Abstract
A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction.
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Data availability
All sequencing data generated as a part of this study have been deposited to NCBI GEO under accession numbers GSE207268 and GSE207269 (NCBI tracking system no. 23081142). Source data are provided with this paper.
Code availability
All pipelines and customized codes are available on GitHub (https://github.com/Berialim/PROMPTs_analysis_visualization)77. Further information and requests for resources and reagents generated in this study are available upon request.
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Acknowledgements
We thank the Lai and Dang lab members for constructive discussions and help with genome-wide sequencing. We also acknowledge the Chen lab at the Institute of Biomedical Research at Yunnan University for their help with high-throughput sequencing. Thanks to Z. Xue for the technical help and discussions. This work was supported by grants from the Yunnan Province Science and Technology Department (202201BF070001-015 to F.L.), the National Natural Science Foundation of China (32070626 to F.L., 32171262 and 31871254 to Y.D.), the Open Research Program of State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan (2021KF002 to F.L.) and startup funds from Yunnan University for F.L. and Y.D.
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Contributions
F.L. and Y.D. conceived and supervised the overall project. J. Yang performed most of the experiments under the supervision of G.R. J.L. performed bioinformatics analysis. J.L. and J. Yang prepared the figures under the supervision of F.L. L.M. and X.G. generated the cell line with 3×U1 sequences. W.S. generated the CPSF73-AID2 cell lines and performed the ChromRNA-seq. L.M. performed the TT-seq. S.L. generated the HCT116:TIR1(F74A) TET-ON cell line. B.P. performed the cut-tag experiments for RNAPII and helped with the INTS11 overexpression experiments. B.P., Z.L. and W.S. generated the INTS9 cell line and performed the INTS9 ChromRNA-seq. S.C., X.G., B.W., A.D., D.H. and J. Yuan prepared all the samples for the high-throughput sequencing. F.L., Y.D., J. Yang and J.L. wrote the paper with critical feedback from all co-authors.
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Extended data
Extended Data Fig. 1 Correlation of PROMPT accumulation under INTS11 or INTS9 depletion.
a. Western blot representing the protein levels of INTS11/9 in wild-type and engineered cells from three biological independent experiments with the similar results. * represents the unspecific bands. b-c. Heatmap (b) and metagene analysis (c) showing HCT116-TIR1 untagged cell ChromRNA-seq results for 10428 genes and their PROMPTs in a 10 kb window around TSS site. “CTRL” represents the untreated control, and “+IAA” represents the treatment with 500 µM IAA. d. Box plots of the expression levels of 7070 PROMPTs and pre-mRNA with/without IAA treatment in HCT116-TIR1 cell line. The two-sided Mann-Whitney test was used for statistical analyses. n.s., not significant. Boxplot shows the minimum and maximum values, the median, and the interquartile range (IQR). The IQR is represented by the box, with the lower quartile (25th percentile) at the bottom and the upper quartile (75th percentile) at the top. Whiskers extend from the box, indicating the range of data points within a certain distance from the IQR. Outliers, values outside the whiskers, were excluded for better visualization. e. Quantitative RT-PCR of RNU11, MYC and CCND1 genes in HCT116-TIR1 cell line treated with 500 µM IAA (top) or 10 µM 5-ph-IAA (bottom). Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). n.s., not significant. (*) p < 0.05. (**) p < 0.01. f-g. IGV tracks of RNU11 and RNU12 loci from INTS11 (f) or INTS9 (g) ChromRNA-seq results. h. Heatmap of 7070 genes and their PROMPTs from ChromRNA-seq with or without INTS9 depletion (left). An overlaying heatmap of +IAA/CTRL is shown on the right. Upregulation is shown in red, and down-regulation is shown in blue, the control group was treated with equal volume DMSO. i. Correlation map of the log2-fold change in PROMPTs after INTS11 or INTS9 depletion calculated with the Spearman method. j. Histogram showing the length distribution of PROMPTs (PROMPTs were identified by PROMPT-finder.) in CTRL, INTS9 and INTS11 depletion conditions.
