RBBP6 loss disrupts PAS-dependent termination of sense transcription.

A. Western blot demonstrating the depletion of dTAG-RBBP6 over a time course of dTAGv-1 addition. SPT5 serves as a loading control.

B. Genome browser track of NEDD1 in POINT-seq data from dTAG-RBBP6 cells treated or not (2hr) with dTAGv-1. RBBP6 depletion induces a termination defect in the protein-coding direction (downstream of the indicated PAS) but not the upstream antisense direction. The y-axis shows Reads Per Kilobase per Million mapped reads (RPKM).

C. Metaplot of POINT-seq data from dTAG-RBBP6 cells treated (RBBP6 dep) or not (Ctrl) (2hr) with dTAGv-1. This shows 1316 protein-coding genes selected as separated from any expressed transcription unit by ≥10kb. Signals above and below the x-axis are sense and antisense reads, respectively. The y-axis scale is RPKM. TSS=transcription start site; TES=transcription end site (this marks the PAS position). This is an average of two biological replicates.

D. Heatmap representation of the data in C, which displays signal as a log2 fold change (log2FC) in RBBP6 depleted versus un-depleted conditions. This is an average of two biological replicates.

INTS11 loss disrupts the termination of antisense transcription.

A. Western blot demonstrating homozygous tagging of INTS11 with dTAG and the depletion of INTS11-dTAG after 1.5hr treatment with dTAGv-1. INTS8 serves as a loading control.

B. Genome browser track showing POINT-seq signal over RNU5A-1 and RNU5B-1 in INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1. Note, that INTS11 depletion induces a termination defect in each case. Y-axis shows RPKM following spike-in normalisation.

C. Genome browser track showing POINT-seq signal over PGK1 and its upstream antisense region in INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1. Y-axis shows RPKM following spike-in normalisation.

D. Metaplot of POINT-seq data from INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1. This shows the same 1316 genes used in Figure 1C. Signals above and below the x-axis are sense and antisense reads, respectively. Y-axis shows RPKM following spike-in normalisation. This is an average of three biological replicates.

E. Heatmap representation of the data in D, which displays signal as a log2 fold change (log2FC) in INTS11 depleted versus undepleted conditions over a region 3kb upstream and downstream of annotated TSSs. This is an average of three biological replicates.

Sense direction transcription initiation is more efficient and focused.

A. Schematic of sPOINT-seq protocol. The POINT-seq protocol is followed, in which chromatin is isolated and engaged RNAPII is immunoprecipitated. Short transcripts are preferentially amplified during library preparation (see Experimental Procedures for full details).

B. Comparison of POINT-(top trace) and sPOINT-seq (lower trace) on PGK1. Y-axis units are RPKM.

C. Metaplot comparison of POINT-(top plot) and sPOINT-seq (lower plot) profiles across the 684 highest expressed protein-coding that are separated from expressed transcription units by ≥10kb (top ∼50%). Signals above and below the x-axis are sense and antisense reads, respectively. Y-axis shows RPKM following spike-in normalisation.

D. Top metaplot shows full read coverage for sPOINT-seq performed in INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1 at the promoters of the top expressed 20% of protein-coding genes. The lower metaplot is the same data but only the 5’ end of each read is plotted. The y-axis signals are RPKM following spike-in normalisation. Two biological replicates of sPOINT were performed.

E. Genome browser track of PGK1 promoter region in sPOINT-seq. This showcases the focused sense TSS (black arrows) and the dispersed antisense reads (brackets). Note the higher y-axis scale (RPKM) for sense vs. antisense.

F. Metaplot zoom of the antisense TSS signals deriving from the lower plot in D. This makes clear the dispersed sites of initiation. The Y-axis scale is RPKM following spike-in normalisation. Two biological replicates of sPOINT were performed.

Some promoter directionality is lost when CDK9 is inhibited.

