Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density

Abstract

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress–induced transcription of Heat Shock Factor 1–regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1–target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription. Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress–induced transcription of Heat Shock Factor 1–regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1–target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription. Biomolecular condensates are self-organized membraneless intracellular structures that lack a fixed stoichiometry and are stabilized by weak, primarily hydrophobic, multivalent interactions between proteins or between proteins and either RNA or DNA. They can form under physiological conditions (e.g., nucleolus, nuclear pore complex, Balbiani bodies) or in response to stress (e.g., P bodies, stress granules). Such structures play important roles in regulating various cellular processes, including RNA metabolism, protein synthesis, ribosome biogenesis, DNA damage repair, and cell type-specific gene transcription, thereby enabling cellular differentiation or permitting cells to evade stressful environments (1Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell. Biol. 2017; 18: 285-298Crossref PubMed Scopus (2648) Google Scholar, 2Iserman C. Desroches Altamirano C. Jegers C. Friedrich U. Zarin T. 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Liquid phase condensation in cell physiology and disease.Science. 2017; 357eaaf4382Crossref PubMed Scopus (1778) Google Scholar)), underscoring their critical evolutionarily conserved roles. In the nucleus, condensation of various regulatory factors has been implicated in gene control. These include gene-specific transcription factor (GSTF)-, coactivator-, and Pol II-containing condensates that assemble at super-enhancers (SEs) (6Boija A. Klein I.A. Sabari B.R. Dall'Agnese A. Coffey E.L. Zamudio A.V. et al.Transcription factors activate genes through the phase-separation capacity of their activation domains.Cell. 2018; 175: 1842-1855.e16Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 7Cho W.K. Spille J.H. Hecht M. Lee C. Li C. Grube V. et al.Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.Science. 2018; 361: 412-415Crossref PubMed Scopus (719) Google Scholar, 8Chong S. Dugast-Darzacq C. Liu Z. Dong P. Dailey G.M. 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L. et by the domain Full Text Full Text PDF PubMed Scopus Google for bodies, nuclear speckles, and stress or but not either or and for in of from heterochromatic but not histone A.V. M. separation domain 2017; PubMed Scopus Google and B.R. Dall'Agnese A. Boija A. Klein I.A. Coffey E.L. Shrinivas K. et al.Coactivator condensation at super-enhancers links phase separation and gene control.Science. 2018; 361eaar3958Crossref PubMed Google for for for for and of and condensates by by a loss of and at as as a loss of RNA Pol II at and genes. of all proteins also at enhancers the at for by of and Pol II condensates in a W.K. Spille J.H. Hecht M. Lee C. Li C. Grube V. et al.Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.Science. 2018; 361: 412-415Crossref PubMed Scopus (719) Google and and for and in also and at the same S. Dugast-Darzacq C. Liu Z. Dong P. Dailey G.M. Cattoglio C. et al.Imaging dynamic and selective low-complexity domain interactions that control gene transcription.Science. 2018; 361eaar2555Crossref Scopus (544) Google and for and and but not T. S. T. S. et of through phase Cell. 2018; Full Text Full Text PDF PubMed Scopus Google and II C-terminal domain and liquid to and M. Dugast-Darzacq C. M. T. et polymerase II through domain phase Struct. Mol. Biol. 2018; PubMed Scopus Google and and and to domain phase separation but not thereby D. S. Liu A. et for C-terminal of RNA polymerase 2018; PubMed Scopus Google 2,5-HD, and to used for either or and strongly RNA from enhancers and or observed in cells either or to enhancers of and S.J. Yang L. Meluzzi D. Oh S. Yang F. Friedman M.J. et al.Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.Nat. Struct. Mol. Biol. 2019; 26: 193-203Crossref PubMed Scopus (190) Google for condensates splicing Sabari B.R. 