Fig. 5: The methyltransferase activity of SMYD5 is regulated by its C-terminal domain.

a Schema showing the functional domains and mutations of SMYD5. b Deletion of the C-terminal domain of SMYD5 reduced its interaction with octamers. Recombinant HIS tagged SMYD5 and SMYD5 mutants were incubated with an equal amount of histone core octamers. NI-NTA Seflnose Resin beads were used to pull down SMYD5. Two independent experiments were performed. c Same as in (b), except H3/H4 tetramer was used to incubate with WT and mutant SMYD5. Two independent experiments were performed. d Removal of the C-terminal domain repressed the interactions among SMYD5, Pol II, and H3. FLAG-tagged SMYD5 full length (FL) or C-terminal deletion (ΔC) over-expressed HEK 293 T were subjected to FLAG IP. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. HEK 293 T transfected with empty vectors were used as the negative controls. Two independent experiments were performed. e HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates. Upper panel, H3K36me3 was detected by Western blotting. Lower panel, input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Asterisk indicates non-specific proteins. f End-point HMT assays of full length SMYD5 and mutant SMYD5 against core octamers. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. Each assay was repeated at least three times with similar results. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. g End-point HMT assays of full length and C-terminal deleted SMYD5 against H3K36M mutant octamers. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. h N-terminal domain is important for the interaction between SMYD5 and phosphorylated Pol II CTD. GST tagged Pol II CTD was purified and then phosphorylated by the CDK7-Cyclin H complex. Recombinant HIS-tagged full length and mutant SMYD5 were purified and incubated with phosphorylated Pol II CTD, respectively. Ni-NTA Seflnose Resin beads were used to pull down SMYD5. The input and beads-bound proteins were analyzed by Western blotting using the indicated antibodies. Unphosphorylated Pol II CTD was used as the negative control for the mobility shift of phosphorylated Pol II CTD. CTD, Pol II CTD. CTD-p, phosphorylated Pol II CTD. Two independent experiments were performed. i HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates in the presence of Pol II CTD. Upper panel, H3K36me3 was detected by Western blotting. Lower panel, the input of the reactions was shown by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Star indicated the non-specific proteins. CTD, Pol II CTD. Two independent experiments were performed. j End-point HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates in the presence of Pol II CTD. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. CTD, Pol II CTD. Source data are provided as a Source Data file.