Figure 3

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.