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PubMed Scopus Google Scholar). typically increased nucleosome density at genes and and nuclear the for the increase in nucleosome from of cells to the by a in chromatin of cells under conditions to to or to both to for exposure for the at of nuclear cells and as in A. by in cell the in are of to not transcription of both Pol II and Pol III genes abolished as a of the we of the cells by the capacity of the or to form on at temperature to form either or exposure to at and to a to that observed for cells cells to thermal stress or to cells to and or their to form and results that the loss of transcription of and RNA genes loss of as cells and the of the their to the genes for their Here, we that cells are to either or in but Pol II transcription and transcription are strongly but so are genes that do not detectably coalesce and transcription not as to condensates. that the impact on transcription from of or in biomolecular condensates. that a the loss of transcription chromatin Our as we that H3 are to within both regulatory and coding regions of all genes in to not Pol II genes but also to quiescent and silent heterochromatic genes. to Pol III genes. these that the of and elicits a increase in nucleosome a density that to transcription. that exposure to HDs under conditions not have the same a increase in Pol II has on H3 has on Pol II occupancy increase H3 at genes we observed that the in in chromatin as by of as a observed in cells to nuclear to the increased nucleosome et have shown through that both and cell chromatin under conditions to used in the or at for Y. S. S. K. T. et and chromatin in Sci. 2021; Google Scholar). the in observed that of increase in nucleosome in the same the results of these that in evolutionarily cell and of chromatin cells are at between and that chromatin to transcription by In the of H3 at expressed genes as and in to their of transcription that in H3 and of chromatin the that the elicits a increase in H3 at genes in silent transcription L. J. transcription from histone Scopus Google Scholar) the that has been observed to nucleosome condensation in Y. S. S. K. T. et and chromatin in Sci. 2021; Google a of RNA These do not that the of RNA polymerase and of are to the regions of genes to to under conditions in their and coding regions are to Pol of factors within these regions D. J. P. M.J. A.M. and a of occupancy and the Biol. Cell. 2018; PubMed Scopus Google Scholar) the of Pol II within that a of Pol II of either Pol II from chromatin under conditions. the increase in we in cells to the of A. B. J. Friedman N. nucleosome in by Full Text Full Text PDF PubMed Scopus Google the of increased chromatin in cells Y. S. S. K. T. et and chromatin in Sci. 2021; Google Scholar, S. L. J. L. et of 1,6-hexanediol on biomolecular condensates and chromatin Biol. 2021; PubMed Scopus Google Scholar). results provide a caveat to that have used HDs in the study of nuclear condensates in that have the use of a negative control to et J.H. S. et al.Phase separation chromatin and 2021; PubMed Scopus Google Scholar) used to the role of transcriptional condensates the protein in a cell the that condensates and DNA and transcriptional of the transcription of chromatin have to et S. Y. Y. et phase separation of for transcriptional response Biol. PubMed Scopus Google Scholar) that forms phase-separated condensates in response to and that condensates, are for transcriptional cells are to transcription abolished these the that of condensates for transcriptional that also results in chromatin on chromatin play a role in transcription in cells as we have observed budding yeast. we that the have the M. C. J. RNA polymerase that Cell. Biol. PubMed Scopus Google Scholar) used to the role of Pol II in as chromatin occupancy of a not as Pol II occupancy to at genes a to the temperature (e.g., D. C. A. N. et of and in their on and RNA polymerase by a to as results in to loss of Pol II occupancy at both constitutively and genes. In have and to the use of in studying biomolecular condensates Y. M. S. L. et by the domain Full Text Full Text PDF PubMed Scopus Google Scholar, D.T. 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Full Text Full Text PDF PubMed Scopus Google Scholar). for in the of from the Pol III used as a control. are in used in either the or In by to a effective of that in a stoichiometry to form to for to the at either or for to at and at chromatin and immunoprecipitation as S. D. Heat factor of gene Rep. 2019; 26: Full Text Full Text PDF PubMed Scopus Google Scholar). from a of chromatin from the used for used as C-terminal H3 and for at and as previously and DNA by DNA in of In to as of DNA used in and as are in cells of and to at in for of conditions and a control in to a by the to cell for at the either or for to to by the temperature at at for of a by we used the in J. V. M. 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