Protein arginine methyltransferase 5 represses tumor suppressor miRNAs that down-regulate CYCLIN D1 and c-MYC expression in aggressive B-cell lymphoma
ABSTRACT
Protein arginine methyltransferase-5 (PRMT5) is overexpressed in aggressive B-cell non-Hodgkin’s lymphomas, including mantle cell lymphoma and diffuse large B-cell lymphoma, and supports constitutive expression of CYCLIN D1 and c-MYC. Here, we combined ChIP analysis with next- generation sequencing to identify microRNA (miRNA) genes that are targeted by PRMT5 in aggressive lymphoma cell lines. We identified enrichment of histone 3 dimethylation at Arg-8 (H3(Me2)R8) in the promoter regions of miR33b, miR96, and miR503. PRMT5 knockdown de-repressed transcription of all three miRNAs, accompanied by loss of recruitment of epigenetic repressor complexes containing PRMT5 and either histone deacetylase 2 (HDAC2) or HDAC3, enhanced binding of co-activator complexes containing p300 or CREB- binding protein (CBP), and increased acetylation of specific histones, including H2BK12, H3K9, H3K14 and H4K8 at the miRNA promoters. Re-expression of individual miRNAs in B-cell lymphoma cells down-regulated expression of PRMT5, CYCLIN D1, and c-MYC, which are all predicted targets of these miRNAs, and reduced lymphoma cell survival. Luciferase reporter assays with wild-type and mutant 3’-UTRs of CYCLIN D1 and c-MYC mRNAs revealed that binding sites for miR33b, miR96, and miR503 are critical for translational regulation of the transcripts of these two genes. Our findings link altered PRMT5 expression to transcriptional silencing of tumor-suppressing miRNAs in lymphoma cells and reinforce PRMT5’s relevance for promoting lymphoma cell growth and survival.
Tightly regulated epigenetic modifications of chromatin structure affect recruitment of both repressor and activator transcription factors and remodeling complexes, which govern gene expression. Consequently, any disruption impacting the function or interaction of transcription factors with chromatin-remodeling enzymes can trigger cellular transformation (1-5).Among the various modifications that can alter gene expression is methylation of arginine residues in histone tails catalyzed by protein arginine methyltransferases (PRMTs). PRMTs are classified either as type I, II, or III based on the type of methylation being catalyzed (6-8). All PRMTs carry out monomethylation at the ω-NH2 of arginine; however, they differ in their ability to add the second methyl group in either asymmetric (type I) or symmetric (type II) fashion. Depending on the type of histone arginine methylation introduced, transcription can either be inhibited or induced (6,7). PRMT5 is a type II arginine methyltransferase that utilizes S-adenosyl-L-methionine as a cofactor to catalyze symmetric dimethylation of arginine residues in histone proteins H3 arginine 8 (H3(Me2)R8) and H4(Me2)R3 (5-7). Ourgroup and others have shown that PRMT5 functions as a chromatin remodeler by interacting with hSWI/SNF complexes to repress transcription of tumor suppressor genes such as suppressor of tumorigenicity 7 (ST7), non-metastatic 23 (NM23), retinoblastoma like 2 (RBL2), phosphotyrosine phosphatase receptor (PTPROt), and cell cycle regulator genes such as CYCLIN E1, P14ARF and P16INK4a (9-13).
Both Mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL) are aggressive subtypes of B- cell non‐Hodgkin’s lymphoma (NHL) with a broad spectrum of clinical, pathological, and biological features. Patients with relapsed/refractory MCL and DLBCL have an overall poor prognosis despite aggressive multimodal therapy (14, 15). Thus, identification of novel therapeutic targets and development of targeted treatment strategies remain a top priority for these patients. The genetic hallmark of MCL is the translocation t(11;14)(q13;q32), which leads to juxtaposition of the proto-oncogene CCND1 at 11q13 to the immunoglobulin heavy chain enhancer (IGH) on chromosome 14q32 (16). This translocation leads to constitutive over- expression of CYCLIN D1 protein and cell cycle dysregulation through direct binding to CDK4/6 and phosphorylation of RB (17). While the t(11;14) is very rare in DLBCL, CYCLIN D1 is overexpressed in a subset of DLBCL and associated with more aggressive behavior (18).c-MYC is a transcription factor with oncogenic function that is overexpressed through a variety of mechanisms in many cancers including a subset of DLBCL and MCL. c-MYC is an immediate early gene involved in promoting transition from the G0/G1 phase to the S phase, activating both directly and indirectly the expression of CCND2 and CDKs as well as many other genes required for onset of the S phase, and down-regulation of cell cycle inhibitors (19).
