Pituitary tumor-transforming gene 1 (PTTG1) is an oncogene that is frequently overexpressed in tumors. The encoded PTTG1 protein has two well-known functions throughout the cell cycle. While PTTG1 acts as an inhibitory chaperone of separase for regulating sister chromatid segregation during mitosis, it also serves as a transcription factor during interphase. Whether the defects caused by the loss-of-function of PTTG1 are due to either one or both of its functions is unclear. Since previous studies of PTTG1 using permanent knock-out and post-transcriptional gene silencing approaches were limited by multiple drawbacks, this study aimed to reassess the role of PTTG1 in the cell cycle using a conditional knock-out strategy. By combining the CRISPR/Cas9 genome editing system, the tetracyclin...[ Read more ]

Pituitary tumor-transforming gene 1 (PTTG1) is an oncogene that is frequently overexpressed in tumors. The encoded PTTG1 protein has two well-known functions throughout the cell cycle. While PTTG1 acts as an inhibitory chaperone of separase for regulating sister chromatid segregation during mitosis, it also serves as a transcription factor during interphase. Whether the defects caused by the loss-of-function of PTTG1 are due to either one or both of its functions is unclear. Since previous studies of PTTG1 using permanent knock-out and post-transcriptional gene silencing approaches were limited by multiple drawbacks, this study aimed to reassess the role of PTTG1 in the cell cycle using a conditional knock-out strategy. By combining the CRISPR/Cas9 genome editing system, the tetracycline-controlled transcriptional activation (Tet-off) system, and the mini-auxin-inducible degron (mAID) system, PTTG1 can be tightly and rapidly depleted at specific time points, allowing the investigation of PTTG1 in mitosis and interphase independently. In this study, a conditional PTTG1 knock-out HeLa cell line was generated. Knocking out PTTG1 did not cause any significant cell-cycle arrest but decreased the proliferation rate. Notably, knockout of PTTG1 during mitosis resulted in prolonged mitosis, possibly due to abnormal separase expression, which affected both mitotic entry and exit. On the other hand, knockout of PTTG1 during interphase resulted in prolonged interphase, possibly due to abnormal gene expression involved in regulating cell proliferation during the G₁/S transition. This study also showed that the lengthening of mitosis and interphase in PTTG1 knock-out cells is independent of each other. Collectively, this study highlights the importance of PTTG1 in regulating both mitosis and interphase through independent mechanisms.