The negative elongation factor NELF promotes active transcription of Drosophila ecdysone-dependent genes

The current work describes molecular mechanism used by the Negative elongation complex (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data 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 the described interaction of its NELF-A subunit with EcR receptor. The NELF complex, consisting of at least three subunits, is formed at the 20E-dependent promoters and participates in their active transcription and maintenance of their repressed state. NELF depletion causes a significant decrease in transcription induced by 20E. We associate this effect with the disruption of Pol II elongation complexes formation. A considerable reduction in the promoter-bound level of Spt5 subunit of DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that participation in stabilization of the Pol II-DSIF complex can be an important function of NELF with a big impact on transcription of its target genes. Part of our study was to directly link NELF to regulation of 20E-dependent genes in development. We showed presence of NELF at the promoters of 20E-dependent genes during their active transcription both in embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters remain in a Pol II paused state at the larva stage, where they are temporarily repressed. ChIP-Seqs by anti-NELF-A, anti-NELF-B, Rpb3, Pol II S2P, Pol II S5P, Spt5 antibodies were performed after 1 hour treatment with 20-hydroxyecdysone (20E 1h) and in DMSO (Control) treated S2 Schneider cells (both states was treated by dsRNA against NELF-A/NELF-B or mock treated by dsRNA to GFP). ChIP-Seq by anti-NELF-A, Rpb3, Pol II S2P antibodies were performed on embryos of various stages (2-4h, 6-8h, 10-12h after egg laying AEL) and on larvae L3 PS 1-3 (full gut) and prepupae 0-1h after puparium formation APF (Oregon-Modencode). ChIP-Seqs by anti CBP/Neijre and NELF-E antibodies were performed after 1 hour treatment with 20E (20E 1h) and in DMSO (Control) treated S2 Schenider cells. RNA-Seq were performed after 1 hour treatment with 20-hydroxyecdysone (20E 1h) and in DMSO (Control) treated S2 Schneider cells. RNA-Seq were performed after 1 hour treatment with 20-hydroxyecdysone (20E 1h) and in DMSO (Control) treated S2 Schneider cells.