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Figure 2 | Molecular Cancer

Figure 2

From: Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex

Figure 2

Smyd2 dimethylates Histone H3 lysine 36. (A) Smyd2 methylates histone H3 in an in vitro histone methyl transferase (HKMT) assay using mixed histones from calf thymus as substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with Histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Lanes 1 and 3 show positive HKMT activity at H3 by myc tagged Smyd3 and myc tagged Smyd2, respectively. Lanes 2 and 4 indicate that neither the Smyd3 (Y239F) nor the Smyd2 (Y240F) catalytic mutants have HKMT activity. It is concluded that the HKMT activity of Smyd3 depends on Y239 and Y240 for Smyd2. (B) Smyd2 methylates Histone H3 in an in vitro histone methyl transferase assay using recombinant octamers as substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with Histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Histone H3 was found methylated by Smyd2 using recombinant octamers as substrate in an in-vitro HKMT assay. (C) Smyd2 methylates histone H3 in an in vitro histone methyl transferase assay using recombinant histone H3 as a substrate. Fluorograms are shown in the upper panel; the 17 kD band, corresponding with histone H3, is indicated; Coomassie stained SDS-PAGE gels were used to verify equal loading and are depicted in the lower panels. Histone H3 was found methylated by Smyd2 using recombinant octamers as substrate in an in-vitro HKMT assay (lane 1). The catalytically defective mutant Smyd2 (Y240F) failed to methylate recombinant histone H3 (lane 2). It is concluded that the HKMT activity of Smyd2 depends on Y240. (D) Smyd2 does not dimethylate histone H3 at lysine 4 using recombinant histone H3 as a substrate in an in-vitro HKMT assay. Western results, using antibodies, specifically reactive with dimethylated histone H3, lysine 4, are shown; the 17 kD band, corresponding with histone H3, is indicated. Lanes 1 and 4 indicate that immunoprecipitated and myc-tagged Smyd3, but not myc-tagged Smyd2, dimethylates histone H3 at lysine 4. Lanes 2 and 5 show that neither Smyd2 (Y240F) nor Smyd3 (Y239F) dimethylate histone H3 at lysine 4. We conclude that Smyd2 does not dimethylate histone H3 at lysine 4. (E) Smyd2 dimethylates histone H3 lysine 36 using recombinant histone H3 as a substrate in an in-vitro HKMT assay. Western results, using antibodies, specifically reactive with dimethylated histone H3, lysine 36, are shown; the 17 kD band, corresponding with histone H3, is indicated. Lanes 1 and 3 indicate that Smyd2 dimethylates recombinant histone H3 at lysine 36, independent of the myc or Gal4 tag. The catalytically inactive mutant Y240F does not dimethylate recombinant histone H3 at lysine 36 (lane 2). Smyd3, as well as the catalytically defective mutant Y239F, do not dimethylate recombinant histone H3 at lysine 36 (lanes 4 and 5). We conclude that Smyd2 dimethylates recombinant histone H3 at lysine 36, whereas Smyd3 does not display this activity.

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Molecular Cancer

ISSN: 1476-4598