In addition, c-MYC up- regulates the oncogenic miR 17-92 cluster, but most microRNAs directly regulated by c-MYC have tumor suppressor function and are usually repressed (20).We have previously shown that PRMT5 is overexpressed in MCL and DLBCL cell lines and primary lymphoma samples (10, 21). Our data show that PRMT5 knock-down with shRNA antagonizes CYCLIN D1-CDK4/6 signaling in MCL and DLBCL (21). In addition to the primary translocation event, other mechanisms that further increase CYCLIN D1 expression are frequently observed in MCL. These mechanisms include secondary chromosomal rearrangement at the 3′ end of the CCND1 locus or mutations in the 3′-untranslated region (3′-UTR) that lead to expression of truncated CYCLIN D1 transcripts missing part of the 3′-UTR (22, 23). These shorter transcripts, depleted of the destabilizing AU-rich elements and the binding sites for different microRNAs, have an extended half-life resulting in higher CYCLIN D1 protein levels, more aggressive disease phenotype, and poor clinical outcome (22, 23).c-MYC overexpression can occur by translocation t(8;14)(q24;q32), amplification or dysfunction of the pathways regulating c-MYC expression (24, 25). c-MYC overexpression is associated with more aggressive disease behavior in patients with MCL and DLBCL (26-31). Prior work showed that c-MYC promotes malignant cell survival and proliferation through direct upregulation of PRMT5 transcription(32).
More recently, we have shown that PRMT5 overexpression is critical for initiation and maintenance of Epstein- Barr virus-induced B-cell transformation, and that its inhibition using a first-in-class small-molecule PRMT5 inhibitor induces lymphoma cell death without affecting normal B cell survival or viability (12, 21). In this report, we shed light on the mechanisms by which PRMT5 regulatesCYCLIN D1 and c-MYC expression in aggressive B-cell NHL. Screening experiments utilizing chromatin immunoprecipitation combined with next generation sequencing (ChIP-Seq) analysis showed that PRMT5 overexpression is associated with enrichment of the H3(Me2)R8 epigenetic mark on promoter regions of over 9,000 genes in DLBCL cells, which was distinct from the enrichment observed in normal control B cells. We used this data set to extract potential micro-RNAs (miRNAs) predicted to target the 3’ untranslated region (UTR) of CYCLIN D1 and c-MYC transcripts. Following experimental validation of the ChIP-Seq data, we identified 3 miRNAs (miR33b, miR96, and miR503) as direct targets of PRMT5 repressive complexes. PRMT5 inhibition triggered derepression of all three miRNAs and resulted in reduced expression of both CYCLIN D1 and c- MYC. Luciferase reporter analysis using 3’UTR of both wild type and mutant CYCLIN D1 and c-MYC mRNAs showed that miR33b, miR96 and miR503 contribute differently to translational regulation of CYCLIN D1 and c-MYC. Furthermore, we show that re-expression of individual miRNAs reduces lymphoma cell survival and promotes cell death. These results demonstrate that PRMT5 epigenetically represses specific tumor suppressor miRNAs in B-cell lymphoma cells, and provide additional rationale for targeting this enzyme in aggressive lymphomas.
RESULTS
Genome-wide PRMT5 recruitment in B-cell lymphoma cells as determined by H3(Me2)R8 enrichmentTo identify genome-wide recruitment of PRMT5, cross-linked chromatin from tonsillar normal human B cells and tworespectively). Top molecular functions of genes with differential PRMT5 load in B cells include transcription DNA-binding activity, transcription regulatory region sequence-specific DNA binding, and RNA polymerase II regulatory region sequence-specific DNA binding (Supplementary Table 2).We have previously reported that decreased expression of miR92b and miR96 promotes efficient PRMT5 translation, over-expression, and methylation of histone epigenetic marks in MCL (10). PRMT5 promotes MCL and DLBCL cell growth and proliferation through enhanced CYCLIN D1-CDK4/6 signaling (21). In light of these results and our ChIP-Seq data, we reasoned that there might be a common set of miRNAs targeted by PRMT5, which are directly involved in regulating pro- survival factors such as CYCLIN D1 and c-MYC.To address this hypothesis, we examined expression of CYCLIN D1 and c-MYC in three different types of NHL lymphoma cell lines including DLBCL cell lines (Pfeiffer and SUDHL-2) and a MCL cell line (JeKo-1) (Figure 1B). Our findings show that both CYCLIN D1 and c-MYC are overexpressed in lymphoma cell lines compared to control normal B cells (Figure 1B). In addition, PRMT5 genetic knock-down via short hairpin (sh)-RNA or pharmacologic inhibition via a small molecule inhibitor (CMP5) lead to reduced CYCLIN D1 and c-MYC expression (Figure 1C and Supplementary Figure 2) (12).
