Protein arginine methyltransferase 5 (PRMT5) activates WNT/β-catenin signalling in breast cancer cells via epigenetic silencing of DKK1 and DKK3

Protein arginine methyltransferase 5 (PRMT5) activity is dysregulated in many aggressive cancers and its enhanced levels are associated with increased tumour growth and survival. However, the role of PRMT5 in breast cancer remains underexplored. In this study, we show that PRMT5 is overexpressed in breast cancer cell lines, and that it promotes WNT/β-CATENIN proliferative signalling through epigenetic silencing of pathway antagonists, DKK1 and DKK3, leading to enhanced expression of c-MYC, CYCLIN D1 and SURVIVIN. Through chromatin immunoprecipitation (ChIP) studies, we found that PRMT5 binds to the promoter region of WNT antagonists, DKK1 and DKK3, and induces symmetric methylation of H3R8 and H4R3 histones. Our findings also show that PRMT5 inhibition using a specific small molecule inhibitor, compound 5 (CMP5), reduces PRMT5 recruitment as well as methylation of H3R8 and H4R3 histones in the promoter regions of DKK1 and DKK3, which consequently results in reduced expression CYCLIN D1 and SURVIVIN. Furthermore, CMP5 treatment either alone or in combination with 5-Azacytidine and Trichostatin A restored expression of DKK1 and DKK3 in TNBCs. PRMT5 inhibition also altered the growth characteristics of breast cancer cells and induced their death. Collectively, these results show that PRMT5 controls breast cancer cell growth through epigenetic silencing of WNT/β-CATENIN pathway antagonists, DKK1 and DKK3, resulting in up-regulation of WNT/β-CATENIN proliferative signalling.     

Arginine methylation: Making its mark on AP-1 gene activation

Oncogene-induced senescence (OIS) is characterised by a stable cell cycle arrest triggered by activated oncogenes and tumour suppressors. Whilst the in vivo relevance of OIS as a mode of tumour suppression is now beyond doubt many key questions with regard to the underlying mechanisms remain unanswered. To address these questions, we first review current knowledge of the essential players and pathways in OIS focussing our discussions mainly on murine cell systems and the paradigm of Ras-induced senescence. We then update experimental evidence for the involvement of the Runx genes that have recently emerged as important mediators of OIS. Of particular interest is the observation that Runx2 disruption renders primary murine embryonic fibroblasts (MEFs) refractory to Ras-induced senescence despite induction of a cascade of growth inhibitors and senescence markers. We suggest that Runx acts downstream of p53 in the "execution phase" of senescence specifically through deregulation of cyclin gene expression. We speculate how this might operate and consider the implications of these findings for the emerging role of the Runx family as tumour suppressors.     

Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement

Engagement of membrane Ig (mIg) on WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells, results in growth arrest and subsequent apoptosis. Of the several dozen genes upregulated greater than two-fold by anti-IgM treatment through DNA microarray analysis, we focused on B cell translocation gene 1 (Btg1) and Btg2, member of Btg/Tob family of proteins. WEHI-231 cells were infected with the Btg1/EGFP or Btg2/EGFP retroviral vectors, and those expressing either Btg1 or Btg2 accumulated in G1 phase at significantly higher proportions than that seen for cells expressing control vector. Btg1 or Btg2 bound to protein arginine methyltransferase (PRMT) 1 via the box C region, an interaction required for anti-IgM-induced growth inhibition. The arginine methyltransferase inhibitor AdOx partially abrogated growth inhibition induced by Btg1, Btg2, or anti-IgM. The Btg1- or Btg2-induced growth inhibition was also abrogated in PRMT1-deficient cells via introduction of small interference RNA. In addition, we observed anti-IgM-induced arginine methylation of two proteins, a 28-kDa and a 36-kDa protein. Methylation, detected by a monoclonal antibody specific for asymmetric, but not symmetric methyl residues, was observed as early as 1 h-2 h after stimulation and was sustained for up to 24 h. The anti-IgM-induced p36 arginine methylation was abrogated in the PRMT1-deficient cells, suggesting that PRMT1 induces p36 methylation. Together, these results suggest that anti-IgM-induced growth inhibition is mediated via upregulation of Btg1 and Btg2, resulting in the activation of arginine methyltransferase activity and culminating in growth inhibition of WEHI-231 cells.     

The RGG domain in hnRNP A2 affects subcellular localization

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.