|
| Status |
Public on Jun 23, 2017 |
| Title |
Genome-wide single molecule footprinting reveals high RNA polymerase II turnover at paused promoters |
| Organism |
Drosophila melanogaster |
| Experiment type |
Methylation profiling by high throughput sequencing
Non-coding RNA profiling by high throughput sequencing
|
| Summary |
Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA Polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here we harnessed single molecule footprinting to quantify distinct steps of initiation in vivo throughout the drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpected high turnover of polymerases at paused promoters--an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat-shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single molecule resolution and refines concepts of transcriptional pausing.
|
| |
|
| Overall design |
S2 and OSC cell lines. Flavopiridol inhibition, triptolide inhibition, and/or heat shock. McviPI and SssI footprints. Amplicon bisulfite sequencing, short RNA sequncing and whole-genome bisulfite sequencing.
|
| |
|
| Citation(s) |
28735898 |
| |
| Submission date |
Jan 28, 2016 |
| Last update date |
Jul 25, 2021 |
| Contact name |
Dirk Schuebeler |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platforms (3) |
| GPL16479 |
Illumina MiSeq (Drosophila melanogaster) |
| GPL17275 |
Illumina HiSeq 2500 (Drosophila melanogaster) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
|
| Samples (76)
|
|
| Relations |
| BioProject |
PRJNA310152 |
| SRA |
SRP069038 |
Less...
More...