Figure 5.

Two Mechanisms for Reb1-Nucleosome Interactions Establish Promoter Unidirectionality

(A) We used auxin-induced degradation to examine how promoter organization changes following depletion of Sth1 (the catalytic unit of the RSC complex). Showing average nucleosome occupancy before auxin addition (Sth1 is intact) and 120 min after addition (Sth1 is depleted for >90 min). Cluster 1 promoters exhibit the expected shift of nucleosomes −1 and +1 into the nucleosome-depleted region. Cluster 4 promoters show stable nucleosome −1, which is consistent with its co-occurrence with the bound Reb1 as suggested by the V-plot.

(B) Average transcribing Pol II occupancy (NET-seq) for clusters 1 and 4 aligned by the Reb1 site and oriented according to the nearest TSS. Top: transcription in the orientation of the TSS. Bottom: anti-sense transcription to the TSS. Both clusters show transcription start at the downstream TSS, but differ in the distance to anti-sense TSS. Cluster 4 also shows aborted short upstream transcripts terminating at the Reb1 site.

(C) Similar to (B) for two subclusters of cluster 4 divided according to presence of aborted upstream transcript (Figure S5D).

(D) Similar to (A) for the two subclusters, showing that the −1 nucleosome in the first subcluster is insensitive to RSC (locked), and in the other subcluster, the −1 is pushed toward Reb1 site by RSC.

(E) Examples of three sites belonging to cluster 1 (isolated), cluster 4a (locked), and cluster 4b (pushed), showing nucleosomes as in (A) and (D) and transcription as in (B) and (C).

(F) Schematic model of how Reb1 binding and RSC activity affect transcription in different promoter architectures.

See also Figure S5.