Figure 2: Rv1988 is a histone methyltransferase (HMTase).
(a) Autoradiogram showing histone methylation activity of MBP-Rv1988 using tritiated SAM as a methyl group donor and recombinant histone H3 as a substrate. MBP-LacZ and SUV39H1 proteins were used as negative and positive control, respectively. Ponceau staining of histone H3 was used as a loading control. (b) In vitro HMTase assay result analysed by scintillation counting. The error bars represent s.d. *Student’s t-test, *P<0.05. (c) Western blot for in vitro Rv1988 HMTase assay (as in a but using cold SAM) with dimethyl arginine (upper panel) and histone H3 (lower panel) antibody. SAM-binding domain was mutated in MBP-Rv1988Adomet (Methods). (d) MS/MS spectra of histone H3 peptide (amino acids 41–49) YRmePGTVALR (Arg42-methylated). In the spectra, the ‘b’ ions are labelled in blue and ‘y’ ions are labelled in red. Evidence of Arg42 methylation is provided by b2 ion at 334.187 m/z and y8 ion at 883.547 m/z. (e) Western blot for in vitro HMTase assay with Rv1988 and recombinant histone H3 and its mutants H3R2A, H3R17A, H3R42A and H3R83A as substrates and cold SAM as methyl group donor, probed with the indicated antibodies. (f) HMTase assay with histone proteins isolated from HEK293 cells, cold SAM and MBP-Rv1988 or MBP-Rv1988Adomet. The western blot was probed with the indicated antibodies. M: Marker (19 kDa marker band is visible); 1: histones+Rv1988; 2: histones+Rv1988Adomet; 3: histones alone.
