BACKGROUND: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin. RESULTS: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. CONCLUSION: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.

Centre de Recherche en Biologie Cellulaire de Montpellier University of Montpellier CNRS 1919 Route de Mende 34293 Montpellier Cedex 05 France

Department of Cell Biology Faculty of Science Charles University Vinicna 7 12800 Prague Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic

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Uhlmann F. SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol. 2016;17(7):399–412. doi: 10.1038/nrm.2016.30. PubMed DOI

Davidson IF, Peters JM. Genome folding through loop extrusion by SMC complexes. Nat Rev Mol Cell Biol. 2021;22(7):445–464. doi: 10.1038/s41580-021-00349-7. PubMed DOI

Ransom M, Dennehey BK, Tyler JK. Chaperoning histones during DNA replication and repair. Cell. 2010;140(2):183–195. doi: 10.1016/j.cell.2010.01.004. PubMed DOI PMC

Morrison AJ, Shen XT. Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes. Nat Rev Mol Cell Biol. 2009;10(6):373–384. doi: 10.1038/nrm2693. PubMed DOI PMC

Helmlinger D, Tora L. Sharing the SAGA. Trends Biochem Sci. 2017;42(11):850–861. doi: 10.1016/j.tibs.2017.09.001. PubMed DOI PMC

Palecek JJ, Gruber S. Kite proteins: a superfamily of SMC/Kleisin partners conserved across bacteria, archaea, and eukaryotes. Structure. 2015;23(12):2183–2190. doi: 10.1016/j.str.2015.10.004. PubMed DOI

Wells JN, Gligoris TG, Nasmyth KA, Marsh JA. Evolution of condensin and cohesin complexes driven by replacement of Kite by Hawk proteins. Curr Biol. 2017;27(1):R17–R18. doi: 10.1016/j.cub.2016.11.050. PubMed DOI PMC

Kolesar P, Stejskal K, Potesil D, Murray JM, Palecek JJ. Role of Nse1 subunit of SMC5/6 complex as a ubiquitin ligase. Cells. 2022;11(1):13. doi: 10.3390/cells11010165. PubMed DOI PMC

Andrews E, Palecek J, Sergeant J, Taylor E, Lehmann A, Watts F. Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage. Mol Cell Biol. 2005;25(1):185–196. doi: 10.1128/MCB.25.1.185-196.2005. PubMed DOI PMC

Zhao X, Blobel G. A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci USA. 2005;102(13):4777–47782. doi: 10.1073/pnas.0500537102. PubMed DOI PMC

Gligoris T, Löwe J. Structural insights into ring formation of cohesin and related smc complexes. Trends Cell Biol. 2016;26(9):680–693. doi: 10.1016/j.tcb.2016.04.002. PubMed DOI PMC

Hassler M, Shaltiel IA, Haering CH. Towards a unified model of SMC complex function. Curr Biol. 2018;28(21):R1266–R1281. doi: 10.1016/j.cub.2018.08.034. PubMed DOI PMC

Nasmyth K, Haering CH. The structure and function of SMC and kleisin complexes. Annu Rev Biochem. 2005;74:595–648. doi: 10.1146/annurev.biochem.74.082803.133219. PubMed DOI

Alt A, Dang HQ, Wells OS, Polo LM, Smith MA, McGregor GA, et al. Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nat Commun. 2017;8:14011. doi: 10.1038/ncomms14011. PubMed DOI PMC

Hallett ST, Campbell Harry I, Schellenberger P, Zhou L, Cronin NB, Baxter J, et al. Cryo-EM structure of the Smc5/6 holo-complex. Nucleic Acids Res. 2022 doi: 10.1093/nar/gkac692. PubMed DOI PMC

Adamus M, Lelkes E, Potesil D, Ganji SR, Kolesar P, Zabrady K, et al. Molecular insights into the architecture of the human SMC5/6 complex. J Mol Biol. 2020;432(13):3820–3837. doi: 10.1016/j.jmb.2020.04.024. PubMed DOI

Yu Y, Li S, Ser Z, Kuang H, Than T, Guan D, et al. Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes. Proc Natl Acad Sci USA. 2022;119(23):e2202799119. doi: 10.1073/pnas.2202799119. PubMed DOI PMC

Ganji M, Shaltiel IA, Bisht S, Kim E, Kalichava A, Haering CH, et al. Real-time imaging of DNA loop extrusion by condensin. Science. 2018;360(6384):102–105. doi: 10.1126/science.aar7831. PubMed DOI PMC

Davidson IF, Bauer B, Goetz D, Tang W, Wutz G, Peters JM. DNA loop extrusion by human cohesin. Science. 2019;366(6471):1338–1345. doi: 10.1126/science.aaz3418. PubMed DOI

