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CDK9 sequestration in mir430 transcription bodies inhibits transcription elongation elsewhere. a, Visualization of CDK9 and RNAPII-Ser2P by immunofluorescence in WT nuclei at 256-cell stage. Nuclei are labelled with DAPI. b–d, Quantification of the enrichment of CDK9 and RNAPII-Ser2P in the two large transcription bodies. The enrichment (observed versus expected signal intensity) (b), the percentage of total nuclear signal present in the two large transcription bodies (c) and the percentage of the total nuclear area occupied by the two large transcription bodies (d) are shown. N = 3 biologically independent experiments, n = 214 nuclei. Wilcoxon rank-sum test was performed to assess if the log2 of the ‘Enrichment of signal in transcription bodies (two-sided)’, the ‘percentage of nuclear signal in two transcription bodies’ (one-sided) and the ‘percentage of nuclear area occupied by two transcription bodies (one-sided)’ are significantly different than zero (V = 23,005 for all tests). A test was considered significant when it had a P value <0.05. Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. See Methods for a detailed description of quantification. e, Volcano plots showing upregulated and downregulated genes in WT + inj cdk9 mRNA (n = 3 biologically independent samples) versus WT (n = 3 biologically independent samples) embryos at the 256-cell stage (top) and the 1,024-cell stage (bottom). Wald test with Benjamini–Hochberg correction was performed, and genes with adjusted P values <0.01 were considered significantly differentially expressed. f, Visualization of RNAPII-Ser2P with Fabs, and miR430 RNA with MOVIE in WT, WT + inj cdk9 mRNA and mir430−/− + inj MiR430 embryos across stages. Shown are representative micrographs of individual nuclei at 256-cell and 1,024-cell stages, extracted from a spinning disk confocal microscopy timelapse. See Extended Data Figs. 7–9 for complete cell cycles from 128-cell to 1,024-cell stages. g, Quantification of ectopic transcription bodies as shown in b. N = 3 biologically independent experiments; n = 101, 184, 290 and 378 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in WT; n = 43, 82, 127 and 179 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in WT + inj cdk9; n = 45, 65, 97 and 152 at 128-cell, 256-cell, 512-cell and 1,024-cell stages in mir430−/− + inj MiR430, respectively. Boxplots show median, quartiles, minimum and maximum, and 1.5× interquartile range. Individual points represent outliers. See Extended Data Fig. 10 and Methods for a detailed description of quantification. h, Model for the role of transcription bodies in transcription regulation. In WT nuclei, two large transcription bodies are nucleated by the mir430 locus. They sequester CDK9 and potentially other factors that are required for pause release, thereby stalling transcription elsewhere in the nucleus in the initiation state. The specific disruption of the two mir430 transcription bodies leads to a redistribution of CDK9, which results in pause release and the upregulation of genes elsewhere in the nucleus. Source numerical data are available in Source data and on GEO (GSE248237). Source data
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