Since its discovery as a CIN suppressor, the mechanism by which the loss of the GPI-AP biosynthesis protein, PIGN leads to CIN has remained elusive. Moreover, conflicting opinions exist in the literature with regards to the origins of CIN, whether pre-mitotic or mitotic 3,18,36. In our prior study, PIGN loss triggered genomic instability and was linked to leukemic transformation in AML-MRC patients and an MDS transformation cell line model 15. CIN has been implicated in leukemia progression and has been proposed as a therapeutic target 28,37−40. In this study, we investigated the mechanistic basis of PIGN in CIN suppression and consequently, leukemic transformation. The cell cycle-dependent expression of PIGN in the context of SAC protein expression and relatively higher expressions of SAC proteins during the S-phase (Fig. 1) as well as interactions with PIGN from early S-phase through mitosis (Fig. 3e) confirmed previous studies that have demonstrated that the SAC can be activated before to mitosis and as early as during the S-phase 41,42. These results offered evidence of spatiotemporal interactions between PIGN and SAC components and may also reveal the order in which these interactions occur (Fig. 5).
PIGN and MAD1 are necessary for proper chromosomal alignment and segregation 3,43. The suppression or loss of PIGN resulted in MAD1 downregulation and vice versa, accompanied by an increased frequency of segregation errors (Fig. 2a-j). We have previously reported increased PIGN gene expression in AML-MRC patient CD34 + PBMCs with partial intron retentions in the PIGN gene compared to normal non-leukemic control cells 15. In this current study, a significant increase in PIGN expression was associated with a significant increase in MAD1 expression in AML-MRC patients known to possess partial intron retentions in the PIGN gene. Additionally, we observed complex formation and/or colocalization involving PIGN and the SAC proteins, MAD1, MAD2, and MPS1. Thus, it is important to investigate the relevant protein sequences involved in these interactions and to establish how PIGN depletion impacts the recruitment of SAC proteins to the kinetochore. This is vital because of the dynamic localization of MAD1 between the spindle attached-kinetochore and the spindle poles for the activation and silencing of the SAC respectively 17,20,44. Nevertheless, our findings reveal the complementary role that PIGN and MAD1 could play in the co-stabilization of the SAC and regulating the availability and migration of SAC proteins to disrupted k-Mt. The interaction of PIGN with MAD1, reciprocal regulation between them as well as their similar pattern of cell cycle-regulated expression (Fig. 1,2) may indicate a direct effect related to the stability of SAC-related proteins, with MAD1 being the limiting factor in SAC activation 20,43,45.
A degree of regulatory control seems to exist between PIGN and other SAC proteins including MAD2, BUBR1, and MPS1. PIGN ablation resulted in the suppression (Fig. 2a) or loss (Fig. 2c,f,i) of MAD2 which was rescued by ectopic overexpression of PIGN. PIGN ablation impacted BUBR1 expression, leading to an upregulation in BUBR1 expression in HEK293 PIGN KO cells similar to RNAi-mediated suppression of PIGN expression K562 cells (Fig. 2c,g). Conversely, CRISPR/Cas9-mediated PIGN ablation resulted in BUBR1 downregulation in the K562 myeloid leukemia cells (Fig. 2i). These variable observations could be cell context-related and/or attributable to PIGN availability (i.e. stable loss vs. transient suppression) in the cells under consideration. Regardless, the downregulation of PIGN expression modulated BUBR1 expression. Aside from their role in the SAC, MAD2, and BUBR1 have been implicated in the timing of mitotic regulation 46. Thus, PIGN regulation of MAD1 and BUBR1 expression may be indicative of regulatory control on mitotic timing. This was corroborated by our observation that, PIGN KO cells and cells ectopically overexpressing the intron-retaining mutant form of PIGN (MUT), presented with a significantly lower cell cycle frequency and mitotic index compared to wild-type PIGN (WT)-expressing cells (Fig. 4e,f). MPS1 gene expression seemed to be slightly upregulated despite a reduction in protein expression following PIGN loss in HEK293 cells (Fig. 2c,d). This disparity between gene and protein expression may be linked to the post-translational regulation of MPS1 47–49. Nonetheless, PIGN loss modulated MPS1 protein expression in HEK293 cells.