Wang XD, Hughes AC, Brandao HB, Walker B, Lierz C, Cochran JC, et al. In vivo evidence for ATPase-dependent DNA translocation by the Bacillus subtilis SMC condensin complex. Mol Cell. 2018;71(5):841–847. doi: 10.1016/j.molcel.2018.07.006. PubMed DOI PMC

Pradhan B, Kanno K, Igarashi MU, Baaske MD, Wong JSK, Jeppsson K, et al. The Smc5/6 complex is a DNA loop extruding motor. bioRxiv. 2022 doi: 10.1101/2022.05.13.491800. PubMed DOI

Zabrady K, Adamus M, Vondrova L, Liao C, Skoupilova H, Novakova M, et al. Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA. Nucleic Acids Res. 2016;44(3):1064–1079. doi: 10.1093/nar/gkv1021. PubMed DOI PMC

Piazza I, Rutkowska A, Ori A, Walczak M, Metz J, Pelechano V, et al. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits. Nat Struct Mol Biol. 2014;21(6):560–568. doi: 10.1038/nsmb.2831. PubMed DOI

Toselli-Mollereau E, Robellet X, Fauque L, Lemaire S, Schiklenk C, Klein C, et al. Nucleosome eviction in mitosis assists condensin loading and chromosome condensation. EMBO J. 2016;35(14):1565–1581. doi: 10.15252/embj.201592849. PubMed DOI PMC

Shaltiel IA, Datta S, Lecomte L, Hassler M, Kschonsak M, Bravo S, et al. A hold-and-feed mechanism drives directional DNA loop extrusion by condensin. Science. 2022;376(6597):1087–1094. doi: 10.1126/science.abm4012. PubMed DOI

Shi ZB, Gao HS, Bai XC, Yu HT. Cryo-EM structure of the human cohesin-NIPBL-DNA complex. Science. 2020;368(6498):1454. doi: 10.1126/science.abb0981. PubMed DOI

Bürmann F, Funke LFH, Chin JW, Löwe J. Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites. Mol Cell. 2021;81(23):4891–906.e8. doi: 10.1016/j.molcel.2021.10.011. PubMed DOI PMC

Lopez-Serra L, Kelly G, Patel H, Stewart A, Uhlmann F. The Scc2-Scc4 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions. Nat Genet. 2014;46(10):1147–1151. doi: 10.1038/ng.3080. PubMed DOI PMC

Munoz S, Minamino M, Casas-Delucchi CS, Patel H, Uhlmann F. A role for chromatin remodeling in cohesin loading onto chromosomes. Mol Cell. 2019;74(4):664. doi: 10.1016/j.molcel.2019.02.027. PubMed DOI PMC

Munoz S, Passarelli F, Uhlmann F. Conserved roles of chromatin remodellers in cohesin loading onto chromatin. Curr Genet. 2020;66(5):951–956. doi: 10.1007/s00294-020-01075-x. PubMed DOI PMC

Roguev A, Wiren M, Weissman JS, Krogan NJ. High-throughput genetic interaction mapping in the fission yeast Schizosaccharomyces pombe. Nat Methods. 2007;4(10):861–866. doi: 10.1038/nmeth1098. PubMed DOI

Kim DU, Hayles J, Kim D, Wood V, Park HO, Won M, et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol. 2010;28(6):617–623. doi: 10.1038/nbt.1628. PubMed DOI PMC

Wagih O, Usaj M, Baryshnikova A, VanderSluis B, Kuzmin E, Costanzo M, et al. SGAtools: one-stop analysis and visualization of array-based genetic interaction screens. Nucleic Acids Res. 2013;41(Web Server issue):W591–W596. doi: 10.1093/nar/gkt400. PubMed DOI PMC

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–2504. doi: 10.1101/gr.1239303. PubMed DOI PMC

Palecek JJ. SMC5/6: multifunctional player in replication. Genes. 2019;10(1):E7. doi: 10.3390/genes10010007. PubMed DOI PMC

Aragon L. The Smc5/6 complex: new and old functions of the enigmatic long-distance relative. In: Bonini NM, editor. Annual review of genetics. San Mateo: Palo Alto: Annual Reviews; 2018. pp. 89–107. PubMed

Lee KM, Nizza S, Hayes T, Bass KL, Irmisch A, Murray JM, et al. Brc1-mediated rescue of Smc5/6 deficiency: requirement for multiple nucleases and a novel Rad18 function. Genetics. 2007;175(4):1585–1595. doi: 10.1534/genetics.106.067801. PubMed DOI PMC

Lehmann AR, Walicka M, Griffiths DJF, Murray JM, Watts FZ, McCready S, et al. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol Cell Biol. 1995;15:7067–7080. doi: 10.1128/MCB.15.12.7067. PubMed DOI PMC