PRMT5 epigenetically represses miRs33b, 96, and 503, and indirectly leads to enhanced CYCLIN D1 and c- MYC expressionTo determine if there is a PRMT5- silenced set of miRNAs, which regulate expression of CYCLIN D1 and c-MYC, we analyzed the 3’-UTRs of each transcript using TargetScan prediction algorithm (www.targetscan.org) for the presence of miRNA binding sites. Furthermore, analysis of the ChIP-Seq list of target genes with enriched H3(Me2)R8 in their promoter region allowed us to identify miRNAs targeted by PRMT5 with average TSS load of 50 or more across the entire cellular genome. Comparison of the list of potential miRNAs binding to the 3’-UTR of CYCLIN D1 and c-MYC with the list of miRNAs targeted by PRMT5 resulted in identification of a small set of candidate miRNAs for each gene (Table 1 and Supplementary Figure 3). To assess if PRMT5 was involved in transcriptional silencing of the identified miRNAs, we measured their expression in both resting and activated normal B cells as well as in JeKo-1, Pfeiffer, and SUDHL-2 lymphoma cell lines (Figure 2A, 2B and Supplementary Figure 4A). Real-time RT-PCR analysis showed that 6 out of 9 predicted CYCLIN D1-specific miRNAs exhibit 1.4 to 5-fold reduced expression in JeKo-1 cells compared to normal B cells (Figure 2A). Similarly, 9 out of 10 c- MYC-specific miRNAs showed 1.4 to 10- fold reduced expression in JeKo-1 (Figure 2B).
Similar CYCLIN D1-specific miRNA repression was seen in Pfeiffer and SUDHL-2 cell lines (Supplementary Figure 4A). As a control, we analyzed the levels of an unrelated miRNA, miR197, which is not a direct PRMT5 target as determined from the ChIP-Seq data, and found that its expression was unaltered in normal B, JeKo-1, Pfeiffer and SUDHL-2 cells. Comparison of miRNA levels between resting and activated B lymphocytes did not show any significant difference.PRMT5 knock-down in all three lymphoma cell lines resulted in de- repression of miR33b (P < 10-4), which is predicted to bind to both CYCLIN D1 and c-MYC 3’UTRs, and miR96 (P < 10-4),and miR503 (P < 10-4), which are predicted to be CYCLIN D1-specific miRNAs (Figure 2C, 2D and Supplementary Figure 4B-F), confirming the direct role played by PRMT5 in miR33b, miR96 and miR503 transcriptional regulation. We also measured expression of CYCLIN D1 and c-MYC-specific miRNAs in primary MCL tumor cells (n = 6), and found that expression of miR33b (P = 0.043), miR96 (P = 0.118) and miR503 (P = 0.0393)were reduced 1.8 to 2.3-fold compared to control normal B cells, demonstrating that expression of these three miRNAs is also suppressed in primary lymphoma cells (Figure 2E). Reduced expression of CYCLIN D1 and c-MYC-specific miRNAs is in agreement with enhanced CYCLIN D1 and c-MYC protein expression in all primary tumors examined, except for patient 3 where there was no detectable CYCLIN D1 expression (Figure 2F). While our data convincingly show that miRs levels are significantly lower in malignant B cells compared to normal B cells, our results suggest that additional factors may play a role in regulating PRMT5, c-Myc, and Cyclin D1 expression in MCL. To confirm if miR33b, miR96 and miR503 were direct PRMT5 targets, we analyzed H3(Me2)R8 methylation recruitment profiles obtained by ChIP- Seq data for promoter regions of each of the three miRNA (Figure 3A). We found that there was enrichment of the PRMT5- induced H3(Me2)R8 methylation mark in the miR33b, miR96 and miR503 promoter regions in both normal and transformed B cells compared to input, and that there were clear qualitative differences between normal B and lymphoma cell lines. Consistent with the ChIP-Seq results, quantitative ChIP-PCR assays showed that PRMT5 and its epigenetic marks, H3(Me2)R8 and H4(Me2)R3, were enriched 2.5 to 4-fold (P = 2 x 10-4 for PRMT5 and for H3[Me2]R8, P = 5 x 10-4 for H4[Me2]R3)at the miR96 promoter (Figure 3B), 2.5 to 6-fold (P = 10-3 for PRMT5, P = 10-4 for H3[Me2]R8, P = 2 x 10-3 for H4[Me2]R3)at the miR33b promoter in JeKo-1 cells (Figure 3C), , and 2 to 3-fold (P = 8 x 10- 4 for PRMT5, P = 6 x 10-4 for H3[Me2]R8,P = 4 x 10-4 for H4[Me2]R3) at the miR503 promoter (Figure 3D). Same experiment was performed in Pfeiffer and SUDHL2 with similar results (Supplementary Figure 5A and B). PRMT5 knock down with shRNA led to loss of recruitment of PRMT5 and associated epigenetic marks at the miR33b (Figure 3E) (P < 10-4), miR96 (Figure 