Figure 3. Swi6 overexpression allows siRNAs to establish de novo H3K9 methylation and silencing in trans in a locus-specific manner, but is only transiently required for hairpin-induced silencing.

A, Swi6 overexpression allows ura4h-5 to silence trp1⁺::ura4⁺ but not endogenous ura4⁺. Cells harboring empty vector or the Swi6 overexpression plasmid were plated on the indicated medium to assess growth and silencing. swi6-W104A contains a point mutation in the chromodomain and does not support ura4-h5-mediated silencing. Asterisk (*) indicates a 5-FOA-resistant clone isolated from trp1⁺::ura4⁺ ura4h-5 cells with Swi6 overexpressing plasmid. B, ChIP assays showing ura4-h5-dependent histone H3K9 methylation and Swi6 recruitment at trp1⁺::ura4⁺ locus (bottom). The centromeric dg repeat is shown as a positive control (top). Double asterisk (**) shows 5-FOA-resistant clone grown in 5-FOA containing medium as in A followed by growth on rich medium lacking 5-FOA. C, Schematic diagrams of isolation and characterization of the trp1⁺::ura4⁺ ura4h-5 epiallele. 5-FOA-resistant clones generated by Swi6 overexpression (i) were cultured in non-selective medium to wean the Swi6-expressing plasmid (ii). All the clones without plasmids could generate 5-FOA resistant cells (iii) and were grown on 5-FOA medium (iv). 5-FOA resistant cells were grown for ten or more generations under non-selective conditions (v). D, Silencing assays showing that hairpin induced silencing was stably inherited in the absence of Swi6 overexpression but required the continuous expression of hairpin RNA. 5-FOA resistant cells (0G) and non-selectively cultured cells (10G) were used for silencing assays. The hairpin deleted derivatives from 5-FOA resistant clone are shown in the lower panel.