Fig. 1: Rv2067c in vitro methylates histone H3 at lysine 79. | Nature Communications

Fig. 1: Rv2067c in vitro methylates histone H3 at lysine 79.

From: The Mycobacterium tuberculosis methyltransferase Rv2067c manipulates host epigenetic programming to promote its own survival

Fig. 1

a Autoradiograph of methylation by Rv2067c with two different concentrations of histones salt extracted from THP-1 monocytes as substrate and tritiated S-Adenosyl-L-methionine (3H-SAM) as a methyl group donor (Lane 1 and 2). BSA (lane 3) was kept as negative control. Lower panel shows ponceau staining (n = 2 independent experiments). b, c Bar graph and autoradiograph showing methyltransferase (MTase) activity of Rv2067c against recombinant H3, H2A, H2B and H4 as substrate and tritiated SAM as methyl group donor. Recombinant Rv2067c was kept as negative control for scintillation counts and ponceau staining shows the loading of histones for autoradiograph. (n = 2 independent experiments; p = 0.002). d MS/MS spectra of H3K79 trimethylation performed by Rv2067c on recombinant H3. The table shows peptide EIAQDFK^TDLR is trimethylated (^ represents trimethylation) when the assay was performed with H3 and Rv2067c. H3 alone serves as control (n = 1). e Blots probed with H3K79me3 and H3K4me3 antibodies respectively depict MTase assays with synthetic H3 peptide 73–83 aa and 1–8 aa (negative control) as substrates, Rv2067c as MTase and non-radioactive SAM as methyl donor. f Scintillation counts for Rv2067c MTase activity with recombinant H3 and H3A79 mutant as substrate (n = 3 independent experiments; p = 0.002, 0.003). g Graph shows MTase assays with Rv2067c and its SAM-binding mutant (GxG to RxR; “Methods”) using recombinant H3 as substrate (n = 3 independent experiments; p = 0.006, 0.008). h Comparison of MTase activity of Rv2067c and DOT1L. Rv2067c methylates free H3, whereas DOT1L methylates H3 in nucleosomal core particle (NCP). Recombinant H3, reconstituted octamers and NCPs were kept as controls; Rv2067c and DOT1L were kept as control to check auto-methylation (p = 0.006, 0.0005). The data for MTase assays is plotted as mean. Error bars correspond to SD and P-value was calculated using unpaired two-tailed Student’s t-test. **, P-value < 0.01, ***, P-value < 0.001.

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