These frozen noodles were lysed utilizing a Retsch PM100 cryomill (Retsch) with continuous liquid nitrogen cooling. physique supplement. elife-68918-fig7-data1.zip (921K) GUID:?E82E9B7A-E7B4-4BF9-8222-30BF54EE222D Transparent reporting form. elife-68918-transrepform1.docx (112K) GUID:?5D8E92DB-BDAE-4A96-9954-70C9FAFE2E5C Data Availability StatementHi-C sequencing data have been deposited in GEO under an accession HQ-415 code “type”:”entrez-geo”,”attrs”:”text”:”GSE164434″,”term_id”:”164434″GSE164434. All other data generated or analyzed during this study are included in the manuscript and supporting source data files. The following dataset was generated: Choppakatla HQ-415 P, Dekker B, Cutts EE, Vannini A, Dekker J, Funabiki H. 2021. Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome compaction and individualization. NCBI Gene Expression Omnibus. GSE164434 Abstract MGC33570 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 this 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 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 HQ-415 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. (Guacci et al., 1994; Umesono et al., 1983). During early embryogenesis in and nuclei (Rosin et al., 2018; Bauer et al., 2012) and drives sister chromatid decatenation by topo II (Nagasaka et al., 2016). It has been proposed that condensin II acts first in prophase to anchor large outer DNA loops, which are further branched into shorter inner DNA loops by condensin I (Gibcus et al., 2018). This proposal is usually consistent with their localization as determined by super-resolution microscopy (Walther et al., 2018). In chicken DT40 cells, condensin II drives the helical positioning of loops around a centrally located axis, thus controlling the organization of long distance interactions (6C20 Mb), whereas condensin I appears to control shorter distance interactions (Gibcus et al., 2018). This organization of the condensin I and II loops is also consistent with their roles in maintaining lateral and axial compaction, respectively (Green et al., 2012; Samejima et al., 2012; Bakhrebah et al., 2015). In egg extracts, in the presence of wildtype condensin I levels, condensin II depletion does not appear to HQ-415 change mitotic chromosome length, suggesting a reduced role for condensin II on these chromosomes (Shintomi and Hirano, 2011). The prevailing model suggests that mitotic chromatin loops are formed by the dynamic loop extrusion activity of condensins (Riggs, 1990; Nasmyth, 2001; Alipour and Marko, 2012), although the molecular details of the process remain unclear (Banigan and Mirny, 2020; Cutts and Vannini, 2020; Datta et al., 2020). Single-molecule experiments using purified recombinant yeast and human condensin complexes exhibited ATP-dependent motor activity and loop extrusion by yeast and human condensins (Terakawa et al., 2017; Ganji et al., 2018; Kong et al., 2020). Condensin-dependent loop extrusion in a more physiological extract system has also been shown (Golfier et al., 2020). In silico experiments further suggest that a minimal combination of loop extruders (like condensin) and strand passage activity (such as topo II) can generate well-resolved rod-like sister chromatids from entangled, interphase-like DNA fibers (Goloborodko et al., 2016a). However, it remains unclear if loop extrusion can proceed on chromatin since condensins prefer to bind nucleosome-free DNA (Kong et al., 2020; Zierhut et al., 2014; Shintomi et al., 2017; Toselli-Mollereau et al., 2016; Piazza et al., 2014). Human and yeast condensin complexes are capable of loop extrusion through sparsely arranged nucleosomes in vitro (Kong et al., 2020; Pradhan et al.,.