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Linker Histone H1.8 Inhibits Chromatin-Binding of Condensins and DNA Topoisomerase II to Tune Chromosome Length and Individualization

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Linker Histone H1.8 Inhibits Chromatin-Binding of Condensins and DNA Topoisomerase II to Tune Chromosome Length and Individualization bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423657; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Choppakatla et al., August 2021-preprint copy- BioRxiv Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome length and individualization Pavan Choppakatla1, Bastiaan Dekker2, Erin E. Cutts3, Alessandro Vannini3,4, Job Dekker2, 5and Hironori Funabiki1, * 1Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, NY 10065, USA. 2Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA. 3Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK. 4Fondazione Human Technopole, Structural Biology Research Centre, 20157 Milan, Italy 5Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. *Correspondence: [email protected] Summary DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase. Keywords Chromosome compaction, chromatin, mitosis, linker histone, nucleosome, condensin, DNA topoisomerase, Hi-C, Xenopus Introduction information in interphase, mitotic compaction of DNA enables efficient distribution of genetic Genomic DNA in eukaryotes is compacted information to daughter cells. In addition, by orders of magnitude over its linear length. The chromosome individualization during mitosis extent and mode of the packaging change between ensures that all duplicated chromosomes are interphase and mitosis to support the cell cycle independently moved by microtubules and are dependent functions of the DNA. While loosely equally distributed to daughter cells. Despite these packed DNA allows efficient decoding of genetic common functional requirements of mitotic bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423657; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Choppakatla et al., August 2021-preprint copy- BioRxiv chromosomes and evolutionary conservation of of the condensin I and II loops is also consistent with major known regulators of mitotic chromosome their roles in maintaining lateral and axial structures, the size and shape of chromosomes vary compaction respectively (Green et al. 2012; among species and developmental stages. For Samejima et al. 2012; Bakhrebah et al. 2015). In example, rod-like individual mitotic chromosomes Xenopus egg extracts, in the presence of wildtype can be readily visualized in fission yeast condensin I levels, condensin II depletion does not Schizosaccharomyces pombe, but not in budding appear to change mitotic chromosome length, yeast Saccharomyces cerevisiae (Guacci, Hogan, and suggesting a reduced role for condensin II on these Koshland 1994; Umesono et al. 1983). During early chromosomes (Shintomi and Hirano 2011). embryogenesis in Xenopus and C. elegans, mitotic The prevailing model suggests that mitotic chromosome lengths become shorter (Ladouceur, chromatin loops are formed by the dynamic loop Dorn, and Maddox 2015; Micheli et al. 1993). The extrusion activity of condensins (Riggs 1990; mechanistic basis of mitotic chromosome shape Nasmyth 2001; Alipour and Marko 2012), although regulation remains largely speculative (Heald and the molecular details of the process remain unclear Gibeaux 2018). (Banigan and Mirny 2020; Cutts and Vannini 2020; Classical experiments on mitotic Datta, Lecomte, and Haering 2020). Single molecule chromosomes indicated that DNA is organized into experiments using purified recombinant yeast and loops around a central protein scaffold (Earnshaw human condensin complexes demonstrated ATP- and Laemmli 1983; Paulson and Laemmli 1977). dependent motor activity and loop extrusion by yeast Two major chromosome-associated ATPases play and human condensins (Terakawa et al. 2017; Ganji pivotal roles in mitotic chromatid formation: DNA et al. 2018; Kong et al. 2020). Condensin dependent topoisomerase II (topo II) and the structural loop extrusion in a more physiological Xenopus maintenance of chromosomes (SMC) family extract system has also been shown (Golfier et al. complex, condensin (Kinoshita and Hirano 2017; 2020). In silico experiments further suggest that a Kschonsak and Haering 2015). Data obtained with minimal combination of loop extruders (like chromosome conformation capture assays are condensin) and strand passage activity (such as topo consistent with the model that mitotic chromosomes II) can generate well resolved rod-like sister are arranged in a series of loops organized by chromatids from entangled, interphase like DNA condensins (Naumova et al. 2013; Gibcus et al. 2018; fibers (Goloborodko et al. 2016). However, it Elbatsh et al. 2019). Vertebrate cells express two remains unclear if loop extrusion can proceed on forms of the condensin complex, condensin I and chromatin, since condensins prefer to bind condensin II (Ono et al. 2003; Hirano, Kobayashi, nucleosome-free DNA (Kong et al. 2020; Zierhut et and Hirano 1997). Condensin I is loaded onto al. 2014; Shintomi et al. 2017; Toselli‐Mollereau et chromatin exclusively during mitosis, whereas al. 2016). Human and yeast condensin complexes are condensin II retains access to chromosomes capable of loop extrusion through sparsely arranged throughout the cell cycle(Hirota et al. 2004; Ono et nucleosomes in vitro (Kong et al. 2020; Pradhan et al. al. 2004; Walther et al. 2018). Condensin II plays a 2021), but mitotic chromatin adopts a more compact role in maintaining chromosome territories in fiber structure (Grigoryev et al. 2016; Ou et al. 2017). Drosophila nuclei (Rosin, Nguyen, and Joyce 2018; Furthermore, large protein complexes such as RNA Bauer, Hartl, and Bosco 2012) and drives sister polymerases are able to limit loop extrusion by SMC chromatid decatenation by topo II (Nagasaka et al. protein complexes, such as bacterial condensins and 2016). It has been proposed that condensin II acts eukaryotic cohesins (Brandão et al. 2019; Hsieh et al. first in prophase to anchor large outer DNA loops, 2020; Krietenstein et al. 2020). Nucleosomes also which are further branched into shorter inner DNA restrict the diffusion of cohesin in vitro (Stigler et al. loops by condensin I (Gibcus et al. 2018). This 2016). Therefore, the effect of higher order proposal is consistent with their localization chromatin fiber structure and other mitotic chromatin determined by super-resolution microscopy (Walther proteins on processive loop extrusion by condensin et al. 2018). In chicken DT40 cells, condensin II remains unknown. drives the helical positioning of loops around a Interphase nuclei are segmented to centrally located axis thus controls the organization chromosome territories, each of which contain highly of long distance interactions (6- 20 Mb), whereas entangled sister chromatids after replication (Cremer condensin I appears to control shorter distance and Cremer 2010; Sundin and Varshavsky 1981; interactions (Gibcus et al. 2018). This organization Farcas et al. 2011). Replicated pairs of chromatids bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423657; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Choppakatla et al., August 2021-preprint copy- BioRxiv are linked by cohesin during interphase, and cohesin binding of topo II and condensins to nucleosome removal during prophase (except at centromeres and arrays. Through a combination of chromosome other limited protected loci) promotes resolution of morphological analysis and Hi-C, we show that H1.8 sister chromatids, together with actions of condensin reduces chromosome length by limiting condensin I II and topo II (Nagasaka et al. 2016; Gandhi, loading on chromosomes, while H1.8 limits Gillespie, and Hirano 2006). Different chromosomes chromosome individualization by antagonizing both are also largely unentangled in interphase HeLa cells condensins and topo II. This
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