title: In vivo live imaging of RNA polymerase II transcription factories in primary cells.
abstract: 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.
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, 3015GE Rotterdam, the Netherlands.
authors.forename: Alireza
authors.initials: A
authors.lastname: Ghamari
authors.forename: Mariëtte P C
authors.initials: MP
authors.lastname: van de Corput
authors.forename: Supat
authors.initials: S
authors.lastname: Thongjuea
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.forename: Wilfred
authors.initials: W
authors.lastname: van Ijcken
authors.forename: Jeffrey
authors.initials: J
authors.lastname: van Haren
authors.forename: Eric
authors.initials: E
authors.lastname: Soler
authors.forename: Dirk
authors.initials: D
authors.lastname: Eick
authors.forename: Boris
authors.initials: B
authors.lastname: Lenhard
authors.forename: Frank G
authors.initials: FG
authors.lastname: Grosveld
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D059008:Time-Lapse Imaging; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D042863:Cyclin-Dependent Kinase 9; D008164:Luminescent Proteins; D012319:RNA Polymerase II; D000097573:Red Fluorescent Protein