 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 13, 2013 |
| Title |
In vivo live imaging of RNA polymerase II transcription factories in primary cells |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as ‘‘transcription factories,’’ but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9- mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.
|
| |
|
| Overall design |
Examination of genome occupancy of CDK9 and RNAPII that was performed by ChIP-seq in the MEL cell line as described (Soler et al. 2010, 2011) using CDK9 C20 antibody (Santa Cruz Biotechnology, C20, sc-484) and RNA Pol II antibody (Santa Cruz Biotechnology, N20, sc899),
|
| |
|
| Contributor(s) |
Ghamari A, van de Corput MP, Thongjuea S, van Cappellen WA, van IJcken W, van Haren J, Soler E, Eick D, Lenhard B, Grosveld F |
| Citation(s) |
23592796 |
| |
| Submission date |
May 12, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Supat Thongjuea |
| E-mail(s) |
supat.thongjuea@ndcls.ox.ac.uk
|
| Organization name |
The Weatherall Institute of Molecular Medicine
|
| Department |
MRC Molecular Haematology Unit
|
| Street address |
Headington
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platforms (1) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
|
| Samples (4)
|
|
| Relations |
| BioProject |
PRJNA202479 |
| SRA |
SRP022370 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE46849_RAW.tar |
436.1 Mb |
(http)(custom) |
TAR (of BIGWIG) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...