Figure 3. Comparison of ZC3H4 and Integrator effects.

(a) Metagene analysis of chromatin-associated RNA-seq performed on cells treated with control or INTS1-specific siRNA. The plot shows signals upstream, across, and downstream of protein-coding genes. Y-axis scale is RPKM. Positive and negative values represent sense and antisense reads, respectively. (b, c) IGV traces of HAP1 and NWD1 genes derived from chromatin-associated RNA-seq in control and INTS1 siRNA treated samples and nuclear RNA-seq from control or ZC3H4 siRNA treatment. NWD1 transcripts are affected by ZC3H4 but not INTS1, whereas the opposite is true for HAP1 RNAs. Y-axes scales are RPKM. (d) Venn diagram showing the number of mRNAs upregulated ≥2-fold, padj ≤0.05 following ZC3H4 depletion versus INTS1 loss and the overlap between the two sets. Genes that showed increased expression due to transcription read-through from an upstream gene were filtered by assessing coverage over a 1 kb region preceding the TSS, relative to untreated cells. (e) Graphs demonstrating the expression level of mRNA transcripts upregulated (log2FC >1) following ZC3H4 or INTS1 depletion by comparison with transcripts unaffected by loss of either factor. Y-axis shows normalised gene counts (i.e. expression level). (f) Comparison of chromatin-associated RNA-seq in control and INTS1 siRNA treated samples with nuclear RNA-seq derived from control or ZC3H4 siRNA treatment. The ITPRID2 PROMPT is displayed and y-axes are RPKM (note the different scales between ZC3H4 and INTS1 samples). (g) Comparison of chromatin-associated RNA-seq in control and INTS1 siRNA treated samples with nuclear RNA-seq derived from control or ZC3H4 siRNA treatment. The MSRB3 SE is displayed and y-axes are RPKM (note the different scales between INTS1 and ZC3H4 samples). (h) Metaplot of RNA-seq profile over super-enhancers following INTS1 depletion (log2 fold depletion/control over 111 super-enhancer as line graphs). The bed file detailing super-enhancer coordinates in HCT116 cells was taken from dbSUPER.org. RPKM = reads per kilobase of transcript, per million mapped reads, TSS = transcription start site.

Figure 3—figure supplement 1. Comparison of ZC3H4 and INTS1 depletion on mRNA and PROMPT transcripts.

(a) IGV snapshots showing examples of protein-coding genes selectively upregulated by ZC3H4 (PJVK and ENO3) or Integrator (TM7SF2 and GFPT2). Y-axis represents RPKM. (b) Venn diagram representing the number of protein-coding transcripts (determined by DESEQ2) that show increased levels in previously published 4sU labelling experiments performed on HeLa cells depleted of INTS11 or ZC3H4 (Austenaa et al., 2021; Lykke-Andersen et al., 2020). Gene lists are provided in Supplementary file 5. Notably, manual curation of this list revealed the presence of false positive hits, especially in the INTS11 data, due to DESEQ2 scoring interference of transcription from neighbouring genes as upregulation. (c) Venn diagram showing the number of PROMPTs showing upregulation (log2 fold of 1 or more) following ZC3H4 or INTS1 loss from HCT116 cells and those that are common between the two conditions. (d) Graph showing the number of PROMPTs enhanced by greater than 0.5, 1, 2, or 3 on a log2 fold scale following the loss of ZC3H4 or INTS1. This illustrates that the effects of ZC3H4 loss are both widely spread and larger than those associated with depletion of INTS1. The list of targets in each case is provided in Supplementary file 6. RPKM = reads per kilobase of transcript, per million mapped reads, PROMPT = promoter upstream transcripts.