Synthetic dropout medium

Plasmid vectors pGAD424 and pGBT9 (Clontech) containing the cDNAs of BRM, SWI3A, SWI3B, BSH, and SWP73B were described previously (Buszewicz et al., 2016 (link)). The full-length cDNAs of BRD1, BRD2, BRD13, SWI3C, SWI3D, SWP73A, ARP4, and ARP7 and a cDNA encoding the N-terminal region of SYD (1–1500 bp of the coding sequence) were cloned into pGAD424 and pGBT9 by ligation with T4 DNA ligase (Thermo Scientific) or using the SLIC protocol (Li and Elledge, 2007 (link)). Cells of the yeast strain AH109 were transformed with plasmid DNA as described previously (Buszewicz et al., 2016 (link)). Serial 10-fold dilutions of the transformant cell suspensions were plated on synthetic dropout medium (Sigma) lacking tryptophan, leucine, and histidine without additions or supplemented with 3-amine-1,2,4-triazole (3-AT) at 0.25–1 mM. The growth of each strain was assessed after incubating the plates for 3 days at 28°C. Three technical replicates were performed for each strain in each growth assay.

The Y2H system was modified by introducing a third plasmid expressing SWI3C, BRD1, or BRD13. To generate these constructs, the full-length coding sequence of each gene was PCR-amplified using primers encoding an SV40 NLS added to the N terminus to ensure nuclear localization in yeast cells (Sun et al., 2011 (link)) and cloned into the BamHI/SalI sites of the plasmid p426 carrying the URA3 nutritional marker to permit selection on uracil-deficient medium (Mumberg et al., 1995 (link)). Yeast strain PJ64-4A was co-transformed with the indicated combinations of plasmids (James et al., 1996 (link)), and transformants were selected on synthetic dropout medium (Sigma) lacking tryptophan, leucine, and uracil. Serial 10-fold dilutions of the transformant cell suspensions were plated onto synthetic dropout medium lacking tryptophan, leucine, uracil, and histidine (W0 medium) without additions or supplemented with 3-AT at concentrations of 1–10 mM. The growth of each strain was assessed after incubating the plates for 3–4 days at 28°C. Three technical replicates were performed for each strain in each growth assay.