Extended Data Fig. 2 Depletion of Integrator leads to altered transcription patterns in the sense and antisense directions.
a. Heatmap representing highly expressed genes and their PROMPTs from ChromRNA-seq results with EV or ectopic expression of INTS11 protein. “+” represents IAA treatments. The corresponding differences by log2-fold change are shown in the right panel. Upregulation is shown in red, and downregulation is shown in blue. b-c. Boxplots representing the transcript levels from PROMPT (b) and pre-mRNA (c) for 2121 active genes. P values were calculated by two-sided Mann-Whitney test. The boxplots was plotted as Extended Data Fig. 1. d. IGV tracks of the MYC and BMP4 loci from ChromRNA-seq results in INTS11 (top) or INTS9 depletion (bottom). e. Heatmap representing 2121 genes and PROMPTs from ChromRNA-seq before and after INTS9 depletion(left). Heatmap representing 2121 genes and PROMPTs from ChromRNA-seq before and after INTS11 depletion and rescue (right). f. Metagene analysis of the average density of ChromRNA-seq signals after INTS9 depletion. g. Boxplots representing the transcript levels from PROMPT (left) and pre-mRNA (right) for 2121 active genes with or without INTS9 depletion. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots. h. Gene by gene transcription ratio plots representing PROMPTs (red) and gene (blue) FPKM values for high or low expressed genes. Genes categorized as Fig. 1g. Top 3 quantiles (8,9,10; 2,121 genes) for highly expressed genes. The bottom quantiles (1,2,3; 2,121 genes) for lowly expressed genes. The yellow line representing the transcription ratio as the log2 fold change of gene FPKM compared to PROMPT FPKM. i. The scatterplot illustrates the change in the transcription ratio for lowly expressed genes calculated using ChromRNA-seq. The transcription ratio represents the log2 ratio of gene FPKM versus PROMPT FPKM, indicating the directionality of transcription.
Extended Data Fig. 3 Validation of altered transcriptional changes upon INTS11 depletion by 4SU-seq and TT-seq.
a-b. IGV tracks of RNU11, RNU12, and TERC loci from TT-seq (a) and 4sU -seq (b) results before and after INTS11 depletion. c. Heatmap representing the identified 2121 active genes and PROMPTs from GSE223265 TT-seq results (left) and the overlaying heatmap of +IAA/CTRL (right). d. IGV tracks of 4sU-seq representing PROMPT accumulation at MYC or BMP4 loci. e-f. Heatmap (e) and Metagene analysis (f) showing accumulated transcripts in the PROMPT direction and reduced transcripts in the pre-mRNAs direction for 2121 active genes upon INTS11 depletion. g. Quantitative boxplots of transcription distribution at PROMPTs (left) and pre-mRNA (right) from 4sU-seq results. P values were calculated by two-sided Mann-Whitney test. Boxplots were plotted as before. h-i. The IGV track comparison of 4sU-seq (h) and TT-seq (i) at the XPO1 or EP300 loci upon INTS11 depletion.
Extended Data Fig. 4 Integrator depletion leads to the upregulation of unbiased bidirectional transcription in the enhancer RNA loci.
a. Enhancer and Super enhancer distribution map calculated by RNAPII ChIP-seq signal in units of reads per million. Enhancers are ranked by the increase in the RNAPII ChIP-seq signal. b. IGV tracks of a super-enhancer locus from ChromRNA-seq and RNAPII ChIP-seq results before and after INTS11 depletion. c. IGV tracks of ChromRNA-seq, 4sU-seq, and TT-seq of two enhancer RNA loci. d. Heatmap of 3,259 enhancer loci. from ChromRNA-seq and 4sU-seq results. e. Metagene analysis showing the average occupancy of ChromRNA-seq and 4sU-seq signals at 3259 enhancer RNA loci.
