Abstract
For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.
Highlights
For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression
In a recent work we revealed an interaction between the ecdysone receptor (EcR)/Usp sensor and the Negative ELongation Factor (NELF)-A subunit of the Negative elongation factor (NELF) and hypothesized that the latter may be responsible for Pol II pausing at 20E-dependent p romoters[3]
Drosophila Schneider cell line 2 (S2) cells were shown to be suitable for the investigation of the ecdysone response as most ecdysone cascade genes are expressed upon 20E treatment[1]
Summary
For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). In a recent work we revealed an interaction between the EcR/Usp sensor and the NELF-A subunit of the Negative elongation factor (NELF) and hypothesized that the latter may be responsible for Pol II pausing at 20E-dependent p romoters[3]. The exact functions of NELF at the promoters of active genes are currently only poorly understood, but there is evidence for its role in mRNA processing and loading of the Cap-associated c omplex[15,16] as demonstrated by the interaction between the NELF-E subunit and RNA in the Pol II paused c omplex[17]
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