Heterochromatic transcripts accumulate on chromatin. (A) H3-RIP sequencing reads in indicated cells are plotted over the subtelomeric region. Total RNA sequencing reads on top as control. Reads from + and − strands are depicted in orange and gray, respectively. Scale bars on the right denote RNA read numbers per total number of reads mapping to protein coding genes. Compared with total RNA, tlh is enriched at the chromatin in wild-type and caf1Δdcr1Δ cells. Region where DNA:RNA hybrids are enriched is highlighted. Scale bars on the right denote RNA read numbers normalized to total number of reads mapping to protein coding genes. (B) Scatter plot showing H3-associated RNA relative to RNA Pol II–bound nascent RNA. A subset of genes is more enriched in H3 RIP than bound to RNA Pol II, indicating post-transcriptional retention on chromatin. Lowly expressed genes show stronger enrichment on chromatin. RNAs enriched on chromatin are shown in orange or red for heterochromatic RNA. (C) Box plot analysis of H3-associated RNA relative to nascent RNA bound to RNA Pol II in wild-type cells. (lncRNA) Euchromatic noncoding RNA with annotation SPNCRNA, n = 1354; (mRNA) all protein coding genes without genes in heterochromatic areas, n = 5014; (heterochromatic) genes which are located in constitutive heterochromatin areas, n = 62. Relative to their transcript levels, ncRNAs and especially heterochromatic RNA are more likely to be bound to chromatin than mRNA. (***) P < 0.001. (D) Box plot analysis of nascent RNA bound to RNA Pol II relative to RNA Pol II ChIP in wild-type cells. Genes that have RNA enriched on chromatin show reduced transcription compared with the quantity of RNA Pol II on the chromatin. (all) All mRNA and lncRNA, n = 6345; (chromatin enriched) euchromatic genes (mRNA and lncRNA) with greater than fourfold enrichment in H3-RIP over nascent RNA, n = 258. (***) P < 0.001. (E) Box plot analysis of H3-associated RNA in caf1Δdcr1Δ cells relative to wild-type cells. In caf1Δdcr1Δ cells, heterochromatic RNA and lncRNA are even more associated with chromatin than in wild-type cells. (mRNA) n = 5058; (lncRNA) n = 1467; (heterochromatin) n = 90. (***) P < 0.001. (F) DNA:RNA hybrid sequencing in wild-type and caf1Δdcr1Δ cells plotted over the subtelomeric region. DNA:RNA hybrids are formed with same sequences, which are also enriched in H3-RIP (highlighted). Scale bars on the right denote the ratio of “sample not treated” over “sample treated” with RNase H (−RNase H/+RNase H) before immunoprecipitation.