Extended Data Fig. 5 Depletion of INTS11 reduces immediate early responsive gene expression and induces PROMPT accumulation.
a. Flow diagram illustrating the EGF / IFN-β induced experiments in INTS11-AID cells. b. Selection of immediate responsive genes (FDR < 0.05). Volcano plots showing the IEG genes b. c. Venn diagram showing the overlap between EGF-induced IEG genes and genes with PROMPT after INTS11 depletion. d. Volcano plots showing the PROMPT regions with significantly upregulated RNA levels and IEG genes with downregulated pre-mRNA levels upon EGF treatment. In total 1026 IEG genes were included in the analyses. e. Quantitative boxplots of transcription distribution for 1026 EGF-induced IEG genes and their PROMPTs upon INTS11 depletion. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots. f. Quantitative RT-PCR results of the EGF-induced NR4A1 gene and PROMPT locus upon INTS11 depletion. Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). (*) p < 0.05. (***) p < 0.001. g. Like in (a) but for IFN-β. h. Like in (b) but for IFN-β genes. i. IGV tracks of DDX58 and IFIT1 gene loci before and after IFN-β induction with or without INTS11 depletion. j. Quantitative RT-PCR results of the IFN-β induced IFIT1 gene and its PROMPT locus before and after the depletion of INTS11. Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). (***) p < 0.001. k-l. IEG genes and their PROMPTs before and after INTS11 depletion. The total represents the combined levels of pre-mRNA and PROMPT fold change for each locus. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots.
Extended Data Fig. 6 Integrator depletion alters the total RNAP II occupancy at actively transcribed genes.
a. IGV tracks of RNU11 and RNU12 loci from total RNAPII ChIP-seq results after INTS11 depletion. b. IGV tracks of EP300 and EZH2 loci from RNAPII ChIP-seq results upon INTS11 depletion. c. Boxplots of 2121 highly expressed gene RNAPII ChIP-seq signals in PROMPTs (top) and gene bodies (bottom). P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots. d. Western blot of TAF1 and TAF3 protein levels after INTS1 and INTS11 depletion in whole cells and chromatin, for INTS1-AID2 cell line “-” represents equal-volume DMSO treatment as a control, and “+” represents 10 µM 5-Ph-IAA treatment, representing from three independent experiments with similar results. e. Heatmap of TAF1 ChIP-seq in a 2 kb window around TSS sites of 10428 genes and 2121 active genes. f. IGV tracks of KLF2 and NR4A1 loci from RNAP II ChIP-seq results upon EGF induction and INTS11 depletion. g. Box plots of RNAPII ChIP-seq signals at the 1026 EGF-induced gene loci (PROMPTs, left; gene bodies, right). P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots.
Extended Data Fig. 7 Integrator depletion alters the dynamics of RNAPII-CTD phosphorylation.
a. Western blot of total RNAP II and different CTD phosphorylation states after INTS1 depletion in whole cells, cytoplasm, nucleoplasm and chromatin, representing from three biological independent experiments with similar results (“-” equal-volume DMSO treatment, and “+” 10 µM 5-Ph-IAA treatment). b. IGV tracks of RNU11 and RNU12 loci from Tyr1P and Ser2P ChIP-seq results in INTS11-AID cells. c-d. Box plots representing the 2121 genes Tyr1P(c) and Ser2P(d) ChIP-seq results in genes and PROMPTs upon INTS11 depletion. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots. e. The IGV tracks of total RNAPII, Ser2P, and Tyr1P ChIP-seq results in two enhancer loci upon INTS11 depletion. f. Heatmap representing Tyr1P ChIP-seq (left) and Ser2P ChIP-seq (right) at 3259 enhancer sites. g. Metagene analysis of Tyr1P and Ser2P ChIP-seq signals (log2) across 2121 highly expressed genes. h. Metagene analyses of the Tyr1P / RNAP II and Ser2P / RNAP II ChIP-seq ratios. i. The comparison of metagene analysis between genes and enhancers.
