RSC and GRFs confer promoter directionality by restricting divergent noncoding transcription

(A) Approach for calculating directionality score for each promoter. In short, the nascent RNA-seq signals 500 bp upstream (antisense strand) and 500 bp downstream (sense strand) of the coding gene transcription start site were taken. Subsequently, the ratio of sense over antisense signals (log₁₀) was computed, which was defined as the directionality score. Another approach was also described in Jin et al (2017). (B) Density plots of directionality scores for tandem non-overlapping genes (n = 2,609) and Rap1-regulated genes (n = 141). (C) Relative changes in divergent (red) or sense (blue) transcription levels in cells depleted (dpl) for Rap1 or Sth1 (RAP1-AID or STH1-AID) in comparison to the corresponding mock-treated cells (IAA/DMSO) for Rap1-regulated gene promoters (n = 141). The nascent RNA-seq signals in the regions upstream (500 bp on the antisense strand, red) and 500 bp downstream (sense strand, blue) of the transcription start site were quantified and compared. Each dot represents one gene, for which the average log₂(FC) value between replicates is shown. (D) Density plot representing promoter directionality of control (RAP1-AID +DMSO) and Rap1-depleted cells (RAP1-AID + IAA) for Rap1-regulated genes (n = 141). Average values between biological replicates (n = 3) are shown. (E) Similar as (D), except comparing control (STH1-AID + DMSO) and Sth1-depleted (STH1-AID + IAA) cells.

(A) Volcano plots of nascent RNA-seq data comparing Sth1-depleted to control cells (log₂(FC) IAA/DMSO) for different classes of transcription units. Displayed are plots for protein-coding genes and noncoding transcripts: CUTs, SUTs, XUTs, and NUTs. Numbers of loci with significantly increased (UP) or decreased (DOWN) levels of transcription are displayed (Padj < 0.05 and log₂(FC) > 1). Average values between biological replicates (n = 3) are shown. (B) Violin plots displaying the relative changes in divergent (red) or sense (blue) nascent RNA-seq signal of tandem non-overlapping protein-coding genes (n = 2,609). Comparisons between Sth1 depletion (STH1-AID + IAA) and control (STH1-AID + DMSO) or control versus WT are shown. The nascent RNA-seq signals in the regions upstream (500 bp on the antisense strand, red) and 500 bp downstream (sense strand, blue) of the transcription start site were quantified and compared. Average log₂(FC) values between replicates (n = 3) are shown. (C) Promoter directionality score of tandem non-overlapping protein-coding genes (n = 2,609) in WT, control (STH1-AID + DMSO), and Sth1-depleted (STH1-AID +IAA) cells. Average values between replicates (n = 3) are represented.

(A) Violin plots representing the distribution of changes in divergent transcription (left), sense transcription (centre), and directionality score (right) in Sth1-depleted versus control cells (log₂(FC) IAA/DMSO), in sets of tandem genes stratified according to their directionality in WT cells (x-axis, Q1 to Q5). The range for directionality scores for each group are the following: Q1(−3.11–0.85), Q2(0.86–1.49), Q3(1.50–1.97), Q4(1.98–2.48), Q5(2.49–4.20). Average values between replicates (n = 3) are represented. (B) Examples of loci that display increased divergent transcription and changes in chromatin structure upon Sth1 depletion. The data of a representative replicate of the nascent RNA-seq, mRNA-seq, and MNase-seq are shown. (A, C) Metagene analysis of MNase-seq data for Q1 and Q5 (directionality in WT cells) obtained as described in (A). Plots are centred on annotated gene transcription start site, and they display MNase-seq signals for chromatin extracts treated with low MNase or high MNase concentrations, obtained from Kubik et al (2015, 2018) (n = 1). Marked are the regions representing the +1, −1 nucleosome, and −2 nucleosome positions. (C, D) Heatmap of MNase-seq data represented in (C), centred on the annotated gene transcription start site.

(A) Metagene analysis and heatmaps of CUT&RUN data of Sth1 (RSC) and the general regulatory factors Abf1 and Reb1. In addition, signals of Sth1 CUT&RUN followed by histone H2B immunoprecipitation are displayed. Data from Brahma and Henikoff (2019). A comparison between genes belonging to directionality Q1 (purple), the group of gene promoters with lowest directionality score, and Q5 (green), the group with the highest directionality score, is shown. The data represent one of n = 2 biological repeats. (A, B) Distribution of the RSC-associated A-track motif for both the sense and antisense strand for Q1 and Q5 overlaid with the RSC CUT&RUN data from panel (A). (C) Data from RNA Pol II CRAC and Reb1-ChEC-seq datasets described previously (Zentner et al, 2015; Candelli et al, 2018), representing RNA Pol II transcription in the presence or absence of Reb1 (top) and binding of Reb1 (bottom), respectively. Displayed are the BAP2 and SCO1 loci, which show increased divergent transcription upon Reb1 depletion. The positions of the Reb1-binding sites (BS) are indicated.

(A) Schematic overview of the reporter construct. The transcription factor binding sites were cloned 20 nucleotides upstream of the YFP (SUT129) transcription start site (TSS). (B) YFP, noncoding, signal for constructs harbouring binding sites for Gal4, Gcn4, Cat8, Gcr1, Cbf1, Abf1, and Reb1 cloned into the WT PPT1 promoter (FW6407). The YFP activity was determined in WT control cells and in gene deletion strains of matching transcription factor binding site reporter constructs (FW6404, FW6306, FW6401, FW6300, FW6402, FW6302, FW6403, FW6424, FW6405, and FW6315). For Abf1 and Reb1 reporter constructs, Abf1 and Reb1 were depleted using the auxin-inducible degron (ABF1-AID and REB1-AID) (FW6415 and FW6411). Cells were treated for 2 h with IAA or DMSO. Displayed is the mean signal of at least 50 cells. The error bars represent 95% confidence intervals. (C) Same analysis as B except that coding direction is shown. (B, D) Same analysis as (B) except that directionality was calculated by taking the ratio of coding over noncoding. (E) Scheme depicting the targeting of dCas9 to repress the divergent transcript IRT2. (F) Design of gRNAs targeting the IRT2 TSS. Displayed are TSS-seq data from after Rap1 depletion (Wu et al, 2018). (G) Western blot of Rap1 and dCas9 detected with anti-V5 and anti-FLAG antibodies, respectively. Cells harbouring RAP1-AID were treated with IAA to deplete Rap1 and induce IRT2 expression in either cells with empty vector (EV) or cells harbouring a dCas9 construct and a construct expressing the gRNA targeted to the Rap1-binding site (FW8477, FW8531). Hxk1 was used as a loading control, detected using anti-Hxk1 antibodies. (E, H) IRT2 expression detected by Northern blot for strains and treatments described in (E). As a loading control, the membrane was re-probed for SNR190. (D, I) IRT2 expression as detected in by qRT-PCR of as described in (D). A control gRNA was included in the analysis targeted to the TSS of TEF1 (FW8527). Displayed are the mean signals of n = 3 biological repeats and SEM. (J) Model for promoter directionality.

Source data are available for this figure.