Protein Arginine Methyltransferase 6 regulates transcription and alternative splicing

Matthew J. Harrison (2010). Protein Arginine Methyltransferase 6 regulates transcription and alternative splicing PhD Thesis, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, The University of Queensland.

       
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s41121908_PhD_abstract.pdf final abstract application/pdf 14.17KB 6
s41121908_PhD_finalthesis.pdf final thesis application/pdf 2.67MB 23
Author Matthew J. Harrison
Thesis Title Protein Arginine Methyltransferase 6 regulates transcription and alternative splicing
School, Centre or Institute Diamantina Institute for Cancer, Immunology and Metabolic Medicine
Institution The University of Queensland
Publication date 2010-09
Thesis type PhD Thesis
Supervisor Dr. Dennis Dowhan
Assoc. Prof. Brian Gabrielli
Total pages 281
Total colour pages 20
Total black and white pages 261
Subjects 11 Medical and Health Sciences
Abstract/Summary Transcription and alternative splicing are tightly controlled events that occur simultaneously during gene expression. Here, we report that Protein Arginine Methyltransferase 6 (PRMT6) regulates both of these processes, and so can determine both the abundance and final composition of gene transcripts. Specifically, we found that PRMT6 can function as a coactivator for steroid hormone nuclear receptors (SHRs), but not several non-steroidal nuclear receptors (NRs). It coactivates the progesterone (PR), glucocorticoid (GR) and estrogen receptors (ER and ER) in a hormone-dependent manner in luciferase reporter assays, but not the thyroid hormone receptor- (TR), peroxisome proliferator-activated  (PPAR) or retinoic acid (RAR) receptors. A mutant PRMT6 with disrupted enzymatic activity cannot function as a coactivator, demonstrating that transcription is promoted by PRMT6-dependent arginine methylation. In addition, we demonstrated that PRMT6 is required for maximal estrogen-stimulated expression of GREB1 and PR, and progesterone-dependent expression of HSD11B2 and FKBP4 in breast cancer cells. This confirms that PRMT6 is an integral component of the estrogen and progesterone signalling pathways, and that it is not redundant with other PRMTs in its regulation of steroid hormone signalling. In addition, we found that PRMT6 co-operates with PRMT4 (known as CARM1) but not PRMT1 to stimulate estrogen-dependent transcription, demonstrating how a complex interplay between different arginine methylation events occurs during transcription. Coimmunoprecipitation experiments demonstrated that PRMT6 forms a complex with ER in mammalian cells. In addition, GST-pulldown and mammalian-2-hybrid analysis demonstrated that PRMT6 interacts with Steroid receptor coactivator-1 (SRC-1). SRC-1 is a member of the p160/SRC family of proteins that link secondary coactivators to SHRs. PRMT6 co-operates with all three members of the p160/SRC family of proteins to stimulate estrogen-dependent transcription, indicating that PRMT6 is recruited to active promoters of steroid hormone-dependent genes by p160/SRC proteins. Furthermore, we demonstrated that PRMT6 is recruited to promoter and enhancer regions of the GREB1 and PR genes in a cyclical manner in response to estrogen. This suggests a model of PRMT6 transcriptional coactivation whereby estrogen-bound ER recruits p160/SRC proteins to estrogen-activated gene promoters and enhancers. The p160/SRC proteins then transiently recruit PRMT6, which stimulates transcription by methylating components of the transcriptional machinery. In addition to its role in transcription, we also found that PRMT6 can regulate alternative splicing. We demonstrated that PRMT6 affects alternative splicing of the endogenous VEGF, Syk, Ninein, BCCIP, THEM4 and CTNND1 genes in breast cancer cells. Amongst these splicing decisions were examples of PRMT6-dependent exon inclusion and skipping, demonstrating that PRMT6 can promote a myriad of splicing changes. The alternative splicing regulation of all of these genes was steroid hormone-independent, demonstrating that PRMT6-mediated pre-mRNA processing can occur independently of recruitment to active transcription sites by SHRs. Similar to its role as a SHR coactivator, we found that PRMT6 requires its enzymatic activity to regulate alternative splicing. This suggests that the most likely mechanism of PRMT6-mediated alternative splicing is by the methylation of splicing factors. The methylation activity of CARM1 can also regulate alternative splicing, and we demonstrated that PRMT6 and CARM1 are not redundant in pre-mRNA processing. Therefore PRMT6 is integral to multiple components of the gene expression pathway, and can direct both gene expression levels and the composition of the final protein product. In order to gain an insight into the genes regulated by PRMT6 in breast cancer cells, we combined RNA interference (RNAi) with exon array analysis. This revealed that PRMT6 can determine the transcription of SHR-independent genes, and can both activate and repress gene expression. In addition, we identified a number of novel alternative splicing decisions regulated by PRMT6. A number of genes were regulated at both the transcription and splicing levels, allowing PRMT6 to severely impact on their function. In addition, a number of genes were regulated at only one of these levels by PRMT6, showing that PRMT6-mediated transcription and alternative splicing can also occur independently. Pathway analysis revealed that genes regulated by PRMT6 at both the transcription and alternative splicing levels are over-represented by genes involved in cancer. We verified that PRMT6 promotes the transcription of the proliferative genes of NCOA4, YWHAE, ARL6Ip1 and MINA, and represses transcription of the protective genes PTEN and IGFPB3. In addition, we confirmed that PRMT6 generates the more oncogenic spliced isoforms of Syk, VEGF, CTNND1 and BCCIP, whilst identifying novel PRMT6-dependent alternative splicing of the cancer-related genes Ninein and THEM4. Therefore PRMT6 has the potential to drive tumorigenesis by activating or repressing gene transcription or modulating alternative splicing. Indeed, we found that PRMT6 is required for estrogen-stimulated proliferation of breast cancer cells. Therefore this thesis identifies PRMT6 as a master regulator of gene expression that can potentially regulate tumorigenesis at a number of different levels.
Keyword PRMT
CARM1
transcription
splicing
cancer
methylation
Additional Notes Colour pages (page numbers refer to PDF page number, not number as displayed in thesis): 23, 24, 32, 33, 34, 40, 41, 42, 115, 171, 172, 178, 179, 183, 184, 185, 189, 190, 191, 202

 
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Created: Tue, 08 Feb 2011, 11:54:57 EST by Mr Matthew Harrison on behalf of Library - Information Access Service