Morikawa H, Morishita T, Kawane S, Iwasaki H, Carr AM, Shinagawa H. Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast. Mol Cell Biol. 2004;24(21):9401–9413. doi: 10.1128/MCB.24.21.9401-9413.2004. PubMed DOI PMC

Dixon SJ, Fedyshyn Y, Koh JLY, Prasad TSK, Chahwan C, Chua G, et al. Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes. Proc Natl Acad Sci USA. 2008;105(43):16653–16658. doi: 10.1073/pnas.0806261105. PubMed DOI PMC

Ryan CJ, Roguev A, Patrick K, Xu J, Jahari H, Tong Z, et al. Hierarchical modularity and the evolution of genetic interactomes across species. Mol Cell. 2012;46(5):691–704. doi: 10.1016/j.molcel.2012.05.028. PubMed DOI PMC

Pebernard S, McDonald WH, Pavlova Y, Yates JR, Boddy MN. Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis. Mol Biol Cell. 2004;15(11):4866–4876. doi: 10.1091/mbc.E04-05-0436. PubMed DOI PMC

Helmlinger D, Marguerat S, Villén J, Swaney DL, Gygi SP, Bähler J, et al. Tra1 has specific regulatory roles, rather than global functions, within the SAGA co-activator complex. EMBO J. 2011;30(14):2843–2852. doi: 10.1038/emboj.2011.181. PubMed DOI PMC

Verkade HM, Bugg SJ, Lindsay HD, Carr AM, O'Connell MJ. Rad18 is required for DNA repair and checkpoint responses in fission yeast. Mol Biol Cell. 1999;10(9):2905–2918. doi: 10.1091/mbc.10.9.2905. PubMed DOI PMC

Hudson JJR, Bednarova K, Kozakova L, Liao CY, Guerineau M, Colnaghi R, et al. Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID families. PLoS ONE. 2011;6(2):14. doi: 10.1371/journal.pone.0017270. PubMed DOI PMC

Duan X, Sarangi P, Liu X, Rangi GK, Zhao X, Ye H. Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex. Mol Cell. 2009;35(5):657–668. doi: 10.1016/j.molcel.2009.06.032. PubMed DOI PMC

Clouaire T, Rocher V, Lashgari A, Arnould C, Aguirrebengoa M, Biernacka A, et al. Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol Cell. 2018;72(2):250–62.e6. doi: 10.1016/j.molcel.2018.08.020. PubMed DOI PMC

Deshpande GP, Hayles J, Hoe KL, Kim DU, Park HO, Hartsuiker E. Screening a genome-wide S. pombe deletion library identifies novel genes and pathways involved in genome stability maintenance. DNA Repair. 2009;8(5):672–679. doi: 10.1016/j.dnarep.2009.01.016. PubMed DOI PMC

Pan X, Lei B, Zhou N, Feng B, Yao W, Zhao X, et al. Identification of novel genes involved in DNA damage response by screening a genome-wide Schizosaccharomyces pombe deletion library. BMC Genomics. 2012;13:662. doi: 10.1186/1471-2164-13-662. PubMed DOI PMC

Helmlinger D, Marguerat S, Villén J, Gygi SP, Bähler J, Winston F. The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8. Genes Dev. 2008;22(22):3184–3195. doi: 10.1101/gad.1719908. PubMed DOI PMC

Oravcová M, Gadaleta MC, Nie M, Reubens MC, Limbo O, Russell P, et al. Brc1 promotes the focal accumulation and SUMO ligase activity of Smc5-Smc6 during replication stress. Mol Cell Biol. 2018 doi: 10.1128/MCB.00271-18. PubMed DOI PMC

Oravcová M, Boddy MN. Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase. Curr Genet. 2019;65(3):669–676. doi: 10.1007/s00294-018-0922-9. PubMed DOI PMC

Pebernard S, Schaffer L, Campbell D, Head SR, Boddy MN. Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively. EMBO J. 2008;27(22):3011–3023. doi: 10.1038/emboj.2008.220. PubMed DOI PMC

Lindroos HB, Strom L, Itoh T, Katou Y, Shirahige K, Sjogren C. Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. Mol Cell. 2006;22(6):755–767. doi: 10.1016/j.molcel.2006.05.014. PubMed DOI

Irmisch A, Ampatzidou E, Mizuno K, O'Connell MJ, Murray JM. Smc5/6 maintains stalled replication forks in a recombination-competent conformation. EMBO J. 2009;28(2):144–155. doi: 10.1038/emboj.2008.273. PubMed DOI PMC

Peng XP, Lim S, Li SB, Marjavaara L, Chabes A, Zhao XL. Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites. PLoS Genet. 2018;14(1):20. doi: 10.1371/journal.pgen.1007129. PubMed DOI PMC

