Fig 2: INTS11 regulates pause-release and transcription dependent on H3K4me3.a, The strategy for CRISPR-based Flag–APEX2–RPB1 (RNAPII–APEX2) tagging. b, Validation of H3K4me3-dependent INTS11 chromatin interaction in DPY30–mAID cells. c, Western blot analysis of HA-tagged INTS11 and actin in INTS11–FKBP cells. d, HA-tagged INTS11 and total RNAPII ChIP–seq profiles and heat maps in INTS11–FKBP cells. e, mNET–seq profiles and heat maps in INTS11–FKBP degron cells. f, The RNAPII pausing index was determined using mNET–seq in INTS11–FKBP cells. The box plots indicate the median (centre line), the third and first quartiles (box limits) and 1.5 × IQR above and below the box (whiskers). P values were calculated using two-sided Wilcoxon tests. n = 10,332 genes. g, Metagene transcriptional profiles were acquired using TTchem-seq in INTS11–FKBP degron cells. h, Metagene analyses of mNET–seq and TTchem-seq signals at single-nucleotide resolution acquired in INTS11–FKBP cells. MNase-seq, micrococcal nuclease sequencing. i, Analysis of elongation velocities (TTchem-seq/mNET–seq) after acute loss of INTS11. j, Immunoblot analysis of integrator subunits in the indicated cell lines treated with or without auxin or dTAG-13 as shown. k, Box plot comparing the log2-transformed fold change in SLAM-seq at the indicated timepoints. The box plots indicate the median (centre line), the third and first quartiles (box limits) and 1.5 × IQR above and below the box (whiskers). n = 13,776 genes. l, The INTS11 signal at TSSs (± 2 kb) in the indicated groups. The box plots indicate the median (centre line), the third and first quartiles (box limits) and 1.5 × IQR above and below the box (whiskers). n = 4,700 genes. m, The average distribution of INTS11 and H3K4me3 ChIP–seq signals at INTS11-bound genes (n = 8,712) versus INTS11-unbound genes (n = 14,955) in mES cells. n, 2D kernel density plot showing the relationship between SLAM-seq changes in INTS11–FKBP and DPY30–mAID degron cells. The colour bar reflects the intensity.

Fig 3: DPY30 and RBBP5 are required for cell proliferation.(a) Cell proliferation assays of DPY30–mAID and RBBP5–FKBP cells grown either with or without Auxin/dTAG-13. The line graph represents the mean ± SD of the numbers of cells counted at each time point. Parental represents the parental E14 ES cells without CRISPR editing. Ectopic expression (EE) of DPY30 or RBBP5 rescues the proliferation of defect observed by degrading endogenous DPY30 and RBBP5, respectively. Data are from three biological replicates (n = 3) and were analysed using Two-way ANOVA (n = 3) and represented as mean ± s.d., ***P < 0.001. (b) Colony formation assay for DPY30–mAID and RBBP5–FKBP cells grown either with or without Auxin/dTAG-13 for two weeks. (c) Quantification of colony formation assays from two independent experiments. Data in bar plots are represented as mean and n = 2 replicates. (d) Cell proliferation assays of DPY30–mAID cells after treatment with Auxin at the indicated times with or without Kdm5ab dKO. (e) RT-qPCR analyses of mRNA expression of DPY30–mAID cells with or without Kdm5ab dKO. Two independent dKO clones were chosen for downstream analysis. The values are normalized to 18S rRNA. Graph shows mean values from technical triplicates (n = 3), from one representative out of two independent experiments. (f) ChIP-qPCR enrichment for WDR5, SETD1B and MLL1 in DPY30–mAID cells system after treatment with Auxin at the indicated points. Graph shows mean values from technical triplicates (n = 3), from one representative out of two independent experiments.