A. Genome browser track of TARDBP in POINT-seq data derived from INTS11-dTAG cells either untreated, dTAG treated, NVP-2 treated or dTAG and NVP-2 treated (2.5 hr). Signals above and below the Y-axis are sense and antisense reads, respectively. The Y-axis scale shows RPKM following spike-in normalisation.

B. Metaplot of POINT-seq in INTS11-dTAG cells depleted or not of INTS11 and treated or not with NVP-2 (2.5 hr). This uses the same gene set as Figure 1C. The regions 3kb upstream and downstream of genes are included. Y-axis units are RPKM following spike-in normalisation. This is an average of two biological replicates.

C. Metaplot of the same CDK9i + and -dTAG data shown in B but zoomed into the region 3kb upstream and downstream of the TSS. This is an average of three biological replicates.

D. Heatmap representation of the data in C, which displays signal as a log2 fold change (log2FC) in INTS11 depleted versus un-depleted conditions covering a region 3kb upstream and downstream of the TSS. This is an average of three biological replicates.

E. Western blot for total RNAPII and RNAPII phosphorylated on Ser2/5 (Ser2/5p) in INTS11-dTAG cells treated or not with dTAGv-1 and/or NVP-2 (as indicated). dTAG-mediated INTS11 depletion is shown in the anti-HA blot and SPT5 us used as a loading control.

F. qRT-PCR analysis of INTS11-dTAG cells transfected with the HIV reporter construct with or without TAT then depleted or not of INTS11. Quantitation shows signals relative to those obtained in the presence of INTS11 and the absence of TAT after normalising to MALAT1 RNA. n=3. Error bars show standard deviation. ** denoted p=0.01. Note that INTS11 depletion was performed concurrently with transection (14hr in total).

G. Model for promoter directionality depicting higher levels of focused transcriptional initiation in the sense direction together with opposing gradients of CDK9 and INTS11 activity that peak in sense and antisense directions, respectively.

Key Resources Table

A. Western blot showing three separate dTAG-RBBP6 cell clones. In each case, homozygous tagging is demonstrated by the size shift versus endogenous RBBP6 (HCT116 lanes). Tagged RBBP6 is completely depleted by 2hr treatment with dTAGv-1 whereas endogenous RBBP6 is unaffected. SPT5 serves as a loading control. Clone number 2 (red asterisk) was selected for the experiments in Figure 1.

B. Graph plotting the number of AAUAAA sequences in antisense transcripts derived from a 3kb window upstream of TSSs. Y-axis is the number of transcripts, and the x-axis shows the AAUAAA count per transcript.

C. Heatmap of control or RBBP6-depleted POINT-seq showing the RBBP6 effect on antisense transcripts without an AAUAAA or those that contain at least one AAUAAA. Most are unaffected by RBBP6 loss. The y-axis is RPKM.

A. Heatmap analysis of the 1316 gene set used in Figure 1C but focused on promoter-proximal transcription of the sense direction. Note the change in scale (Log2FC - INTS11/+INTS11) to reflect the smaller effect sizes vs. those for antisense transcription from the same gene set.

B. Comparison of INTS11-dependent changes in promoter-proximal protein-coding transcription with the expression level of each gene using the 1316 gene set as Figure 1C. X-axis shows Log2FC in levels (-INTS11/+INTS11) and y axis shows expression level. Note that genes with the largest increase in promoter-proximal signal following INTS11 loss are low expressed (coloured orange). Data derives from POINT-seq following treatment or not with dTAGv-1 (1.5hr).

C. Metaplot of POINT-seq data from INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1. These are protein-coding genes arranged head-to-head. Signals above and below the x-axis are sense and antisense reads, respectively. Y-axis shows RPKM following spike-in normalisation.

D. Metaplot of POINT-seq data from INTS11-dTAG cells treated or not (1.5hr) with dTAGv-1. This shows RNAs derived from enhancer clusters, which generally initiate transcription bidirectionally. Signals above and below the x-axis are sense and antisense reads, respectively. Y-axis shows RPKM following spike-in normalisation.

A. Plot showing the size distribution of fragments mapped by sPOINT-seq. This demonstrates its selectivity toward transcripts <150nts.

