CDK9-dependent RNA polymerase II pausing controls transcription initiation
- Max-Planck-Institute for Biophysical Chemistry, Germany
- Helmholtz Center Munich, Center of Integrated Protein Science, Germany
- Ludwig-Maximilians-Universität München, Center of Integrated Protein Science, Germany
Figures
Figure 1 with 2 supplements
CDK9 inhibition decreases RNA synthesis in the 5’-region of genes.
(A) Experimental design. TT-seq was carried out with CDK9as cells after treatment with solvent DMSO (control) or 1-NA-PP1 (CDK9as inhibited). (B) TT-seq signal before (black) and after (red) CDK9 …
Figure 1—figure supplement 1
Model of a paused polymerase positioned up to around 50 bp downstream of the TSS.
Modeling shows that paused Pol II (silver, right) positioned 50 bp downstream of the transcription start site (TSS) allows for formation of the Pol II initiation complex (different colors, left). …
Figure 1—figure supplement 2
CRISPR-Cas9 directed engineering, cellular and biochemical characterization of CDK9as Raji B cell line.
(A) BstUI restriction enzyme recognition site used for screening is indicated in the HDR template sequence (highlighted in red). Agarose gels of screening PCRs followed by restriction digest with …
Figure 2 with 1 supplement
Pol II elongation velocity.
(A) Schematic representation of observed response window of TT-seq signal with CDK9as inhibitor (red) or control (black) for TUs of three different length classes (short TUs < 25 [kbp], …
Figure 2—figure supplement 1
Example genome browser views of TT-seq signals in CDK9as cells with estimated response window and genomic features correlating with elongation velocity.
(A) YWHAQ gene locus (47,042 [bp]) on chromosome 2. The upper panel shows TT-seq signal with CDK9as inhibitor (red) and control (black). Grey box depicts transcript body from transcription start …
Figure 3 with 1 supplement
Distribution and sequence of promoter-proximal pause sites.
(A) Distribution of pause site distance from the TSS for 2135 investigated TUs depicted as a histogram (mean 128 [bp], median 112 [bp], mode 84 [bp]). Two biological replicates were averaged. (B) …
Figure 3—figure supplement 1
Features of underlying DNA sequence around promoter-proximal pause sites.
(A) Distributions of pause site depicted as densities for TUs with a response ratio >75% quantile (574 TUs, red) and TUs with a response ratio <25% quantile (469 TUs, black). (B) Plot showing the …
Figure 4 with 2 supplements
Pol II pausing generally limits transcription initiation (‘pause-initiation limit’).
(A) Schematic representation of polymerase flow in the promoter-proximal region. The mNET-seq signal (top) is the ratio of the initiation frequency I over the elongation velocity v. The TT-seq …
Figure 4—figure supplement 1
A longer pause duration but not promoter-proximal termination of polymerase leads to shortage of labeled RNA in the region between TSS and pause site.
(A) Simulation of labeled RNA fragments synthesized in 5 min labeling duration (TT-seq fragments depicted for polymerases with a distance corresponding to 40 s of elongation, middle panel) for a …
Figure 4—figure supplement 2
Verification of anti-correlation between initiation frequency I and pause duration d including ‘pause-initiation limit’.
(A) Scatter plot comparing the initiation frequency [cell−1min−1] against the pause duration [min] for 2135 common TUs with color encoding according to mNETseq signal strength (weak in white to …
Figure 5
Increasing Pol II pause duration decreases the frequency of transcription initiation.
(A) Schematic representation of observed decrease in TT-seq signal upon CDK9 inhibition, upstream and downstream of the pause site. (B) Distributions of gene-wise mean TT-seq signals in the region …
Figure 6
CDK9 inhibition leads to increased pause duration.
(A) Metagene analysis comparing the average mNET-seq signal before and after CDK9 inhibition. Two biological replicates were averaged. The mNET-seq coverage was averaged for 2538 investigated TUs …
Figure 7 with 1 supplement
Determinants of CDK9-dependent promoter-proximal pausing.
(A) Distribution of gene-wise mean in vivo DMS-seq signals (detecting RNA secondary structure) for a window between −65 and −15 [bp] upstream of the pause site for TUs with long pause durations …
Figure 7—figure supplement 1
Features of promoter-proximal pausing.
(A) Distributions of gene-wise mean minimum free energy (Materials and methods) for a window of [−15,–65] bp upstream of the pause site for TUs with long pause durations (pause duration >75% …
Author response image 1
Example genome browser views of TT-seq signals in CDK9as cells with high responsiveness (~ 90%).
(A) CYB5R4 gene locus (107,781 [bp]) on chromosome 6. The upper panel shows TT-seq signal with CDK9as inhibitor (red) and control (black). Grey box depicts transcript body from transcription start …
Additional files
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Supplementary file 1
Published datasets used for analysis.
Note that the conclusions we draw across different cell-lines are all based on metagene analysis, involving from 500 up to more than 2000 genes. Thus, we assume cell-line specific differences to have an insignificant influence and that the tendencies we observe rather suggest strong conservation.
- https://doi.org/10.7554/eLife.29736.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.29736.019
