Background: Ki-67 has long been regarded as a DNA binding protein. It has distinct staining patterns depending on the cycling state of a cell. The transcription of its RNA, translation of the protein, as well as the degradation of the protein are extremely dynamic, and are highly correlated with cycling state of the cell as well. Although tied to the cell cycle, Ki-67 does not regulate the cell cycle, and its functions are not well understood. One proposed function of Ki67 is to coat mitotic chromosomes during mitosis to protect them from DNA damage as cells traverse from M to G1 of the cell cycle. Investigating this role in the context of SETD2 loss remains an important ask due to well documented roles SETD2 plays in safeguarding mitotic chromosomes from adverse events.
Methods: To investigate how SETD2 loss affects Ki67, I generated inducible knockout SETD2 cell line with an FKBP tag, which upon the addition of small molecule DTAG47, immediately results in degradation of SETD2. To confirm any notable phenotypes, I also used a small molecule SETD2 inhibitor, which maintains the SETD2 protein, but inhibits SETD2's methyltransferase activity. Using this cell-line, and this SETD2 inhibitor, I've performed cell proliferation assay to assess how the cells respond to SETD2 loss. Additionally, I've stained and subsequently characterized Ki67 levels in these cells to assess any functional changes in the Ki67 protein.
Results: When SETD2 is lost, cells proliferate less vs their wildtype counterparts. Additionally, when assessed using flow cytometry and immunofloresence, Ki67 appear to increase across cell cycling stages.
Conclusions: Acute loss of SETD2 and loss of SETD2 methyltransferase activity uncouples Ki67 levels from cellular proliferation. Further characterizing why Ki67 levels change, and what the functional roles of Ki67 are in cells remains and important task due to its crucial prognostic utility in medicine.
