Figure 1. Forward genetics identifies ISWI complex members required for repression of H3K27-methylated genes.

(A) Selection scheme with reporter genes inserted at H3K27 methylation-marked loci to select for genes required for silencing. (B) Schematic of protein domains in ISWI and ACF1 with the changes identified in our selection (L430P and D161fs, respectively; marked with asterisks). The conserved nature of the changed residue in ISWI is highlighted for the designated species. (C) Serial dilution spot-test silencing assay for the indicated strains, which all contain P_NCU07152::nat-1 on media with or without nourseothricin (NTC). (D), Serial dilution spot-test silencing assay for the indicated strains, which contain P_NCU07152::nat-1 and P_NCU5173::hph, on media with or without nourseothricin (NTC) or hygromycin (HYG). For complementation tests, wild-type copies of each gene were inserted at the his-3 locus (indicated at left as +iswi^WTor +acf1^WT). All spot tests were imaged after 48 hr at 32°C and performed at least twice. The number of cells spotted is indicated beneath the images.

Figure 1—figure supplement 1. Genetic mapping and growth rate analysis of mutants identified in the selection.

(A) Whole-genome sequencing of pooled mutant genomic DNA identified a region near the middle of LG VI (indicated by asterisk) that is enriched for Oak Ridge single-nucleotide polymorphisms (SNPs) and contained a point mutation in iswi. Each point represents a running average (window size is 10 SNPs; step size is 1 SNP). (B) Whole-genome sequencing of pooled mutant genomic DNA identified a region near the middle of LG III (indicated by asterisk) that is enriched for Oak Ridge SNPs and contained a two base pair insertion in acf1. Each point represents a running average (window size is 10 SNPs; step size is 1 SNP). (C) Linear growth rates as measured using race tubes for strains with the indicated genotypes. Points represent technical replicates.