3F) (P < 10-4), and miR503 (Figure 3G) (P < 10-4 for PRMT5 and H3[Me2]R8, P = 9 x 10-4 for H4[Me2]R3) promoters, further supporting the direct role played by PRMT5 in regulating miR33b, miR96, and miR503 transcription.Functional analysis of miR33b, miR96 and miR503 binding sites in the 3’UTR of CYCLIN D1 and c-MYC mRNAs. In-silico analysis showed that while miR33b binds to both c-MYC and CYCLIN D1 3’UTRs, miR96 and miR503bind only to CYCLIN D1 3’UTR. To evaluate the contribution of each of these binding sites in translational regulation of both CYCLIN D1 and c-MYC, we performed luciferase assays with constructs containing either wild-type or mutant sequences at predicted bindings sites for each miR in the 3’UTRs. Sequence analysis of the 3’UTR of human CYCLIN D1 mRNA revealed thepresence of conserved miR33b (nucleotides 1465 to 1471), miR96(nucleotides 1284 to 1290), and miR503(nucleotides 2034 to 2040) binding sites; whereas the 3’UTR of c-MYC mRNA harbored only one miR33b binding site (nucleotides 1969 to 1974) (Figure 4A and 4D). To assess the effect of each miRNA on CYCLIN D1 and c-MYC expression, we cloned the wild type 3’UTR region of either CYCLIN D1 mRNA (nucleotide 1126 to 2098) or c- MYC mRNA (nucleotides 1320 to 2340) downstream the coding sequence for the firefly luciferase reporter gene. We also generated mutant constructs where each site was mutated to abolish miRNA binding (Figure 4B and 4E). Normal B cells and lymphoma cells (JeKo-1, Pfeiffer, and SUDHL-2) were transfected with either control pCMV-Luciferase alone, or pCMV-Luciferase fused to wild- type or mutant CYCLIN D1, or wild-type or mutant c-MYC 3’UTR, and luciferase activity was measured 36 hours later.When the construct containing wild-type CYCLIN D1 3’-UTR was electroporated into normal B cells, which express miR33b, miR96, and miR503, a basal level of luciferase activity was observed (Figure 4C). When the same construct was introduced into lymphoma cell lines, (which express lower, albeit detectable levels of miR33b, miR96 and miR503 compared to normal B cells), there was no significant increase in luciferase activity over that seen with normal B cell control. When constructs containing mutant CYCLIN D1 3’UTR were used, there was a slight enhancement (1.5 to 1.8-fold) in luciferase activity in normal B cells compared to a 3.9 to 5-fold increase in luciferase activity in lymphoma cells, suggesting that mutation of miR33b, miR96 and miR503 binding sites prevented downregulation of luciferase activity. Similar results were observed with luciferase constructs containing either wild type or mutant c-MYC 3’UTR (Figure 4F). These results indicate that the identified PRMT5 target miRNAs are involved in CYCLIN D1 and c-MYC translational regulation, and that their suppression in lymphoma cells contributes to enhanced CYCLIN D1 and c-MYC protein expression.Restored expression of miR33b, miR96 or miR503 reduces CYCLIN D1 and c-MYC expression in lymphoma cells.Having shown that specific miRNA binding sites in the 3’UTR of either CYCLIN D1 or c-MYC mRNAs can regulate luciferase translation, we wanted to determine the consequences of re-introduction of either miR33b, miR96 or miR503 on CYCLIN D1 and c- MYC expression. Lymphoma cells were electroporated with either wild-type or mutant miRNAs, and whole cell extracts were analyzed for CYCLIN D1 and c- MYC protein expression (Figure 5A and Supplementary Figure 6C and 6E). In accord with the results of the luciferase assay, re-introduction of wild-type miR33b led to a decrease in CYCLIN D1 and c-MYC protein expression, while both miR96 and miR503 caused a significant drop in CYCLIN D1 protein levels. Since CYCLIN D1-CDK4/6 complexes are responsible for phosphorylation of the retinoblastoma protein (RB1), we checked if the levels of phospho-RB (S795) were altered in JeKo-1 cells upon reintroduction of each miRNA. As expected, re-introduction of miR33b, miR96 or miR503 triggered a reduction in phospho-RB (S795) (Figure 5A). We also analyzed CYCLIN D1 and c-MYC mRNA levels, however, we did not find any significant changes in thepresence of each miRNA (Supplementary Figure 6A, 6B and 6D).Since miR33b, miR96 and miR503 are direct PRMT5 targets, we sought to evaluate the effect of PRMT5 knock down on CYCLIN D1 and c-MYC expression. JeKo-1 cells were infected with lentivirus designed to express a PRMT5-specific short hairpin