Figure 1.
Rpd3S and Rpd3L target overlapping sets of genes.
(A) Self-organizing map (SOM) clustering of Rpd3 binding on promoter and ORFs of the 3693 genes with no missing value. Red indicates enriched (bound) regions and blue represents depleted regions. (B,C) Mapping of Rpd3 (green), Rco1 (red) and Sds3 (blue) on the 954 genes from cluster 1 (solid line, genes where Rpd3 is present both on ORFs and promoters) and on the 1906 genes from cluster 3 (dashed lines, genes where Rpd3 is not bound) (B) and on the 833 genes from cluster 2 (solid lines, genes where Rpd3 is present only on promoters) and cluster 3 (C). (D) SOM clustering of Rco1, H3K36me3 and RNAPII on genes from the cluster 2 from panel “A”. Cluster 4 represents the 222 active genes with no Rco1 binding.
Figure 2.
The recruitment of Rpd3S to transcribed genes does not require Set2-dependent H3K36 methylation in vivo.
(A) SOM clustering of Rco1 enrichment on ORFs of all the 5007 genes with no missing value in WT and various mutants. (B,C) Mapping of RNAPII occupancy (B) and H3K36me3 enrichment (C) on genes contained within clusters 5 (green, 1425 genes) and 6 (blue, 841 genes) from panel “A”. (D) Mapping of Rco1 in WT (solid lines) and set2Δ (dashed lines) cells along genes contained within clusters 5 (green) and 6 (blue) from panel “A”. (E) Western blot showing the Rco1 protein levels in the strains used in panels A–D. Note the decreased level of the PHD-truncated Rco1 protein. The star symbol (*) indicates a degradation product.
Figure 3.
Set2-dependent H3K36 methylation is required for the function of Rpd3S.
(A,B) SOM clustering of H4K5ac enrichment (A) and RNAPII occupancy (B) on ORFs of all genes in WT cells, along with the difference calculated between the enrichment observed in various mutants and WT cells.
Figure 4.
Spt4 negatively regulates the recruitment of Rpd3S.
(A) SOM clustering of Rco1 occupancy on ORFs of all genes in WT, and various mutants, together with Spt4 occupancy in WT on ORFs. The RNAPII ORF occupancy in WT was added after the clustering. (B) Correlation between Rco1 occupancy and RNAPII occupancy in WT (grey) and spt4Δ cells (black). A sliding median window of 300 genes was applied to the data. (C) Correlation between Spt4 occupancy and the difference of Rco1 occupancy in spt4Δ versus WT cells. A sliding median window of 300 genes was applied to the data. (D) Mapping of Rco1 occupancy on the 953 genes contained within cluster 8 in WT (black solid line), spt4Δ (dashed line) and spt4Δ/set2Δ (dotted line) cells. The RNAPII phosphorylated on serine 2 enrichment is also shown (red solid line). (E,F) Mapping of Rco1 occupancy on genes grouped according to their lengths in spt4Δ/ctk1Δ (E) and spt4Δ/set2Δ (F) cells (red line: the 465 genes >3000bp, green line: the 1703 genes between 1500–3000bp, blue line: the 2091 genes between 750–1500bp).
Figure 5.
Phosphorylation of the RNAPII and Spt5 CTD are important for the Rpd3S recruitment on genes.
(A) Mapping of Rco1 occupancy on the 2089 genes contained within clusters 7 and 8 in WT (solid line), ctk1Δ (dashed line), and kin28AS (dotted line) cells. (B) As in ‘A’ using the bur1AS (dashed line) and spt5ΔC (dotted line) cells.
Figure 6.
A model summarizing how Rpd3S is recruited to active genes.
Phosphorylation of the RNAPII CTD by Kin28 and Ctk1 stimulates the recruitment of Rpd3S to the CTD. DSIF (Spt4/Spt5) counteracts the recruitment of Rpd3S but its phosphorylation by Bur1 alleviates its repressing activity.