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. 2021 Jan 8;11:172. doi: 10.1038/s41598-020-80650-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Depletion of NELF affects promoter-bound level of RNA polymerase II and DSIF in both the induced and uninduced states of 20E-dependent genes. (A) Average distribution of NELF-B and NELF-A subunits binding in 1 h 20E treated (20E 1 h) or in DMSO-treated (control) Drosophila S2 cells concomitantly treated with dsRNAs to NELF-B and NELF-A (RNAi) or dsRNA corresponding to GFP transcript (mock). Pile-up profiles were generated for the TSSs of nine 20E-dependent genes whose promoter regions demonstrated more than twofold decrease in NELF-B binding level upon treatment with dsRNA against NELF-A/NELF-B. All profiles were calculated as a median of proteins binding levels. The standard error is displayed on the graphs as lighter area around the main line of the profiles. (B) Binding profiles of NELF-B and NELF-A on TSS of cg1607 and eip78c 20E-dependent genes in 1 h 20E treated (20E 1 h) or in DMSO-treated (control) Drosophila S2 cells concomitantly treated with dsRNAs to NELF-B and NELF-A (RNAi) or dsRNA corresponding to GFP transcript (mock). Protein binding levels were estimated by ChIP-Seq and represent the enrichment of ChIP-Seq signal over input. (C, D) Average binding distribution of Rbp3, Pol II, phosphorylated on Ser2 of CTD (Pol S2P), Pol II, phosphorylated on Ser5 of CTD (Pol S5P) (C) and Spt5 (D) in 1 h 20E treated (20E 1 h) or in DMSO-treated (control) Drosophila S2 cells concomitantly treated with dsRNAs to NELF-B and NELF-A (RNAi) or dsRNA corresponding to GFP transcript (mock). Pile-up profiles were generated for the TSSs of nine 20E-dependent genes whose promoter regions demonstrated more than twofold decrease in NELF-B binding level upon treatment with dsRNA against NELF-A/NELF-B. All profiles were calculated as a median of proteins binding level which was estimated as an enrichment. The standard error is displayed on the graphs as lighter area around the main line of the profiles. (E) Binding profiles of Rbp3, Pol II, phosphorylated on Ser2 of CTD (Pol S2P), Pol II, phosphorylated on Ser5 of CTD (Pol S5P) and Spt5 on cg1607 and eip78c 20E-inducible promoters. Protein binding levels were estimated by ChIP-Seq as an enrichment. ChIP-Seq experiments were performed using Drosophila S2 cells upon 1 h 20E induction (20E 1 h) or DMSO-treated cells (control) concomitantly treated with dsRNAs against NELF-B and NELF-A (RNAi) or dsRNA corresponding to GFP transcript (mock). (F) Proposed model describing a role of NELF complex in transcriptional regulation of an average 20E-dependent gene (image created using BioRender.com). NELF complex is recruited to 20E-dependent promoters, possibly through interaction of NELF-A subunit with EcR/Usp dimer at 20E-dependent enhancers. NELF organizes Pol II pause on uninduced 20E-dependent genes and its depletion causes the decrease in Pol II promoter-bound level. During induced transcription NELF promotes formation of Pol II-elongating complex by stabilizing the complex between Pol II and DSIF.