RNA, and levels of PRMT5 mRNA as well as levels of all three miRNAs were monitored every 12 hours over an 84 hour time span (Figure 5B). Similarly, kinetic analysis of PRMT5, its epigenetic marks, CYCLIN D1 and c-MYC protein expression was also conducted following genetic knock- down of PRMT5 via sh-RNA (Figure 5C) and pharmacologic inhibition with CMP5 (Figure 5E). Our findings showed that as the levels of PRMT5 mRNA began to decrease 36 hours post-infection, the levels of miR33b, miR96 and miR503 increased gradually to reach maximal de- repression 48 to 60 hours post-infection (Figure 5B). Analysis of protein expression revealed that there was kinetic concordance between miRNA derepression and increased repression of CYCLIN D1 and c-MYC protein expression (Figure 5C). These results indicate that PRMT5 knock down leads to derepression of miR33b, miR96 and miR503, which in turn triggers inhibition of both CYCLIN D1 and c-MYC translation.Given the importance of CYCLIN D1 and c-MYC in the control of cell growth and proliferation, we next determined whether re-introduction of individual miRNAs would induce apoptosis (Figure 5D and Supplementary Figure 6F and 6G). Electroporation of each individual miRNA into JeKo-1 cells, followed by fluorescence activated cell sorting (FACS) analysis demonstrated that re-introduction of any of the threemiRNAs results in a 45-55% (P < 10-4 for each miRNA) lymphoma cell death. Similar findings were obtained when miR33b, miR96 or miR503 were electroporated into either Pfeiffer (Supplementary Figure 6F) or SUDHL-2 (Supplementary Figure 6G) cell lines. Collectively, these results show that through its ability to epigenetically silence expression of specific miRNAs, PRMT5 is able to promote lymphoma cell survival by upregulating CYCLIN D1- CDK4/CDK6 proliferative signaling and c-MYC-driven oncogenic gene expression.PRMT5 knock down results in miR33b, miR96 and miR503 derepression through loss of repressive complex recruitment targeting miRNA promoters.We have previously shown that PRMT5 interacts with different repressor complexes to regulate target gene expression (12). In light of these results, we monitored recruitment of transcription factors (NF-kB p65 and Sp1), co- activators (p300 and CBP), co- repressors (HDAC2 and HDAC3), and histone acetylation marks known to be regulated by these chromatin-modifying enzymes in the promoter region of miR33b, miR96 and miR503 (Figure 6 and Supplementary Figure 7). Real time, quantitative ChIP-PCR analysis using cross-linked chromatin from JeKo-1 cells infected with lentivirus that expressed either control sh-GFP or sh-PRMT5 showed that PRMT5 knock down led to a 4-fold (P = 2 x 10-4) increase in NF-B p65 recruitment, loss of HDAC3 binding, a 2.5 to 3-fold (P = 5 x 10-4) enhanced binding of p300 and CBP, as well as a 3 to 7-fold increase in acetylation of histones H2BK12 (P = 3 x 10-4), H3K14 (P < 10-4), and H4K8 (P < 10-4) in themiR96 promoter region (Figure 6A and B). These molecular changes were consistent with restored miR96 transcription, and were reproducible in Pfeiffer and SUDHL-2 lymphoma cell lines (Supplementary Figure 7A and 7B). When we tested recruitment of NF-B p65 and HDAC3 to the promoter region of miR33b and miR503, there were no significant changes (Figure 6C and 6E), suggesting that different complexes were involved in transcriptional control of PRMT5 target miRNAs.Prior work has shown that SP1 and histone deacetylases can physically interact with PRMT5 and repress transcription of miR29b (33, 34). To assess whether SP1 was involved in transcriptional regulation of miR33b and miR503, we tested its recruitment to both miR33b and miR503 promoters in JeKo-1 (Figure 6C and 6E), Pfeiffer (Supplementary Figure 7C and 7E), and SUDHL-2 (Supplementary Figure 7D and 7F) cell lines. While SP1 was not recruited to the miR96 promoter in JeKo-1 cells (Figure 6A), its binding was enriched 8- and 7.5-fold (P < 10-4) on the miR33b and miR503 promoters, respectively (Figure 6C and 6E). Similarly, SP1 binding to these promoters was increased 5.5 and 4.5-fold (P < 10-4) in Pfeiffer and SUDHL-2 cells, respectively (Supplementary Figure 7).Both HDAC2 and HDAC3 have been shown to interact with SP1, and are recruited to transcriptionally repressed SP1 target genes (34, 35). Since there was no significant recruitment of HDAC3 on both miR33b and miR503 promoters, we checked