bioRxiv preprint doi: https://doi.org/10.1101/2020.11.04.367847; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. Developmental regulation of mitotic chromosome formation revealed by condensin reporter mice Gillian C A Taylor1, Lewis A Macdonald1, Matilda Bui1, Lucy Scott1, Ioannis Christodoulou1, Jimi C Wills2, Dimitrios K Papadopoulos1, Andrew J Wood1,* 1 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, EH4 2XU, UK. 2Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK. *Email address for correspondence: [email protected] Germline mutations affecting subunits of condensins I and II cause tissue-specific disease in humans and mice through chromosome segregation failure. However, condensin activity is universally required for chromosome segregation, and hence the developmental basis for these phenotypes is not understood. Using novel transgenic mouse strains, we show that cell-lineage-specific dosage of non-SMC condensin subunits controls the number of catalytically active holocomplexes during different haematopoietic cell divisions in mice. Thymic T cell precursors load significantly higher levels of both condensin I and II subunits onto mitotic chromosomes compared to B cell or erythroid precursors, and undergo elevated mitotic chromosome compaction. Thymic T cells also experience relatively greater chromosome instability in a condensin II hypomorphic strain, indicating that genome propagation requires particularly high condensin activity at this stage of development. Our data highlight developmental changes in the mitotic chromosome condensation pathway, which could contribute to tissue-specific phenotypes in chromosome instability syndromes. Introduction 11,12. Condensins have 5 core subunits: a heterodimer At the onset of mitosis, the entire genome is restructured of structural maintenance of chromosomes proteins in a process involving two condensin complexes and (SMC2 and SMC4), a kleisin subunit and two HEAT- topoisomerase II 1-5. This serves several purposes: to repeat proteins 1,13. The N- and C-termini of the kleisin shorten chromosomes and prevent their breakage in subunit interact with apposing SMC subunits to form the cytokinetic furrow, to provide the rigidity necessary an asymmetric ring-like structure that can tether to withstand traction forces from the mitotic spindle, DNAs at the base of expanding loops 4. SMC proteins and to remove catenations between sister chromatids possess a DNA-dependent ATPase activity encoded that arise during DNA replication. Under conditions within their globular head domains, which also of condensin or topoisomerase II deficiency, persistent requires the kleisin and HEAT-repeat subunits 14,15, and catenations result in the formation of anaphase bridges is central to their loop forming activity 16,17. The HEAT that impair chromosome segregation and trigger repeat subunits create surfaces for DNA binding, and, structural rearrangements such as chromothripsis 6. together with the kleisin, form a ‘safety belt’ structure Accordingly, condensin mutations occur at above- that tethers one side of the loop anchor 18 to ensure that background levels in human tumour genomes 7, and loop extrusion proceeds in a unidirectional manner 4 8 cause haematological malignancy in mice . Mammals have two condensin complexes, condensin I Condensins are DNA-dependent motor protein and II (Figure 1A), which share the same heterodimer complexes 9 that are homologous to cohesins and of SMC subunits but contain a distinct set of three SMC5/6, and play an essential role in cell division in associated proteins 1. These complexes are each eukaryotes, bacteria and archea 10. On mammalian essential for cellular proliferation in most cell types chromosomes, condensins form an axial structure from that have been studied, but show distinct patterns which they extrude linear chromatin fibres into loops of subcellular localisation during the cell cycle. In 3,4. When combined with the strand passage activity cultured cells, condensin II is present in the nucleus of topoisomerase II, this provides a mechanism that throughout the cell cycle 19,20 and is loaded onto allows intertwined DNA molecules to be individualised chromatin following DNA replication 21,22. Condensin 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.04.367847; this version posted November 4, 2020. 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 4.0 International license. I is primarily cytoplasmic during interphase and Crosses of heterozygous transgenic parents produced associates with chromosomes only following nuclear homozygotes at the expected mendelian frequency envelope breakdown during prometaphase 19,20. (Figure 1E). This contrasts with null mutations in each Condensin II therefore drives the early stages of loop gene, which are homozygous embryonic lethal 34,35. extrusion during prophase and is positioned at the The Ncaph transgene did not significantly affect fertility centre of the chromatid axis during prometaphase, (Figure 1F) or growth (Figure 1G) in the homozygous when condensin I binds to form nested loops which state, but Ncaph2 homozygotes showed modest enable maximal levels of compaction during anaphase reductions in both traits (Figure 1F & 1G). No other 3,23. In Xenopus egg extracts, the ratio of condensin developmental abnormalities were apparent in either I to II has been shown to determine the shape of line. In particular, thymocyte development, which metaphase chromosomes. Ratios favouring condensin was previously shown to be perturbed by a missense I lead to longer and thinner chromosomes, whereas mutation in Ncaph2 (I15N, 8,27), was unaffected in ratios favouring condensin II generate chromosomes mice homozygous for either transgenic allele (Figure that are shorter and thicker 24. S1). We conclude that the essential functions of Ncaph and Ncaph2 are retained in the presence of C-terminal We and others have previously shown that germline mClover tags. mutations affecting the non-SMC condensin subunits cause tissue-specific developmental abnormalities Cell lineage-specific differences in Ncaph and in humans 25,26 and mice 8,27. This poses a paradox, Ncaph2 expression because the core chromosome condensation pathway Condensin dynamic behaviours have been well is not thought to change during cellular differentiation. documented in cancer cell lines 1,19,20,38. However, 28-32 However, with few exceptions , our current whether the cellular activity of these complexes is understanding of mitotic chromosome condensation modulated during different physiological cell cycles, originates from studies performed in vitro, or in cell as cells progress along distinct lineage paths within 33 lines kept in long-term culture . The aim of the tissues, is not known. To address this question we current study was to compare the dynamic properties chose to focus on blood development, where cell types of condensins I and II across different cell divisions with high mitotic activity can be identified in several and lineages within a complex tissue, and to establish functionally specialised cell lineages, each descended the relationship between condensin dynamics, mitotic from the same pool of tissue stem cells. chromosome compaction and condensin mutant phenotypes during development. Multiparametric flow cytometry was used to compare steady-state levels of Ncaph and Ncaph2 in Results haematopoietic progenitor and precursor cells that Generation of fluorescent reporter mice for were freshly isolated from the bone marrow and condensin I and II thymus of young adult animals (Figure 2A, Figure S2). DNA content staining was used to selectively assess In order to measure how the expression level and cells during the S/G2/M phases of the cell cycle (Figure molecular dynamics of condensin complexes change 2A), thereby controlling for differences in the fraction during different mitotic cell cycles, we generated of G0 versus G1 cells across different populations. This transgenic reporter mice (Figure 1B, Supplemental analysis revealed that Ncaph protein levels differed by Methods). The kleisin subunits Ncaph and Ncaph2 more than 8-fold between cell types, with lowest levels were selected for tagging as they are known to be in early haematopoietic progenitors (LSK: Lin-/Sca- essential for the function of mouse condensins I and 1+/c-Kit+, including haematopoietic stem cells and II, respectively 34,35, and are expressed at levels that 23 their multipotent derivatives), intermediate levels in are limiting for holocomplex assembly . Ncaph and bone marrow B cell (CD19+) and erythroid lineage Ncaph2 therefore serve as proxies for condensin I and 23 (Ter119+) precursors, and highest levels in thymic II, respectively . T cells (Figure 2B). Ncaph2 protein was also most Using a CRISPR-Cas9-based strategy 36 (Materials abundant during divisions in the T cell lineage (Figure and Methods), the mClover coding sequence was 2C). Importantly, these cell-type differences were not integrated at the endogenous Ncaph and Ncaph2 due to changes