Protein Arginine Methyltransferase 5 Directly Targets and Epigenetically Silences microRNAs miR33b and miR96 to Support Constitutive Cyclin D1 Activity in Non-Hodgkin’s Lymphoma

Epigenetic regulation mediated by arginine-specific methyltransferases enzymes play a central role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 has been associated with increased cell proliferation and survival. We have previously demonstrated that PRMT5 is over expressed in mantle cell lymphoma (MCL) and supports constitutive CYCLIN D1/CDK4/6 activity leading to inactivation of the RB/E2F pathway. PRMT5 is also a driver of PRC2 epigenetic activity and promotes activation of b-CATENIN target genes MYC and SURVIVIN (Chung et al ASH, 2014). While PRMT5 has been characterized as a transcriptional silencer, few genes have been identified as direct targets.

In this study, we have taken advantage of the next generation sequencing (NGS) platform and integrated high throughput Illumina High-seq ChIP sequencing with whole transcriptome analysis to shed light on the global epigenetic role played by PRMT5 in B-cell lymphoma. We have conducted ChIP-sequencing analysis to map genome-wide recruitment of the PRMT5-specific epigenetic mark H3Me2R8 in three patient-derived non-Hodgkin’s lymphoma (NHL) cell lines (pre germinal center (GC) Jeko, GC Pfeiffer, and post GC SUDHL2) as well as control Normal B-cells. Our results indicate that PRMT5 targets as many as 8500 genes in each lymphoma cell line and there are approximately 4600 genes that are common targets between the three different lymphoma cell lines. We next performed genome-wide mRNA sequencing of each lymphoma cell line after treating with a lentivirus expressing PRMT5 shRNA (or GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5, or DMSO vehicle control). Evaluation of direct microRNA (miR) targets common to all three NHL cell lines showed that the PRMT5-driven epigenetic mark H3Me2R8 was enriched on the promoters of miR33b, miR96 and miR506, three miRs predicted to bind the 3’ untranslated region (UTR) of CYCLIN D1. RNA-Seq and ChIP experiments from NHL samples treated with control or PRMT5 inhibition suggested that PRMT5 epigenetically silenced all three microRNAs and validation studies using real-time RT-PCR demonstrated that PRMT5 knock down/inhibition led to transcriptional derepression of miR33b and miR96 in all three cell lines and primary patient mantle cell lymphoma samples. Inhibition of PRMT5 led to loss of recruitment of epigenetic co-repressor complexes containing PRMT5 and HDAC3 and gain of p65 and enrichment of hyperacetylated lysine epigenetic marks H4K8, H3K14 and H2BK12. Interestingly, suppression of both of these miRNAs was also found to augment PRMT5 translation and re-expression of miR33b and miR96 led to simultaneous down regulation of PRMT5 and CYCLIN D1. Using wild-type and mutant CYCLIN D1 3’ UTRs subcloned downstream of a CMV driven luciferase reporter, we show that the binding sites of miR33b and miR96 are critical for translational regulation by these miRNAs.

Our studies link dysregulated PRMT5 expression, which is a common finding in aggressive lymphomas, to aberrant expression of miR33b and miR96 and highlights yet another mechanism by which this arginine methyltransferase can support the expression of CYCLIN D1 in transformed lymphoma cell lines and MCL clinical samples. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own miRNAs and those that regulate CYCLIN D1, and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.