Accumulation of RNA on chromatin disrupts heterochromatin. (A) Scheme showing the constructs used. One half of tlh (5′tlh: first half; 3′tlh: second half) or LEU2 were inserted ∼5.5 kb upstream of tlh under the thiamine-inducible nmt1 promoter. The ade6 reporter gene was inserted 5 kb upstream of the inducible expression system. Cells that silence ade6 are red, while ade6-expressing cells are white when grown on low adenine plates. Heterochromatin maintenance of a silenced, red colony was analyzed for each strain on YE (low adenine, thiamine +) or EMMC low ade (10 mg/L adenine, thiamine −) plates. (B) Growth assay with ade6 reporter gene showing nmt1-5′tlh and nmt1-LEU2 cells. nmt1-5′tlh cells show strong loss of heterochromatic silencing when grown under inducible conditions. Expression of a control LEU2 gene did not lead to increased loss of heterochromatin when expression was induced. Cells were plated in 10-fold dilutions starting with 105 cells. (C) Quantification of different colored cells is shown for the indicated strains and expression, respectively. The total number of colonies counted is indicated above the bars. (D) Quantification of chromatin-associated RNA in indicated strains and conditions by H3 RIP RT-qPCR. In nmt1-5′tlh strains, 5′tlh RNA is enriched at chromatin in EMMC (induced) compared with YES medium (repressed). In nmt1-LEU2 strains, neither tlh nor LEU2 RNA is changed upon induction of LEU2 expression. (E) RNAi or Shelterin complex recruit the CLRC methyltransferase and SHREC deacetylase complexes to establish heterochromatin in wild-type cells. CLRC and SHREC spread into repeats to establish heterochromatin. The Ccr4-Not complex eliminates heterochromatic RNA and promotes spreading over the transcribed region. In the absence of the Ccr4-Not complex and RNAi, heterochromatic transcripts accumulate on the chromatin. This leads to loss of heterochromatin. At the transcribed loci, heterochromatin is lost in caf1Δdcr1Δ cells, indicating that at transcribed regions, RNA degradation is required for heterochromatin assembly.
