RNA polymerase II transcription is concentrated outside replication domains throughout S-phase.
Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5'-triphosphate and digoxigenin-11-deoxyuridine 5'-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.
Wansink
D G
DG
E.C. Slater Institute, University of Amsterdam, The Netherlands.
Manders
E E
EE
van der Kraan
I
I
Aten
J A
JA
van Driel
R
R
de Jong
L
L
D002277:Carcinoma / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000096:biosynthesis; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D012319:RNA Polymerase II / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000096:biosynthesis; D016196:S Phase; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000473:pathology
D002843:Chromatin; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D012319:RNA Polymerase II