Characterization of arginine methyltransferase PRMT8 in cells with increased plasticity

Hernandez, Sarah

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Characterization of arginine methyltransferase PRMT8 in cells with increased plasticity

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Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

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