B. Genome browser track of ACTB promoter region in sPOINT-seq from INTS11-dTAG cells treated or not with dTAGv-1 (1.5 hr). This showcases the focused sense TSS (black arrows) and the dispersed antisense reads (brackets). Note the different y-axis scales (RPKM) for sense vs. antisense.

C. Violin plot of the sum sPOINT 5’end spike-in normalised RPKM signal across a 500bp window from the protein-coding TSSs shown in Figure 3D (n=3060). These samples are untreated with dTAG to quantitate the normal levels of sense vs. antisense initiation/pausing in a window -/+ 500bp from the TSS in respective directions for antisense and sense. This shows more initiation/pausing in sense vs. antisense directions.

D. Genome track of RNU5A-1 and RNU5B-1 in RNAPII ChIP-seq performed on INTS11-dTAG cells treated or not with dTAGv-1 (2hr). INTS11 depletion causes RNAPII build-up beyond both genes indicating a transcription termination defect. The Y-axis scale is RPKM.

E. Metaplot of RNAPII occupancy of protein-coding TSS regions (the same gene set employed for the sPOINT analysis in Figure 3) derived from RNAPII ChIP-seq performed on INTS11-dTAG cells treated or not with dTAGv-1 (2hr). The Y-axis scale is log10 qvalue of peak pileup values normalised to spike in control.

A. Comparison of INTS11-dependent changes in promoter-proximal protein-coding transcription with the expression level of each gene. The gene set is equivalent to the analogous panel in Supplemental Figure 2B. The x-axis shows Log2FC in levels (-INTS11/+INTS11) and the y-axis shows expression level. Note that genes with the largest increase in promoter-proximal signal following INTS11 loss are low expressed (coloured orange). Data derives from POINT-seq following treatment or not with dTAGv-1 (2.5hr).

B. Venn diagram showing the number of protein-coding genes where promoter-proximal POINT-seq signal increases by ≥Log2FC of 1 after INTS11 loss after 1.5h or 2.5h dTAGv-1 treatment. A strong overlap is seen indicating reproducible effects at the two time points.

C. qRT-PCR analysis of PSCA, SLC16A7, INTS6L, and KICS2 pre-mRNAs in INTS11-dTAG cells treated or not with dTAGv-1 for 1.5hr or 2.5hr. To enrich nascent transcripts, primers detect intronic RNA. Quantitation shows fold change versus spliced actin relative to untreated samples. Error bars show standard deviation. n=4. n.s denotes not significant.

D. qRT-PCR analysis of PGK1, ENY2, PLEKHF2, and NUDCD1 pre-mRNAs in INTS11-dTAG cells treated or not with dTAGv-1 and at the same time exposed or not to DRB (all 2.5hr). To enrich nascent transcripts, primers detect intronic RNA. Quantitation shows fold change versus spliced actin relative to samples untreated with dTAGv-1 or DRB. DRB treatment substantially reduces signal, which is restored when INTS11 is co-depleted. Error bars show standard deviation. n=3, * denotes p≤0.05.

E. As for C but following treatment with the CDK7 inhibitor, THZ1, and/or depletion of INTS11 with dTAGv-1 (all treatments 2.5hr). Error bars show standard deviation. n=3, * denotes p≤0.05.

F. qRT-PCR analysis of chromatin-associated (nascent) RNA isolated from INTS11-dTAG cells transfected with the HIV reporter construct together with TAT then treated or not with INTS11 and/or NVP-2. Quantitation shows signals relative to those obtained in untreated cells after normalising to MALAT1 RNA. n=4. Error bars show standard deviation. **denotes p≤0.01.

G. Flag immunoprecipitation performed in INTS11-dTAG cells transfected with flag-tagged wild-type or E203Q INTS11. The gel shows input and co-precipitated samples probed for components of different Integrator modules (tail module – INTS10; backbone module – INTS1; phosphatase module – INTS8 and cleavage module – INTS9). These components are poorly associated with E203Q INTS11 vs. wild type.