Optimal expression, timely activation, modulation, and silencing of the SAC-related factors are very critical for the efficiency and the stepwise time-bound precision of cell cycle progression 17. Thus, we sought to identify at which stage of the cell cycle these interactions occurred and other SAC components that may be involved. Despite its widespread cytoplasmic and ER localization, it is yet to be determined how PIGN may regulate the recruitment of SAC proteins to the kinetochore. Our observation of PIGN interaction with MAD1 and MPS1 from early S-phase revealed that a complex involving PIGN, MAD1, and MPS1 may be involved (Fig. 3e). Moreover, interactions between PIGN, MAD1, and MAD2 were observed in G2/M synchronized cells (Fig. 3c,d). Thus, MAD2 recruitment may be a later event following MAD1 and MPS1 recruitment. Overall, PIGN may interact with MPS1 and MAD1 in a ‘pre-SAC’ complex during early S-phase till late S-phase and form another complex involving MAD1 and MAD2 during mitosis (Fig. 5). While this regulatory interaction and complex formation may occur as revealed in this study, further studies to discriminate between the presence of binary complexes from that of a ternary complex are required.
The complexity of the mechanisms involved in SAC regulation highlights the importance of transcriptional regulation during SAC induction. Aside from PIGN, other proteins that previously had no physical interaction or functional association with the SAC such as WT1, RED, NUP153, YY1, SMURF2, TRRAP, and TAp73 have been reported to interact with and/or regulate the SAC 50–56. However, tumor suppressors and proto-oncogenes have been shown to transcriptionally regulate SAC-related genes 57. The loss of tumor suppressors like TP53, BRD4, and BRCA1 has been associated with altered expression of SAC-related genes, genomic instability, and tumor progression 58,59. We have previously reported PIGN gene expression aberration-driven genomic instability in leukemic cells that was independent of the TP53–regulatory pathway 15. Interestingly, TP53 has been shown in multiple studies to transcriptionally regulate the genes encoding MAD1, CDC20, cyclin A1 and cyclin B 58,60. Moreover, the tumor suppressors, Rb, and VHL transcriptionally regulate the gene expression of the core SAC component MAD2 57,61. Transcription factors like E2F, c-Myc, and FoxM1 and histone deacetylases have been linked to the transcriptional regulation of the SAC-related proteins including MAD2, PLK1 BUB3, and CDC20 57,59,60,62,63. Yet, the possibility of the translocation of PIGN to the nucleus for the regulation of the transcription of cell cycle- or SAC-related genes may be complicated by the dissolution of the nuclear membrane during mitosis 64,65. Thus, it will be necessary to determine whether PIGN regulates that SAC transcriptionally and/or translationally.
The ER membrane absorbs the nuclear membrane and dissolves into the cytosol which could explain the interaction between PIGN and MAD1 during mitosis despite the two proteins occupying separate cellular compartments 64–66. Accordingly, nuclear proteins that are not normally localized at the ER may typically show up in the ER or cytosol during mitosis. However, it remains unclear how and where the interactions between PIGN and MAD1 occurs whether at the kinetochore, cytosol, or the ER. Although, the mitotic interaction between MAD1 and PIGN seemed to involve MAD2. PIGN may be involved in the stabilization of the interaction between MAD1 and MAD2, however, this requires experimental confirmation. The colocalization of the partial-intron retaining mutant of PIGN with MAD1 (Fig. 4d), implies that this intron-retaining mutant could compete with the wild-type protein for colocalization with these SAC proteins. Overall, PIGN maintains chromosomal stability, by interacting with and possibly regulating the SAC (Fig. 6).