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 WT 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.
Highlights
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
We have previously shown that PRMT5-driven symmetric methylation of H3R8 is associated with transcriptional repression of tumor suppressor genes such as retinoblastomalike 2 (RBL2), suppressor of tumorigenicity 7 (ST7), and PTPROt [10,11,12, 21]
We have used a ChIP-Seq data set derived from normal B cells and two diffuse large B-cell lymphoma (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
Summary
Genome-wide PRMT5 recruitment in B-cell lymphoma cells as determined by H3(Me2)R8 enrichment. We studied genome-wide recruitment of the H3(Me2)R8 PRMT5 epigenetic mark (enrichment sites) and differences between normal B cells and DLBCL cell lines. Comparison of PRMT5 enriched genomic sites in the promoter region of identified target genes showed that there was a significant difference between normal and transformed B cells (Fig. 1A). The heatmap of ChIP-Seq read densities in the promoter (Ϫ2.0 kbp upstream from the transcription start site (TSS)) and transcribed genomic regions (up to ϩ2 kbp downstream of the transcription end site (TES)) ranked by decreasing combined occupancy signals showed that maximum PRMT5 occupancy is in the promoter region for both normal and transformed B cells (Fig. S1A). Genome-wide correlation of ChIP signals showed that Pfeiffer and SUDHL-2 cell lines have highly-similar PRMT5-binding profiles that were distinct from normal B cells with average correlation coefficient of 0.98 and 0.8, respectively (Fig. S1B). We have previously reported that decreased expression of miR92b and miR96 promotes efficient PRMT5 translation, overexpression, and methylation of histone epigenetic marks in
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