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      Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster

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          Abstract

          Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.

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          Getting up to speed with transcription elongation by RNA polymerase II.

          Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.
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            Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons

            Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. DOI: http://dx.doi.org/10.7554/eLife.02407.001
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              Pybedtools: a flexible Python library for manipulating genomic datasets and annotations

              Summary: pybedtools is a flexible Python software library for manipulating and exploring genomic datasets in many common formats. It provides an intuitive Python interface that extends upon the popular BEDTools genome arithmetic tools. The library is well documented and efficient, and allows researchers to quickly develop simple, yet powerful scripts that enable complex genomic analyses. Availability: pybedtools is maintained under the GPL license. Stable versions of pybedtools as well as documentation are available on the Python Package Index at http://pypi.python.org/pypi/pybedtools. Contact: dalerr@niddk.nih.gov; arq5x@virginia.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

                Author and article information

                Contributors
                leielissa@niddk.nih.gov
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                24 October 2018
                24 October 2018
                2018
                : 8
                : 15685
                Affiliations
                [1 ]Nuclear Organization and Gene Expression Section, Bethesda, USA
                [2 ]ISNI 0000 0001 2203 7304, GRID grid.419635.c, Laboratory of Cellular and Developmental Biology, , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, ; 9000 Rockville Pike, Bethesda, MD 20892 USA
                Article
                34115
                10.1038/s41598-018-34115-1
                6200757
                aa589e6c-3a56-442b-a568-68587c5caae5
                © The Author(s) 2018

                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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 31 July 2018
                : 8 October 2018
                Funding
                Funded by: This work was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health [DK015602]
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