Histone Mutant Expression and Epigenetic Enzyme Purification

Doxycycline (Sigma; 10,000 × stock solution; 1 mg/ml) was resuspended in 70% ethanol. Biotin (BioShop and Sigma) was prepared as a 1000 × stock solution (50 mM) by dissolving 250 mg in 2 ml 30% v/v NH₄OH on ice and neutralized by slowly adding 18 ml HCl (1 N), before sterile filtration through a 0.22 µm filter and storage at 4 °C. Chaetocin (Selleck) and OTS186935 (MedChemExpress) were dissolved in DMSO, aliquoted, and stored at − 80 °C.
H3.3 (H3-3a) or H3.1 (H3C2) cDNA that was WT or had K27M or G34R mutations was amplified from existing cDNA stocks and cloned between the XbaI/BamHI sites of a pCDH-CMV-MCS-EF1α-Hygro (SystemBioscience) previously modified to encode a FLAG/HA tag between the BamHI/NotI sites. K9M mutations were introduced with the Q5 site-directed mutagenesis kit (NEB). pcDNA5-FLAG-BirA* plasmids to create Flp-In T-REx HEK293 cells were generated as previously described [34 (link)]. Bacterial histone expression vectors were created by cloning cDNA into pET3a [17 (link)]. EHMT2 cDNA was amplified from pLenti6-MK1-EHMT2-V5 (Addgene #31113) and cloned between the KpnI and EcoRI sites of pFastBac (Invitrogen) to create pFastBac_EHMT2. SUV39H2 cDNA was amplified from a HEK293T cDNA library and inserted by Gibson assembly into pDest_pACE1 to create pACE1_SUV39H2. All plasmids were sequenced before use. shRNA targeting H3K9 methylases were from Sigma.