Killing a zombie: a full deletion of the BUB1 gene in HAP1 cells

Abstract

T he exact contribution of the BUB1 kinase to the spindle assembly checkpoint (SAC) in mammalian cells has been under debate since many years. While some studies confirmed a (near)-essential role for BUB1 in the SAC (Meraldi & Sorger, 2005; Klebig et al, 2009), other studies reported no obvious SAC defect in Bub1deficient cells (Johnson, 2004) or showed a SAC defect that could only be observed after sensitization with inhibitors for Mps1 (Vleugel et al, 2015). The arrival of CRISPR technology has thus far also failed to resolve this controversy. We and others showed that deletion of BUB1 in HAP1 or RPE-1 cells resulted in only a minor SAC defect (Currie et al, 2018; Raaijmakers et al, 2018), while another study showed that (acute) deletion of BUB1 in p53-deficient RPE-1 cells results in a more prominent SAC defect (RodriguezRodriguez et al, 2018). The latter study showed that editing of the BUB1 locus by CRIPSR-Cas9 is challenging as alternative splice variants of BUB1 can be expressed. Recently, Zhang et al combined CRISPR/ Cas9-mediated gene editing with siRNA depletion and concluded that BUB1 also makes a prominent contribution to the SAC in HeLa cells (Zhang et al, 2019). In addition, this study suggested that the role of BUB1 in the SAC was overlooked in HAP1 and RPE-1 cells due to residual expression of BUB1 protein (2 and 8%, respectively), and subsequently showed that the SAC defect became more apparent when these cells were further challenged with BUB1 siRNAs. Thus, complete deletion of BUB1 can be challenging. A recent commentary therefore referred to BUB1 as a zombie protein, and it was suggested that the only way to completely delete BUB1 is to remove the entire gene (Meraldi, 2019). Here, we did exactly that. We deleted the BUB1 gene in HAP1 cells using CRIPSR/ Cas9 by using two-guide RNAs targeting the first and last exon of BUB1 (Fig 1A, Appendix Supplementary Methods, Appendix Table S1). Clones that displayed successful loss of the two targeted exons were tested for the absence of several additional exons located in the gene body. We identified two ΔBUB1 clones that met all criteria; none of the tested exons were present in clones 20 and 35 (Fig 1B). This implies that in these clones, the entire BUB1 gene was deleted successfully and the deleted fragment was not integrated somewhere else in the genome. Clone 20 displayed a fusion between exons 1 and 24, involving the break sites induced by Cas9 (Fig 1B and C). In clone 35, we were unable to amplify such repair product (Fig 1B), and therefore, we are uncertain of how the genomic locus in clone 35 was precisely repaired. Nonetheless, it is clear from the loss of all exons that also in this clone, the BUB1 gene locus is deleted in its entirety. As expected, both clones did not display any BUB1 transcripts (Fig 1D). Besides, no BUB1 could be observed by Western blot or immunofluorescence (Fig 1E, H and I). Also, H2A-pThr210, a well-characterized substrate of the BUB1 kinase, was completely absent in the two ΔBUB1 clones (Fig 1F and G). Furthermore, and consistent with our previous observations, BUBR1 levels at kinetochores were severely reduced in the ΔBUB1 clones, although some residual levels could still be observed (Fig 1G and J, Raaijmakers et al, 2018). Taken together, we successfully generated two ΔBUB1 clones in HAP1 cells by completely eradicating the entire gene locus. Next, we assessed the SAC functionality in the full knockout clones (ΔBUB1 c20 and c35). To this end, we performed live cell imaging of cells treated with a high dose of nocodazole to trigger a full SAC response. We observed that both ΔBUB1 clones were able to establish a functional SAC as cells displayed a prominent arrest in response to nocodazole treatment, not significantly different from WT HAP1 cells or our previously published BUB1 KO cells (ΔBUB1 “Ex3”), that were generated by the stable integration of a Blasticidin resistance cassette in exon 3 (Fig 2A, Raaijmakers et al, 2018). Depleting BUB1 with an siRNA did not affect the SAC response in any of the tested clones (Fig 2A). However, we observed previously that our ΔBUB1 “Ex3” cells failed to maintain a prominent SAC when treated with a low dose of the MPS1 inhibitor reversine. Consistently, the ΔBUB1 clones c20 and c35 were unable to maintain a SAC when challenged with a high dose of nocodazole in the presence of a low-dose reversine, while WT HAP1 were still able to arrest (Fig 2B). These data suggest that BUB1 becomes critical to maintain a SAC when the SAC is not fully activated. To test this, we induced a more graded SAC response by treating the cells either with a lower dose of nocodazole or with noscapine, an opium alkaloid that produces minor perturbations in chromosome alignment, to the extent that only one or a few chromosomes misalign (Tame et al, 2016). Consistent with this hypothesis, we found that the full BUB1 knockout, ΔBUB1 c20, was unable to maintain a SAC under conditions where the SAC was