for the presence of HDAC2. We found that HDAC2 binding was enriched 3-fold (P < 3.4 x 10-2) on the miR33b promoter, and 6.5-fold (P < 10-4) on the miR503 promoter in JeKo-1 cells (Figure 6C and 6E). Consistent withthese results, HDAC2 recruitment was enhanced 3-fold (P < 10-4) in Pfeiffer and 4-fold (P < 10-4) in SUDHL-2 cells (Supplementary Figure 7C-F) to miR33b and miR503 promoter regions. PRMT5 knock down resulted in loss of HDAC2 recruitment on both miRNA 33b and 503 promoters in all three lymphoma cell types, which in turn led to increased H3K9 (4-fold for miR33b and 3.5-fold for miR503, P < 10-4) and H3K14 (3-fold for miR33b and 4-fold for miR503, P < 10-4) acetylation in JeKo-1 cells (Figure 6D and 6F). ChIP-PCR analysis also showed that with PRMT5 knockdown, there was an increase in both CBP (4- fold for miR33b and 4.3-fold for miR503, P < 10-4) and p300 (3.2-fold for miR33b, P < 10-3; 3.5-fold for miR503, P < 10-4)binding to the promoter regions of miR33b and miR503 in JeKo-1 cells (Figure 6C and 6E). Similar results were observed in Pfeiffer and SUDHL-2 cell lines (Supplementary Figure 7C-F). These dynamic changes in chromatin binding were very specific, because when we tested for recruitment of the hSWI/SNF ATPase, BRG1, there was no significant change in its binding despite its 4 to 5-fold enrichment in the promoter region of all three miRNAs. These results suggest that PRMT5 knock-down induces changes in the chromatin structure of all three target miRNA promoters to promote their transcription via distinct complexes. To test whether, PRMT5 and the co-activator complexes are co-recruited to promoters of each miRNA, we performed a ChIP-reChIP assay using chromatin from JeKo-1 cells treated with either DMSO or with CMP5 (Figure 6G-I) (12). DNA was first immunoprecipitated with anti-PRMT5 antibody and then re-immunoprecipitated using indicated antibodies in Figure 6G, 6H and 6I. Our results show thatinhibition of PRMT5 causes gain of p65, p300 and acetylated histone marks H2BK12, H3K14, and H4K8 as well as loss of HDAC3 on promoter of miR96 (Figure 6G). Inhibition of PRMT5 also resulted in gain of CBP, p300 and acetylated histones H3K9 and H3K14 and loss of SP1 and HDAC2 on promoters of miR33b and miR503 (Figure 6H and 6I). To confirm inhibition of PRMT5 activity via CMP5, Figure 6J-L show loss of PRMT5 and its epigenetic marks on the promoter of targeted miRs. In a separate control experiment, we showed that PRMT5 inhibition led to loss of the PRMT5 epigenetic histone mark H3(Me2)R8 and not total H3 recruitment (Supplementary Figure 8). Interestingly, PRMT5 inhibition did not result in any significant change to BRG1 recruitment on any of the promoters. DISCUSSION Post-translational modification of conserved histone residues plays an essential role in controlling transcription of genes that govern growth regulatory networks. Recent studies from our group and others have shown that aberrant methylation of histone arginine residues as well as non-histone proteins by PRMT enzymes promotes lymphomagenesis (7, 36). We have previously shown that PRMT5-driven symmetric methylation of H3R8 is associated with transcriptional repression of tumor suppressor genes such as RBL2, ST7 and PTPROt (10-12, 21). While PRMT5 has been mainly shown to repress gene transcription, recent work in acute myeloid leukemia (AML) has implicated PRMT5 in transcriptional activation of FLT3, suggesting that it plays a dual epigenetic role as a transcriptional activator and transcriptional repressor in cancer cells (10, 12, 33). Genome-wide miRNA screens provide valuable information about small, non-coding RNAs that cells utilize to control expression of target genes. Differences in global miRNA profiles also help distinguish normal cells from transformed cells, and help further classify the different subtypes of cancer cells within each tumor. In this study, we have used a ChIP-seq data set derived from normal B cells and two DLBCL cell lines to show that PRMT5 suppresses expression of miR33b, miR96 and miR503, which are involved in regulating CYCLIN D1 and c-MYC translation. CYCLIN D1 is a G1 to S cell cycle regulator that operates through phosphorylation and inactivation of RB1. c-MYC is an established proto-oncogene that functions as a transcriptional activator promoting cell survival and proliferation by controlling expression of key target genes