Fig 4: H3K4me3 regulates the paused RNAPII half-life.(a) Half-lives of paused RNAPII in control and Auxin-treated DPY30–mAID degron cells. The half-life was calculated based on an exponential decay model. Normalized promoter-proximal RNAPII density for each gene is shown over the course of triptolide treatment both for control (DMSO, black) and Auxin (blue) conditions. n = 2 biological replicates. (b) Boxplot showing increased paused RNAPII duration following H3K4me3 loss. P value was calculated with a two-sided Wilcoxon test, n = 4,007 genes. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box. (c) An IGV snapshot comparing mNET–seq signals after triptolide-induced block of transcription in control and Auxin-treated in DPY30–mAID cells at the indicated time points. The decreasing levels of paused RNAPII were observed in the pausing window (shaded areas) over time. (d) Heat maps and profiles showing changes in mNET–seq signals upon acute loss of H3K4me3. Heat maps were plotted as increasing gene length over regions 5 kb upstream to 50 kb downstream of the TSS. The right panel display the difference (?) in mNET–seq between control (+ Auxin 0 h) and Auxin-treated (+ Auxin 8 h) cells. Genes were sorted by gene length. (e–f) Heat maps and profiles showing changes of H3K4me3 ChIP–seq and mNET–seq at promoters, active and inactive enhancers upon Auxin treatment (8 h) in DPY30–mAID degron cells. The signals were plotted over the transcription start sites (TSS ± 5 kb) or the centre of enhancers (centre ± 5 kb). (g) H3K36me3 ChIP–seq profiles in control (+ Auxin 0 h) and Auxin-treated (+ Auxin 8 h) cells in DPY30–mAID cells. Genes were split by their H3K4me3 and H3K27me3 levels around TSS regions. (h) Histogram of RNAPII elongation rates for individual genes between 60 and 300 kb with RPM value =100 across all time points (n = 855) with a 10-min wave peak called beyond 2 kb and sequential increase from the TSS over the 10-, 20- and 30-min time points. (i) Heat maps and profiles showing changes of ChIP–seq, mNET–seq and TTchem-seq at promoters upon Auxin treatment (8 h) in DPY30–mAID degron cells. The downregulated genes and unchanged genes measured by SLAM-seq. The signals were plotted over the transcription start sites (TSS ± 5 kb). (j) Boxplot showing RNAPII elongation rates following H3K4me3 loss. P value was calculated with a two-sided Wilcoxon test. The number of genes (n) from each group are shown at the bottom. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box.

Fig 5: INTS11 is required for nascent transcription.(a) Venn diagram indicating overlap of H3K4me3 interactors and RNAPII-APEX2 dependent interactors from ChIP-MS (chromatin proteomic profiling) data. (b) Relative enrichments of selected targets in various ChIP preparations based on ChIP-MS. (c) Validation of INTS11 interaction with H3K4me3 in RBBP5–FKBP degron cells at different times after dTAG-13 addition. Biotinylated proteins within lysates were enriched using Streptavidin-coated magnetic beads and analysed by Western blot. In parallel, sample in which H2O2 was omitted was prepared as negative control. (d) Schematic representation of the dTAG INTS11 targeting strategy for the INTS11–FKBP degron mES cells. (e) Western blot showing the expression of INTS11 and INTS11–FKBP–HA, using antibodies recognizing INTS11 or the HA in parental and knock-in degron cells. The arrow indicates the specific HA-tagged INTS11–FKBP–HA protein. (f) RT-qPCR analysis showing the expression of selected pluripotency and differentiation genes in the parental and INTS11–FKBP knock-in cells. Data are from three biological replicates (n = 3) and are analysed using Two-way ANOVA and represented as mean ± s.d. (g) Growth curve analysis of parental and INTS11–FKBP E14 cells treated with or without dTAG-13. (h) INTS11 enrichment profiles and heat maps as determined by using the HA-tag in control (0 h) and Auxin-treated (2 h) DPY30–mAID; INTS11–FKBP degron cells. Genome-wide binding averages showed enrichments at the TSS regions (TSS ± 2 kb) of protein coding genes. TSS, transcription start site. Rows were sorted by decreasing ChIP–seq occupancy in the control (0 h) cells. (i) Correlations between TTchem-seq replicate experiments in INTS11–FKBP degron cells treated with or without dTAG-13 for the indicated times. (j) Average profiles for TTchem-seq for the upstream anti-sense RNAs of each annotated protein-coding gene in INTS11 degron cells. TSS, transcription start site. (k) RNAPII profiles of various subclasses of annotations in INTS11–FKBP degron cells with or without dTAG-13 treatment. TSS, transcription start site. mRNA, messenger RNA. snRNA, small nuclear RNA. ncRNA, non-coding RNA. eRNA, enhancer RNA.