiography of histone methyltransferase assays of indicated heparin-Sepharose fractions using (middle panel) H2A 4-129 and (lower panel) endogenous acidic extracted histones, full-length recombinant H2A, myelin basic protein (MBP), and glutathione S-transferase (GST)-GAR (glycine- and arginine-rich) as substrates.

Protein arginine methyltransferase (PRMT)1, 5 and 6 can methylate recombinant and endogenous H2A. (A) Histone methyltransferase (HMT) assays with different Flag-hemagglutinin (HA)-tagged PRMTs immunoprecipitated from HEK 293, stably expressing PRMTs on (lane 1) nucleosomes, (lane 2) purified histones and (lane 3) recombinant full-length H2A. (Left) Coomassie stain and (right) autoradiography are shown. (B) HMT assays using recombinant full-length H2A and H2A 4-129. Only PRMT1 and 6 were found to methylate H2A 4-129. (C) HMT assays (left panel) with Flag-Ha-PRMT1 on H2A 4-129, H2A 4-129 R11 mutated to K, and (right panel) with Flag-Ha-PRMT6 on H2A 4-129, H2A 4-129 R11K, H2AR11K R29K. Loading controls are shown below. (D) Mass spectrometry (MS) analysis of H2 Amethylated in vitro by PRMT1 shows methylation of R11. Tandem MS (MS/MS) spectra of a propionylated histone H2A peptide at a mass:charge ratio (m/z) of 876 from trypsin digest of propionylated H2A is shown. (E) MS analysis of H2A, in vitro methylated by PRMT6 shows methylation of R29. MS/MS spectrum of a doubly-charged peptide ion at m/z of 703.901 is shown.

H2AR29 is methylated by protein arginine methyltransferase (PRMT)6 in vitro and in vivo. (A) Immunoblot with H2AR29me2-specific antibody shows the presence of H2AR29me2 in various human and mouse cell lines. (B) Mass spectrometry (MS) analysis of endogenous, purified H2A. Shown in the collisionally activated dissociation (CAD) tandem MS (MS/MS) spectrum of the doubly-charged histone H2A derived tryptic peptide 22AGLQFPVGR(me2)29 ion (mass:charge ratio (m/z) 486.78522; 0.71 ppm mass deviation), identifying R29 as dimethylated. The identity and assignment of the b and y ions of the dimethyl arginine-containing peptide was corroborated by nano liquid chromatography (LC)-MS/MS analysis of the corresponding chemically synthesised peptide (see Additional file 4, Figure S4). (C) (Top panel) H2AR29me2 levels decreased upon knockdown of PRMT6, but not PRMT1. PRMT1 RNA levels (relative to β-actin) measured by reverse transcriptase (RT)-PCR in HEK293 cells were reduced by approximately 80%, and PRMT6 by 50%. (Bottom panel) H2AR29me2 levels were not altered upon knockdown of PRMT1, but they decreased by approximately 50% upon PRMT6 knockdown. The average of the H2AR29me2 levels relative to the control from three independent experiments is shown; error bars correspond to the standard deviation of the means. (D) Overexpression of PRMT6 (top panel) in HEK293 cells resulted in increased H2AR29me2. (Top panel) PRMT6 RNA levels (relative to β-actin) were measured by RT-PCR. (Bottom panel) H2AR29me2 levels increased by approximately 2.5 times in PRMT6 overexpressing cells compared with the wild-type cells. The average of the H2AR29me2 levels relative to the control from four independent experiments is shown; error bars correspond to the standard deviation of the means.

H2AR29me2 is enriched at genes repressed by protein arginine methyltransferase (PRMT)6. (A) H2AR29me2 was found to be enriched in less accessible chromatin fractions. Kinetic of micrococcal digestion of chromatin [28]. Kinetic of micrococcal digestion of chromatin [28] is shown. The abundance of the indicated histones modifications in the fractions was tested by western blotting with specific antibodies. (B) PRMT6 was overexpressed in HEK293 cells. Expression levels of the indicated genes were measured by real-time PCR and normalized to β-actin. The relative expression of the normalized genes in control and PRMT6-overexpressing cells was quantified using the ΔCt method, and the resulting relative quantification (RQ) values were plotted in a bar graph on a logarithmic scale. The cross indicates genes not expressed in HEK293 (GEO: record GSE11892; [20]). (C) Chromatin immunoprecipitation assay with H2AR29me2 antibody. Purified DNA from immunoprecipitated chromatin fragments from HEK293 cells was amplified by real-time PCR with specific primer sets for indicated gene promoters (see Additional file 6, Table S1). The enrichment of precipitated chromatin relative to an intergenic region was determined and plotted in a bar graph. Error (standard deviation) bars are shown.
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