Extended Data Fig. 1: siRNA-induced H3K9me3 at the euchromatic ade6⁺ locus and flanking region.

a, Summary of pathways and factors involved in mRNA 3′-end processing and its coupling to nuclear export. Chp1 and Ago1 are subunits of the RITS. Uap56 and Mlo3 are TREX (transcription–export) complex subunits, and associate with Dss1 to mediate mRNA export. Tho1 is a subunit of the THO/TREX complex, which is responsible for recruiting other subunits of the TREX complex to mRNA. Mex67 is an orthologue of human NXF1, a critical mRNA export receptor, and associates with Nxt1, another mRNA export factor. Puf6 is an mRNA 3′-UTR-binding protein that has been shown to associate with Mlo3. Rhn1 is involved in RNA polymerase II transcriptional termination. Nab2 is a poly(A)-binding protein. The Paf1 complex is required for transcriptional elongation and 3′ end processing, and mutations in Paf1 subunits allow siRNA-mediated heterochromatin formation and silencing of euchromatic genes. Nup132 is a component of the nuclear pore complex (NPC) that has been linked to mRNA export factors. Bdf2 is a histone-binding protein that has been shown to inhibit the spreading of centromeric heterochromatin. Epe1 is a JmjC-domain containing putative demethylase that also promotes spreading of heterochromatin. Mst2 is a histone aceyltransferase. See main text for references. b–c, Around 1,000 cen::ade6⁺ mst2∆ (b) or cen::ade6⁺ leo1∆ (c) cells were plated on low adenine medium (Low Ade). Most cells formed white colonies, indicating expression of endogenous ade6⁺, although approximately 2% of mst2∆ (b) and 12% of leo1∆ (c) cells formed red or pink colonies, indicating silencing of endogenous ade6⁺ (white arrow). Upon replating, the resulting red colonies formed mostly red colonies, indicating efficient maintenance of the silent state. Experiment was performed twice with similar results for each. d–e, ChIP–qPCR assays showing mean ± s.d. H3K9me2 levels at the vtc4⁺ locus, which is located next to the ade6⁺ gene, in the indicated mutant cells on the basis of two (d) or three (e) independent clones. P values are based on a two-tailed Student's t-test comparing the indicated mutants to wild-type cells. f–h, ChIP–qPCR assays showing mean ± s.d. RNA polymerase II occupancy at the ade6⁺ (purple) or vtc4⁺ (blue) locus in mlo3∆ or leo1∆ clones that have not been selected for silencing (99.5% or 88% white, respectively). P values are based on a two-tailed Student's t-test comparing the indicated mutants to cen⁺ mlo3∆ (f) or to wild-type cells (g). In h, either an ade6⁺ ON (W) or ade6⁺ OFF (R) colony from each of the two clones was picked for analysis. P values are based on a two-tailed Student's t-test comparing the indicated red to white cells for each clone. Three biological replicates were used per sample.