PRMT5 inhibition leads to miR96 transcriptional derepression. ChIP assays were performed on crosslinked chromatin from transformed B cells (60A and SR27) using either preimmune (PI) or the indicated immune antibodies, and the retained DNA was amplified by qPCR using miR96-specific primers and probe. Fold enrichment with each antibody was calculated relative to the PI sample. (A) ChIP findings showed significant enrichment of PRMT5 and its epigenetic marks (S2Me-H3R8 and S2Me-H4R3) at the miR96 promoter. (B-E) ChIP assays and qPCR showed that PRMT5 knockdown (shRNA) or inhibition (CMP5, 40 μM) in EBV-transformed LCL 60A led to loss of recruitment of the enzyme and its epigenetic marks to the miR96 promoter (B and C, respectively) and transcriptional derepression of miR96 (D and E, respectively). miR197 is a nonbinding miR used as control. (F-G) ChIP assays and qPCR showed that PRMT5 knockdown (shRNA) or inhibition (CMP5, 40 μM) in EBV-transformed LCL 60A led to enhanced recruitment of p65 and p300, loss of HDAC3 recruitment, and hyperacetylation of lysine marks on histones H3, H4, and H2B, changes consistent with restored transcriptional activity of miR96. ChIP experiments were repeated in SR27 cells with similar results. *P < .05; **P < .01. Error bars indicate SEM. (H) Nuclear extract (500 µg) from the immortalized cell line (D-22) and the fully transformed cell line 60A was immunoprecipitated using either preimmune (immunoglobulin G [IgG]) or anti-PRMT5 (top panel) or anti-p65 antibody (bottom panel), and bound proteins were analyzed by western blot analysis using anti-HDAC3, anti-PRMT5, anti-p300, or antip65 antibody. Input represents 50 µg of nuclear extract from indicated cell lines. Results indicate that treatment with PRMT5 inhibitor leads to physical deassociation between PRMT5 and HDAC3 with subsequent physical association between PRMT5 and p300. (I) Proposed schema for PRMT5 regulation of miR96 transcription and mir96 regulation of PRMT5 translation: LMP1-driven NF-κB–repressive complex, which includes PRMT5, p65, and HDAC3, binds to the miR96 promoter and leads to miR96 transcriptional silencing with subsequent enhanced PRMT5 translation. Inhibition of PRMT5 leads to: disruption of the PRMT5/p65/HDAC3-repressive complex, loss of recruitment to the miR96 promoter, enhanced association of p65 with the acetyltransferase p300, and recruitment of this activation complex to miR96 promoter. This leads to hyperacetylation of histone marks and miR96 re-expression which in turn leads to inhibition of PRMT5 translation and subsequent re-expression of critical regulatory/tumor suppressor genes.