Mechanisms Of Transcriptional Regulation And Ubiquitination-proteasomal Degradation Of PRMT5 Expression

Protein arginine methyltransferase 5(PRMT5) is a type II methyltransferase that can symmetrically methylate arginine residues of histones and non-histone protein substrates, and regulate a variety of cellular processes by epigenetic regulation of target gene expression and by post-translational modification of critical signaling molecules. Recently, several studies have shown that PRMT5 is overexpressed in human cancers such as lung cancer, ovarian cancer, colorectal cancer, breast cancer, melanoma, leukemia and lymphoma, and glioblastoma, and its overexpression is correlated with disease progression and poor survival. Further, knockdown of PRMT5 can inhibit cell proliferation or induce apoptosis in these human cancer cell lines. These studies collectively suggest that PRMT5 may function as an oncogene. However, how PRMT5 expression in cancer cells is regulated remains largely unknown. My thesis was aimed at investigating the transcriptional and post-translational regulation of PRMT5 expression in cancer cells.We investigated the transcriptional regulation of PRMT5 expression in prostate cancer cells. Based on the observation in the Hu lab that PRMT5 is overexpressed in prostate cancer, the most common prostate cancer cell lines, including LNCaP, PC-3 and DU 145 cells, were chosen for the investigation of the transcriptional regulation of PRMT5 in prostate cancer cells. First, we showed that there are two distinct promoters that harbor 6 SNPs(Single Nucleotide Polymorphisms) and one 13 bp Indel(Insertion/Deletion Polymorphism) within the 1.8 kb promoter region of PRMT5 in LNCaP cells(heterozygous). Second, we mapped the proximal promoter of PRMT5 to the-240 bp region by using the Dual-Luciferase Reporter Gene, and identified nuclear transcription factor Y(NF-Y) as a critical transcription factor that bind to the two inverted CCAAT boxes and regulate PRMT5 expression in multiple cancer cell lines by using EMSA, Ch IP, site-directed mutagenesis, deletion mutagenesis. Moreover, we provided evidence that loss of PRMT5 was responsible for cell growth inhibition induced by knockdown of NF-YA. More importantly, the PKC/c-Fos signaling negatively regulated PRMT5 expression in LNCaP prostate cancer cells through down-regulation of NF-YA transcription. Because down-regulation of several PKC isozymes correlates with human cancer development and progression, we then infer inactivation of PKC-c-Fos signaling pathway may be responsible for PRMT5 overexpression in a subset of human cancer patients, and further analysis of the interplay between PRMT5 and the PKC/c-Fos signaling in human cancer will provide novel insights into the oncogenic role of RPMT5 in human cancers.We investigated the post-translational modification of PRMT5 expression in cancer cells. Based on results described above and our unpublished observation, we hypothesized that PRMT5 expression may be also regulated at the post-translational level. To this end, Co-IP(Co-Immunoprecipitation) and WB(Western blot) were performed, and results showed that PRMT5 indeed undergo polyubiquitination. We also demonstrated that multiple lysine residues in PRMT5 attribute to polyubiquitination of PRMT5 by using IP and site-directed mutagenesis. We provided evidence showing that CHIP(The carboxyl terminus of Hsc70-interacting protein), an E3 ligase that often couples with the chaperone system to regulate protein degradation, interacted with PRMT5, and regulated PRMT5/MEP50 expression through a mechanism of ubiquitin-dependent proteasomal degradation. In addition, we demonstrated that Hsp90 inhibitors, such as GA and 17-AAG, mediated down-regulation of PRMT5 in a CHIP dependent manner. In addition, we also found that 17-AAG enhanced the down-regulation of PRMT5 and cell apoptosis induced by CHIP overexpression. These findings suggest that down-regulation of CHIP in human cancer tissues may contribute to the higher expression of PRMT5 in theses cancers. Finally, we demonstrated that the combination of targeting PRMT5 with 17-AAG treatment is a novel approach for inhibiting cell growth.Taken together, we have provided evidence that PRMT5 expression can be regulated at both transcriptional and post-translational level. We have demonstrated that the transcription factor NF-Y is responsible for PRMT5 transcription in prostate cancer cells, and the NF-Y transcriptional activity is also negatively regulated by the PKC/c-Fos signaling. We have also showed that the E3 ligase CHIP couples with the chaperone system to regulate ubiquitination and proteasomal degradation of PRMT5 at the post-translational level. Continued effort to uncover the regulatory mechanisms of PRMT5 expression in cancer cells may lead to identification of molecular targets for the development of novel anti-cancer agents.