Extended Data Fig. 8 Endonuclease activity of Integrator is specifically required for degrading PROMPTs.
a. Heatmap representing highly expressed genes and their PROMPTs from ChromRNA-seq results with EV or ectopic expression of E203Q mutant protein. “+” represents IAA treatments. The corresponding differences by log2-fold change are shown in the right panel. Upregulation is shown in red, and downregulation is shown in blue. b-c. Boxplots representing the transcript levels from PROMPT (b) and pre-mRNA (c) for 2121 active genes. P values were calculated by two-sided Mann-Whitney test. n.s., not significant. See Extended Data Fig. 1 for description of boxplots. d. Quantitative RT-PCR representing the accumulation of 3’ end extension of RNU11 or RNU12 through the ectopic expression of INTS11 or the E203Q mutant. “+” stands for the IAA treatments. Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). (**) p < 0.01. (***) p < 0.001. e. The accumulation of PROMPTs can be rescued by gapmer ASO specific for PROMPT at the SRRT gene locus, which leads to the recovery of gene expression. (PROMPTs shown in red, gene shown in blue). Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). (**) p < 0.01. (***) p < 0.001. f. Schematic diagram showing the generation of the CPSF73-AID2 cell line (top). Western blot of CPSF73 depletion over time after 10 µM 5-Ph-IAA treatment (bottom), representing from three independent experiments with similar results. g. IGV tracks of the BMP4 and JUN locus revealing 3’ end extension after CPSF73 depletion, the control group was treated with equal volume DMSO. h. Quantitative RT-PCR results representing a 3’ extension of MYC or ACTB genes after CPSF73 depletion. Data are presented as mean ± SD. P values were calculated by unpaired t-test (n = 3 independent experiments). (*) p < 0.05. (**) p < 0.01. i. Volcano plot showing the log2 fold changes in 3847 genes showing significant 3’ extension after CPSF73 depletion (FDR < 0.05, FC > 2). j. Histogram of PROMPTs (PROMPTs were identified by PROMPT-finder.) length distribution in the CTRL, CPSF73 deception, and INTS11 depletion conditions.
Extended Data Fig. 9 The distribution and characterization of U1 sites in the human genome.
a. Density of predicted 5’ U1 splice sites within a 1-kb region flanking gene TSS. Strong and medium sites are defined in the Methods. b. Definition of U1 sites and distance with TSS. The red bar shows the predicted U1 sites. The green box shows the analyzed region. c. Quantitative boxplots of PROMPTs categorized by SOM using predicted U1 site parameters (details in Methods). Left, reads count from the CTRL sample. Right, PROMPT fold change after INTS11 depletion. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots. d. IGV examples of an enhancer locus with a low U1 score together with ChromRNA-seq, TT-seq, RNAPII ChIP-seq, and predicted U1 site results. e. Schematic diagram of the endogenous 3 x U1 sequence in the CTTN gene PROMPT region. f. The sequence of the artificial 3xU1 site sequence. Predicted U1 sites are shown as green boxes below. g. The sequence of the first predicted U1 site sequence in the CTTN gene PROMPT region. The sequence was removed by homologous recombination. h. Boxplots representing the change in transcription levels of PROMPTs (left) and pre-mRNA (right) after blocking U1 snRNA using fully modified ASO. P values were calculated by two-sided Mann-Whitney test. See Extended Data Fig. 1 for description of boxplots.
Supplementary information
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Yang, J., Li, J., Miao, L. et al. Transcription directionality is licensed by Integrator at active human promoters. Nat Struct Mol Biol 31, 1208–1221 (2024). https://doi.org/10.1038/s41594-024-01272-z
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DOI: https://doi.org/10.1038/s41594-024-01272-z
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