Nanog organizes transcription bodies

Abstract

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis, to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription, and independently of RNA accumulation. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies.

Transcription bodies regulate gene expression by sequestering CDK9

“…Zebrafish were maintained and raised under standard conditions, and according to Swiss regulations (canton Vaud, license number VD-H28). Wildtype (TLAB) and mutant ( Mir430 -/- ) ( 9 ) embryos were collected immediately upon fertilization and allowed to develop to the desired stage at 28°C. Mir430 -/- embryos were rescued by injecting 2nL of rescue solution (10μM of MiR430a, MiR430b and MiR430c duplex in 1x siRNA solution (300mM KCl, 30mM HEPES, 1mM MgCl2, brought to pH 7.5 with KOH)), as described before ( 9 , 24 ).…”

“…S12). Even motifs of Nanog, Pou5f3 and Sox19b, transcription factors that have been shown to be required for the activation of transcription during ZGA in zebrafish ( 9 , 29–32 ) did not fulfill these two requirements. Thus, removal of the Mir430 transcription bodies advances expression onset of genes poised to be activated.…”

“…Before transcriptional activity can be seen throughout the nucleus, it is confined to two micron-sized transcription bodies (Fig. 1B) ( 9 , 20–23 ), nucleated by the mir430 locus, which contains multiple copies of the mir430 gene ( 21 , 24 ). These bodies are enriched for RNAPII Serine 5 and Serine 2 phosphorylation (Ser5P, Ser2P), which mark the initiating and elongating form of RNAPII, respectively, as well as the transcription factors Nanog and Sox19b ( 9 , 20–22 ).…”

“…To investigate the role of transcription bodies in development, we generated a fish line in which the mir430 locus is deleted ( 9 ), resulting in the specific disruption of the two mir430 transcription bodies, as observed by the loss of Ser2P signal (Fig. 1C).…”

“…To investigate this possibility, we visualized transcription elongation in WT and Mir430 -/- + inj MiR430 embryos. We injected 1-cell stage embryos with an antigen-binding fragment (Fab) recognizing RNAPII Ser2P (transcription elongation), as described before ( 9 , 22 , 23 ) and imaged them. We also visualized Mir430 transcripts using Morpholino Visualization of Expression (MOVIE, ( 21 )), to be able to distinguish the transcription bodies that are nucleated by the Mir430 locus from other transcription bodies.…”