Genetic Modifications of S. pombe Strains

For S. pombe strains, see Table S1. Deletion and epitope tagging (3xFLAG or GFP) of Stc1 were achieved by homologous recombination with PCR fragments comprising resistance cassettes flanked by sequence homologous to insertion sites (Bähler et al., 1998 (link)). LIM domain mutations were generated with Quickchange II XL Kit (Stratagene) and integrated by replacement of stc1:ura4⁺. The Clr4-GBD tethering system was described previously (Kagansky et al., 2009 (link)). For pDUAL-TetR-2xFLAG-Stc1, stc1⁺ was cloned into a modified pDUAL-HFF81c vector (Matsuyama et al., 2004 (link)), made by swapping His to TetR^off (Promega), so that Stc1 was N-terminally tagged with TetR^off followed by 2xFLAG, under the nmt81 promoter. Not1-digested plasmid was integrated at leu1⁺. For the 4xTetO-ade6⁺ reporter, 4xTetO repeats (TCCCTATCAGTGATAGAGA) were introduced upstream of ade6⁺ inserted at the ura4⁺ locus via the strategy described previously for 3xgbs-ade6⁺ (Kagansky et al., 2009 (link)).