Generation of a stable HEK293 cell line to investigate how DIS3 dysfunction perturbs cell proliferation
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DIS3 gene is frequently mutated in Multiple Myeloma (MM), a yet incurable cancer of the B-lymphocytes lineage. As the major catalytic subunit of the nuclear exosome in human, is responsible for the processing of stable RNA species, mRNA quality control, and decay, as well as degradation of the products of spurious RNA Pol II transcription. Unexpectedly, for a cancer-related gene, disruption of the DIS3 gene affects the sustainability of cells and the rate of mitosis. Such a proliferative phenotype seems to be conserved across species as distant as fungi, insects, and mammals. Studies done in yeast revealed that it could be due to the failure of kinetochore formation correlating with centromere deheterochromatinization. DIS3 dysfunction leads to the accumulation of unstable transcripts, which may disturb the centromere heterochromatinization by deregulating methyltransferases (SUV39H1/H2) action at the centromeres, essential for establishing heterochromatin.The aim of this study was to generate a stable cell line that will enable investigation of disruption of SUV39H1/H2 methyltransferase function, in cells with DIS3 dysfunction and the consequential effect on the cell proliferation. Such a cell line generation required 2 steps: tagging the SUV39H1/H2 methyltransferase with fluorescent protein or FLAG epitope and replacing the endogenous DIS3 gene with a mutated variant.The correct tagging of SUV39H1 was verified at the genomic DNA and mRNA level using PCR or RT-PCR. Moreover, the alignment of sequenced PCR products confirmed the proper integration in the endogenous locus. The tagged protein was visualized by Western Blot. Using flow cytometry, it was confirmed that the cassette was functionally inducible and the generated cell line with an introduced mutated DIS3 gene, had a proliferative phenotype as reported previously. An RTqPCR analysis showed that upon induction, the stable cell line reproduces previously published molecular phenotypes. All together analyses proved the successful generation of a cell model system, which will be utilized in the lab as a platform for various experiments which are designed to explain the link between DIS3 dysfunction and the regulation of SUV39H methyltransferases action resulting in the disruption of mitotic progression. Understanding the molecular mechanism of this process may enable the development of targeted therapy for MM patients.