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      Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

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          Abstract

          Background

          Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive.

          Results

          We employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. We detected mutually exclusive binding patterns for HSF and PRC1 at the hsp70 locus. We found that Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically interacts with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein–protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho.

          Conclusions

          We propose that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus.

          Electronic supplementary material

          The online version of this article (10.1186/s13072-017-0166-9) contains supplementary material, which is available to authorized users.

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          Most cited references35

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          Chromatin compaction by a polycomb group protein complex.

          Polycomb group proteins preserve body patterning through development by maintaining transcriptional silencing of homeotic genes. A long-standing hypothesis is that silencing involves creating chromatin structure that is repressive to gene transcription. We demonstrate by electron microscopy that core components of Polycomb Repressive Complex 1 induce compaction of defined nucleosomal arrays. Compaction by Polycomb proteins requires nucleosomes but not histone tails. Each Polycomb complex can compact about three nucleosomes. A region of Posterior Sex Combs that is important for gene silencing in vivo is also important for chromatin compaction, linking the two activities. This mechanism of chromatin compaction might be central to stable gene silencing by the Polycomb group.
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            Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1.

            Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat shock protein (Hsp) genes. A chaperone/Hsp functioning as repressor of heat shock transcription factor (HSF) could make activation of hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can be activated by nonnative protein, heat, and geldanamycin, addition of Hsp90 inhibits activation. Reduction of the level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo, geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1 complex is present in the unstressed cell and dissociates during stress. We conclude that Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of HSF1.
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              Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo.

              Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC(50) determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.
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                Author and article information

                Contributors
                allwyn.pereira.90@gmail.com
                renato.paro@bsse.ethz.ch
                Journal
                Epigenetics Chromatin
                Epigenetics Chromatin
                Epigenetics & Chromatin
                BioMed Central (London )
                1756-8935
                6 December 2017
                6 December 2017
                2017
                : 10
                : 57
                Affiliations
                [1 ]ISNI 0000 0001 2156 2780, GRID grid.5801.c, Department of Biosystems Science and Engineering, , ETH Zurich, ; 4058 Basel, Switzerland
                [2 ]ISNI 0000 0004 1937 0642, GRID grid.6612.3, Faculty of Sciences, , University of Basel, ; 4056 Basel, Switzerland
                Author information
                http://orcid.org/0000-0003-3308-2965
                Article
                166
                10.1186/s13072-017-0166-9
                5718073
                29208012
                2954f34b-32da-4e64-9f59-9aa7bb30cc1e
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 9 August 2017
                : 29 November 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Genetics
                heat shock,pol ii pausing,polycomb proteins,protein–protein interactions,transcriptional activators

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