Torres-Rosell J, Machin F, Farmer S, Jarmuz A, Eydmann T, Dalgaard JZ, et al. SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions. Nat Cell Biol. 2005;7(4):412–419. doi: 10.1038/ncb1239. PubMed DOI

Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, et al. The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol. 2007;9(8):923–931. doi: 10.1038/ncb1619. PubMed DOI

González-Medina A, Hidalgo E, Ayté J. Gcn5-mediated acetylation at MBF-regulated promoters induces the G1/S transcriptional wave. Nucleic Acids Res. 2019;47(16):8439–8451. doi: 10.1093/nar/gkz561. PubMed DOI PMC

Nugent RL, Johnsson A, Fleharty B, Gogol M, Xue-Franzén Y, Seidel C, et al. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation. BMC Genomics. 2010;11:59. doi: 10.1186/1471-2164-11-59. PubMed DOI PMC

Elías-Villalobos A, Toullec D, Faux C, Séveno M, Helmlinger D. Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast. Nat Commun. 2019;10(1):5237. doi: 10.1038/s41467-019-13243-w. PubMed DOI PMC

Lai J, Jiang J, Wu Q, Mao N, Han D, Hu H, et al. The transcriptional coactivator ADA2b recruits a structural maintenance protein to double-strand breaks during DNA repair in plants. Plant Physiol. 2018 doi: 10.1104/pp.18.00123. PubMed DOI PMC

Jiang J, Ou X, Han D, He Z, Liu S, Mao N, et al. A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair. Plant Cell. 2022 doi: 10.1093/plcell/koac191. PubMed DOI PMC

Chiolo I, Minoda A, Colmenares SU, Polyzos A, Costes SV, Karpen GH. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell. 2011;144(5):732–744. doi: 10.1016/j.cell.2011.02.012. PubMed DOI PMC

Williams JS, Williams RS, Dovey CL, Guenther G, Tainer JA, Russell P. gammaH2A binds Brc1 to maintain genome integrity during S-phase. EMBO J. 2010;29(6):1136–1148. doi: 10.1038/emboj.2009.413. PubMed DOI PMC

Moreno S, Klar A, Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. PubMed DOI

Maere S, Heymans K, Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005;21(16):3448–3449. doi: 10.1093/bioinformatics/bti551. PubMed DOI

Lock A, Rutherford K, Harris MA, Hayles J, Oliver SG, Bähler J, et al. PomBase 2018: user-driven reimplementation of the fission yeast database provides rapid and intuitive access to diverse, interconnected information. Nucleic Acids Res. 2019;47(D1):D821–D827. doi: 10.1093/nar/gky961. PubMed DOI PMC

Paleček JJ, Vondrová L, Zábrady K, Otočka J. Multicomponent yeast two-hybrid system: applications to study protein-protein interactions in SMC complexes. Methods Mol Biol. 2019;2004:79–90. doi: 10.1007/978-1-4939-9520-2_7. PubMed DOI

Sergeant J, Taylor E, Palecek J, Fousteri M, Andrews E, Sweeney S, et al. Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex. Mol Cell Biol. 2005;25(1):172–184. doi: 10.1128/MCB.25.1.172-184.2005. PubMed DOI PMC

Palecek J, Vidot S, Feng M, Doherty AJ, Lehmann AR. The SMC5-6 DNA repair complex: bridging of the SMC5-6 heads by the Kleisin, NSE4, and non-Kleisin subunits. J Biol Chem. 2006;281:36952–36959. doi: 10.1074/jbc.M608004200. PubMed DOI

Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50(D1):D439–D444. doi: 10.1093/nar/gkab1061. PubMed DOI PMC

Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19(6):679–682. doi: 10.1038/s41592-022-01488-1. PubMed DOI PMC

Byska J, Jurcik A, Furmanova K, Kozlikova B, Palecek JJ. Visual analysis of protein–protein interaction docking models using COZOID tool. Methods Mol Biol. 2020;2074:81–94. doi: 10.1007/978-1-4939-9873-9_7. PubMed DOI

Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, et al. The genome sequence ofSchizosaccharomyces pombe. Nature. 2002;415(6874):871–880. doi: 10.1038/nature724. PubMed DOI

Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–360. doi: 10.1038/nmeth.3317. PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Bonfield JK, Marshall J, Danecek P, Li H, Ohan V, Whitwham A, et al. HTSlib: C library for reading/writing high-throughput sequencing data. Gigascience. 2021 doi: 10.1093/gigascience/giab007. PubMed DOI PMC

Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016;44(W1):W160–W165. doi: 10.1093/nar/gkw257. PubMed DOI PMC

Wang H, Dienemann C, Stützer A, Urlaub H, Cheung ACM, Cramer P. Structure of the transcription coactivator SAGA. Nature. 2020;577(7792):717–720. doi: 10.1038/s41586-020-1933-5. PubMed DOI PMC