1. Investigating the regulation and putative interphase function of the condensin II subunit NCAPH2
- Author
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Brisbane, Jennifer M., Wood, Andrew, and Caceres, Javier
- Subjects
condensin II subunit ,NCAPH2 ,mitotic chromatids ,abnormal ploidy ,ploidy ,kleisin subunit ,condensin II assembly ,mitosis ,NCAPH2 protein ,interphase roles ,5'UTR of NCAPH2 ,upstream open reading frame (uORF) ,NCAPH2 uORF ,auxin-inducible degron-tagged NCAPH2 ,STAT5 target gene expression ,subnuclear heterochromatin distribution - Abstract
Condensins are protein complexes imperative for the individualisation and rigidity of mitotic chromatids. The functions of these highly conserved proteins are essential for the successful inheritance of genetic material during cell division. Mutations in condensin subunits cause DNA damage and abnormal ploidy in cells, and tissue-specific phenotypes such as T-cell lymphoma and microcephaly when inherited through the mammalian germline. The kleisin subunit NCAPH2 is dose limiting for condensin II assembly, and has been suggested to function during interphase as well as mitosis. This thesis studies how the level of NCAPH2 protein, and thus presumably condensin II, is regulated, and explores possible interphase roles. Within the 5'UTR of NCAPH2 is an upstream open reading frame (uORF), a potential cis-regulator of NCAPH2 protein synthesis. Many uORFs have been characterised throughout the genome as negative regulators of translation efficiency. These 5' UTR elements have been proposed to act through several mechanisms, which ultimately result in a reduction in the number of ribosomes commencing translation at their main ORF initiation codon. The human NCAPH2 uORF has been studied previously using transient over expression assays, which are limited in their ability to capture the true influence of the uORF in its endogenous context. When looking at ribosome occupancy at this putative translational regulatory element, analysis of public data show that the uORF is robustly occupied by ribosomes in a variety of mammalian cell types. Consequently, I hypothesised that this region could be important in regulating NCAPH2 protein levels. Using transient reporter assays designed to test the regulatory ability of both the mouse and human 5' UTR of NCAPH2, I established that this conserved uORF was capable of regulating downstream translation in cis. Upon this finding, I then performed base-editing of the endogenous uORF locus in a NCAPH2AID:mCherry/AID:mCherry reporter cell line. The resulting ΔuORF cell line revealed that this element does regulate NCAPH2 protein levels at the endogenous locus. Further work to characterise the consequences of its loss on condensin II function found that ΔuORF cells were able to divide at similar rates to the parental cell line, and that the rate of de novo NCAPH2 synthesis was not detectably altered. Studies of condensin function during interphase have historically used RNAi or constitutive mutations to perturb protein function. Previous work in our lab generated novel CRISPR-engineered mice, in which auxin-inducible degron-tagged NCAPH2 can be rapidly depleted upon addition of the small molecule auxin. Using this system, experiments were performed ex vivo and in vivo to monitor the effects of rapid NCAPH2 loss on peripheral T-cells and thymic precursors. This enabled me to characterise the impact of rapid NCAPH2 loss at different stages of cellular differentiation, and at specific cell cycle phases. Contrary to previous reports, acute loss of NCAPH2 did not alter STAT5 target gene expression, ability to exit quiescence, or subnuclear heterochromatin distribution. Rather, it caused abnormalities consistent with known mitotic functions of condensin II. The Ncaph2AID tagged allele was subsequently combined with Ncaph2I15N, a germline mutation which causes a developmental block during thymopoiesis. By generating compound heterozygous animals combining AID-tagged alleles and germline I15N mutations, I conditionally depleted tagged NCAPH2 to study the consequences of the missense mutation in a thymus that had developed normally, without downstream consequences of prior defective cell divisions. Finally, previous work from our lab using Ncaph- and Ncaph2-AID tagged mice revealed that depletion of either subunit in precursor thymic T and bone marrow B cells resulted in a significant decrease in cell division efficiency, but not in mature peripheral lymphocytes. By combining Ncaph- and Ncaph2-AID tagged alleles, I found that simultaneous loss of both kleisin subunits severely impacts cell division efficiency in the periphery. These results support previous findings that different cell types have different vulnerabilities to condensin deficiencies, despite sharing the same genetic load.
- Published
- 2023
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