needed for G1 to S transition. Our studies have identified three specific miRNAs, miR33b, miR96, and miR503, which can directly target the CYCLIN D1 3′-UTR and regulate its expression in aggressive B-cell lymphomas. In MCL, CYCLIN D1 overexpression is driven by the t(11;14) translocation; however, our results show the importance of epigenetic silencing of CYCLIN D1-specific miRNAs to promote translation and over-expression. We have also found that miR33b can specifically target the c-MYC 3’-UTR, and that re-expression of each individual miRNA triggers downregulation of CYCLIN D1 and c-MYC protein expression and leads to enhanced cell death. These findings were further substantiated using MCL primary tumor samples, which showed that miR33b, miR96 and miR503 levels are suppressed, and that both CYCLIN D1 and c-MYC protein levels are over- expressed. Mechanistic studies showed that PRMT5 is involved in transcriptional repression of target miRNAs through recruitment of two distinct repressor complexes (summarized in Figure 7). At the miR96 promoter, PRMT5 promotes binding of HDAC3 to induce deacetylation of promoter histones H2BK12, H3K14 and H4K8, which is associated with PRMT5-driven enhanced symmetric methylation of H3R8 and H4R3 as evidenced by ChIP assays. PRMT5 knock down led to miR96 derepression resulting from decreased HDAC3 recruitment and binding of the transcription activator NF- B p65 and its co-activators CBP and p300. Interestingly, the mechanism by which PRMT5 suppresses transcription of miR33b and miR503 does not involve NF-B p65, but instead relies on removal of the selective protein SP1 and its associated co-repressor, HDAC2. PRMT5 has been shown to symmetrically methylate promoter histone H4R3 in the regulatory region of miR29b in AML patients (33). The mechanism by which PRMT5 silences miR-29b involves both SP1 and HDACs, and PRMT5 inhibition results in decreased H4(Me2)R3 in the miR29b regulatory region (33). Consistent with these results, we have found that PRMT5 is co-recruited with SP1 and HDAC2 to regulate expression of miR33b and miR503, suggesting that PRMT5 cooperates with different transcription factors and associated co-repressors to efficiently silence target gene expression. On both miR33b and miR503 promoters, PRMT5 co-localizes with SP1, BRG1 and HDAC2. PRMT5 knock down leads to dissociation of the repressive complex, and concomitant recruitment of the histone acetyltransferases CBP and p300, which hyperacetylate histones H3K9 and H3K14. Thus, it appears that when PRMT5 activity is inhibited, dynamic changes take place to promote transcriptional derepression through recruitment of transcriptional activators and co-activators. A recent study has shown that miR33b is down-regulated in gastric cancer cells, and hypermethylation of CpG islands located upstream of the miR33b promoter is responsible for its decreased expression (37). Although DNA hyper-methylation is known for its ability to suppress gene expression in cancer cells, our results show that in three different types of aggressive lymphoma cells PRMT5-mediated histone modification contributes to target tumor suppressor miRNA gene repression. Interestingly, in another report, overexpression of miR33b was shown to have no impact on CYCLIN D1 mRNA or protein levels in multiple myeloma (MM) cells (38); however, ectopic expression of miR33b in MM cells induced their growth arrest and death. The involvement of miR503 in the control of cancer cell growth and survival was recently highlighted in a study by Wang et al. where overexpression of miR503 in a hepatocellular carcinoma cell line (HepG2/ADM), known for its resistance to the drug adriamycin, led to downregulation of drug resistance- related proteins including multi-drug resistance 1, multi-drug resistance associated protein 1, DNA excision repair protein ERCC 1, SURVIVIN, and BCL2 (39). The net outcome of miR503 re- expression in HepG2/ADM cells was reversal of adriamycin resistance through drug efflux inhibition. Increased expression of miR503 was also associated with cell cycle blockade at G0/G1 and increased cell death (40). It is conceivable that PRMT5-mediated suppression of miR503 expression may render cancer cells more drug-resistant to therapeutic drugs, and enhance survival by driving expression of growth promoting genes such as CYCLIN D1 and c-MYC. Our results clearly show the direct