Fig 2: TOX4 promotes Pol II recycling and transcriptional reinitiation through facilitating CTD dephosphorylation.a Metagene profiles of TT-seq of protein-coding genes in TOX4 KO versus control cells. b Normalized read distribution of TT-seq within the C11orf21 and TSPAN32 loci in TOX4 KO versus control cells. c A volcano plot of TT-seq showing transcriptional output changes of TOX4-bound genes upon TOX4 loss. d A heatmap of TT-seq showing TOX4 direct targets with transcriptional output fold change ≥ 1.2 or ≥ 1.5 upon TOX4 loss. e Comparison of levels of free (up) and chromatin-bound (down) total, Ser-5 phosphorylated, and Ser-2 phosphorylated Pol II by Western blot in control and TOX4 KO cells. f Correlation plots for biological replicates of CUT&Tag of TBP. g Genome-wide meta-gene profiles and heatmaps of CUT&Tag comparing chromatin occupancies of TBP in TOX4 KO versus control cells. Genes were sorted by total Pol II CUT&Tag signal in control cells. h Nine groups of genes sorted according to their Pol II TR and transcriptional output status upon TOX4 loss. i Pol II metagene profiles of eight out of the nine groups of genes in h. j Cumulative frequency curves comparing mRNA levels of Groups 1, 2, 4, and 5 genes in h in K562 cells. k Working model of TOX4 in transcriptional regulation. TOX4 restricts pause release and early productive elongation by facilitating Pol II CTD Ser-2 and SPT5 Thr-806 dephosphorylation, and promotes reinitiation by facilitating dephosphorylation of serines 2 and 5 of Pol II CTD.

Fig 3: TOX4 may facilitate Pol II CTD dephosphorylation.a Comparison of cellular levels of total, Ser-5 phosphorylated and Ser-2 phosphorylated Pol II by Western blot in control and TOX4 KO cells. Top: Western blot images, Bottom: A bar graph showing relative levels of Ser-5 phosphorylated Pol II and Ser-2 phosphorylated Pol II quantified by ImageJ in control and TOX4 KO cells. Pictures are representative of three independent experiments. Statistical significance was determined with a two-sided Student’s t-test; the centers and the error bars represent the mean and the SD, respectively. *P < 0.05, **P < 0.01. b–d Correlation plots for biological replicates of CUT&Tag of total (b), Ser-5 phosphorylated (c), and Ser-2 phosphorylated (d) Pol II. e Normalized read distribution of CUT&Tag of total, Ser-5 phosphorylated and Ser-2 phosphorylated Pol II within the SERBP1 locus in TOX4 KO versus control cells. f–h Genome-wide meta-gene profiles and heatmaps of CUT&Tag comparing chromatin occupancies of total (f), Ser-5 phosphorylated (g), and Ser-2 phosphorylated (h) Pol II in TOX4 KO versus control (Ctrl) cells. A genome-wide meta-gene profile and a heatmap of TOX4 CUT&Tag in control cells are placed to the left of those of Pol II to facilitate comparison of TOX4 and Pol II occupancies. Genes were sorted by total Pol II CUT&Tag signal in control cells. i A correlation plot of CUT&Tag data of TOX4 and Pol II. j A Venn diagram showing overlap between TOX4-bound and Pol II-bound promoters. Statistical significance of the overlap between them was determined with a Hypergeometric test.

Fig 4: Chromatin occupancy pattern of TOX4 resembles that of Pol II.a Correlation plots for biological replicates of ChIP-seq (left) and CUT&Tag (middle) of TOX4, and for ChIP-seq versus CUT&Tag of TOX4 (right). b Annotation of TOX4 occupancy. Left: a Venn diagram showing overlap between TOX4 peaks identified by ChIP-seq and CUT&Tag, respectively, Right: TOX4 peak distribution across genomic features. c Normalized read distribution of TOX4 ChIP-seq (top), TOX4 CUT&Tag (middle) and Pol II CUT&Tag (bottom) within the SERBP1 locus in K562 cells. d Generation and characterization of a TOX4 knockout K562 cell line. Left: Schematic of TOX4 knockout strategy. Right: Characterization of the TOX4 KO K562 cell line by Western blot. e Comparison of proliferation of control and TOX4 KO cells by MTT assays. Statistical significance was determined with a two-sided Student’s t-test; the centers and the error bars represent the mean and the SD of five independent experiments, respectively. NS: P ≥ 0.05. f Comparison of association of WDR82 or PP1γ with PNUTS by co-IP in control and TOX4 KO cells. g A Venn diagram showing overlaps between TOX4-bound genes and downregulated or upregulated genes of RNA-seq. h A volcano plot showing expression changes of TOX4-bound genes upon TOX4 loss. i A heatmap comparing expression of TOX4 direct targets in TOX4 KO versus control cells. j Pathway analysis results of downregulated genes upon TOX4 loss. k Meta-gene profiles and heatmaps of ChIP-seq (left) and CUT&Tag (right) each showing a positive correlation between TOX4 occupancies and mRNA levels of TOX4 direct targets in K562 cells. Genes were sorted according to TOX4 occupancy level detected by ChIP-seq (left) and CUT&Tag (right), respectively, in control cells. High: top 25% of the non-silent genes (mean TPM ≥ 1); Medium: non-silent genes between top 25% and bottom 25%; Low: bottom 25% of the non-silent genes or genes with mean TPM < 1.

Fig 5: TOX4 facilitates promoter-proximal pause of Pol II.a Schematic representation describing the calculation of traveling ratio (TR) at each Pol II-bound gene. b A cumulative distribution plot comparing Pol II TRs of TOX4-bound genes in control and TOX4 KO cells. c A scatter plot comparing Pol II TRs of TOX4-bound genes in TOX4 KO versus control cells.