<p> FRIcore(μM) 0 0.5 2 Vmax 59.88 116.28 185.19 Km/Vmax 4.57 0.94 0.75 Km 273.63 109.77 139.02</p><p>Supplemental Figure 1. Kinetic analysis of the FRI-mediated activation of the histone methyltransferase activity of EFS. Supplemental Figure 2. FRI specifically stimulates the histone methyltransferase activity of EFS.</p><p>A) FRI stimulates EFS, but not the G9a HMTase. FRI weakly stimulates the HMTase activity of ASH1L. </p><p>B) FRI, but not BSA, stimulates the histone methyltransferase activity of EFS. Supplemental Figure 3. Sequence alignment of human ASH1L_SET (2069–2288 a.a.) and Arabidopsis EFS_SET (955–1174 a.a.). Supplementary materials and methods</p><p>Protein purification – The FRI domain (FRIcore, a.a. 131–432) from Arabidopsis thaliana was cloned into a pET28a vector containing a 6X His tag in its N-terminal region and the Tobacco Etch Virus (TEV) protease cleavage site. The FRI domain was expressed in Escherichia coli BL21 (DE3) and induced with 1 mM Isopropyl beta-D-1-thiogalactopyranoside (IPTG) for 18 h at 18°C. The cells were lysed in a buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, and PMSF. Soluble proteins were separated by centrifugation and the N-terminal His-tagged FRI domains were purified by Ni-NTA (Qiagen) affinity chromatography with an elution buffer containing 50 mM Tris-HCl pH 8.0,</p><p>300 mM NaCl, 5% glycerol, and 100 mM imidazole. EFS-SET850 (a.a. 850–1166) from Arabidopsis thaliana was cloned into a pGEX4T-1 vector containing a GST tag in its N-terminal region. The protein expression conditions were the same as those used for the FRI domain. EFS-SET850 was then purified by GST affinity chromatography with an elution buffer containing 5% glycerol and 10 mM GSH. EFS-</p><p>SET917 (a.a. 917–1263) was cloned into pFastBac HTB, which adds an additional 6X His tag to the N- terminus of the protein, and expressed in an insect cell expression system. EFS-SET917 was purified by Ni-NTA affinity chromatography and the His tag was removed with TEV protease. The protein was further purified via ion exchange (Hitrap Q column, GE healthcare) and size-exclusion chromatography (Superdex 200-26-60, GE healthcare). Then, EFS-SET917 was used for pull-down assays.</p><p>In vitro histone methyltransferase assay - EFS_SET850 (100 nM) was incubated in a reaction mixture containing G5E4 nucleosome arrays (12 nucleosomes reconstituted with Xenopus histone octamers on a G5E4 DNA fragment) and 2 μCi of S-adenosyl-L-[methyl-3H] methionine (3HSAM) (1 mCi, PerkinElmer Life Science, NET 155-3) per 25 μl reaction for 1 hr at room temperature. Reactions were stopped by adding 5X SDS sample buffer and boiling at 95°C. Then, the mixtures were separated by 15% SDS-PAGE and transferred to a PVDF membrane by the semi-dry transfer method. The membrane was exposed to an image plate and the image plate was used to expose FLA-7000 (Fuji Film) for autoradiography. Band intensities were measured with Multigauge.</p><p>Western blot assay - EFS_SET850 and FRIcore were incubated with substrates in the HMTase assay conditions described above using methyl donor SAM (NEB). Then, the reactants were separated using 15% SDS PAGE and transferred to a PVDF membrane by the wet transfer method (100 V, 60 min) in transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. The membrane was blocked with 5% skim milk in Phosphate buffer saline (PBS) containing 0.1% Tween 20 for 1 hr in room temperature. Antibodies specific to H3 (9717, Cell Signaling Technology), H3K4me2 (generated by Dr. Daeyoup Lee), H3K4me3 (07-473, Upstate Biotech Millipore), H3K36me2 (9758S, Cell Signaling Technology), or H3K36me3 (9763S, Cell Signaling Technology) were used to detect histone methylations. Bands were scanned with a Chemidoc station (Biorad) and their intensities were compared with Multigauge.</p><p>Pull-down assay - 6x HIS-tagged FRIcore and EFS_SET917 were used for the Ni-NTA pull-down assay. FRI was incubated with Ni-NTA resin for 1 hour at 4°C. Then, the beads were washed with a wash buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 2 mM DTT for 3 times. The same amounts of beads were aliquoted into each tube along with 10 μg of EFS_SET917. New beads without HIS-tagged</p><p>FRIcore were used as a negative control. Then, the samples were incubated for 1 hour at 4°C and washed 3 times with wash buffer. The samples were analyzed via 10% SDS-PAGE with Coomassie staining. </p><p>Gel retardation assay – Mono-nucleosomes were prepared for the binding assay with FRIcore and</p><p>EFS_SET917. Mono-nucleosomes (0.25 μM) were used for the gel retardation assay and the target proteins were added by increasing their molar ratio with reference to the mono-nucleosomes. The samples were then incubated for 1 hour on ice. After adding 10% sucrose, the samples were separated on a 4% native acrylamide TBE gel (150 V, 60 min). DNA was visualized by Ethidium Bromide (EtBr) staining and proteins were visualized by Coomassie staining.</p>