and inverse relationship between PRMT5 and miR503, and demonstrate the effects of miR503 re-expression on promoting lymphoma cell death. While our findings show that PRMT5 modulates CYCLIN D1 and c- MYC through direct suppression of tumor suppressor miRs in aggressive B cell lymphomas, we speculate that a similar mechanism could be operable in other type of cancers. For example, it is well established that PRMT5 regulates cell- cycle-related proteins such as CYCLIN D1 in hepatocellular carcinoma and oropharyngeal squamous cell carcinoma in addition to modulation c-Myc expression in breast cancer (41,42). Our data support the notion that PRMT5 operates upstream of CYCLIN D1 and c- MYC, and promotes over expression through direct inhibition of regulatory miRNAs. Importantly, a study by Klier et al. has shown that CYCLIN D1 knock down leads to only moderate reduction in cell growth without induction of apoptosis in MCL cells, which was associated with weak induction of p27(Kip1), decreased RB1 (Ser807/811) phosphorylation, and consistent upregulation of CYCLIN D2 mRNA and protein expression (43). Interestingly, simultaneous knock down of CYCLIN D1 and D2 did not intensify the effects observed with CYCLIN D1 knock down alone, suggesting that compensatory cyclin-independent mechanisms governing proliferation are activated. These findings have important implications for MCL and perhaps DLBCL therapy, as strategies targeting only CYCLIN D1 function might be hampered by compensatory regulatory mechanisms, resulting in a low probability of treatment response. Since PRMT5 is overexpressed in MCL and DLBCL, and directly controls expression of tumor suppressor miRNAs that target CYCLIN D1 and c-MYC, our results prompted us to focus on the development of anti-PRMT5 therapy (12). Recent findings indicate that PRMT5 regulates both histones and non-histone proteins, including critical transcription factors, such as NF-B p65, E2F1 and P53, and chromatin remodelers such as PRC2, as well as cellular metabolism and cell migration (7). It is possible that each of these additional biological functions of PRMT5 contribute to tumorigenesis. Therefore, strategies aimed at targeting PRMT5, a pleiotropic enzyme regulating the activity of multiple cancer drivers, will likely be an attractive approach for aggressive B-cell lymphomas and potentially other cancers. Jeko-1 is a cell line derived from the peripheral blood of a patient with blastoid MCL in leukemic phase. DLBCL is classified in two distinct molecular subtypes with different biological characteristics and clinical outcome: germinal center B cell (GCB) and activated B cell (ABC) with the latter associated with a poorer prognosis. Pfeiffer is a GCB-DLBCL cell line while SUDHL2 represents an ABC-DLBCL cell line. The PDX cell line was established from an ibrutinib-resistant MCL PDX mouse model developed in Dr. Alinari’s lab. A20 is a murine DLBCL cell line. B- cell NHL cell lines (JeKo-1, Pfeiffer, SUDHL-2, PDX, A20) were grown in RPMI-1640 medium supplemented with 10% FBS and 1 mM sodium pyruvate. All studies using patient lymphoma samples, which had no patient identifiers, abide by the declaration of Helsinki principles and were approved by the Ohio State University Comprehensive cancer center Institutional Review Board (IRB protocol no. 1997CO194) and conducted in agreement with the approved guidelines (IBC protocol no. 2006R0017-R1-AM6). Similarly, all animal studies were performed in compliance with guidelines approved by the Federal and the Ohio State University Institutional Animal Care and Use Committee (IACUC protocol no. 20009A0094-R3). To isolate normal B cells, tonsillar tissues were minced extensively in RPMI-1640 containing 10% FBS and strained through a collector sieve (Bellco Glass, Inc.) to remove tissue debris. Next, monocytes were removed by adhesion to tissue culture plates, and B cells were isolated by depletion of T lymphocytes. Cells were mixed with 8-fold excess of sheep red blood cells (Colorado Serum Company) and incubated on ice for 1 hr. To separate B cells JNJ-64619178 from rosetted T lymphocytes, 10 ml of ficoll-paque (Amersham, Inc.) was added, and samples were spun at room temperature before collecting the layer containing B cells. B lymphocytes were washed with RPMI-1640 media and purity of the isolated B cells was determined by FACS analysis using anti-CD19-PE antibody.