PRMT5 and MEP50 mediate the repressive activity of the BCL6 RD2 domain. (A) Schematic of plasmids encoding the GAL4 DNA-binding domain (GAL4^DBD) fused to either full-length BCL6 or domains of BCL6 used in panels B and D. (B) GAL4 luciferase reporter assays in 293T cells transfected with plasmids encoding GAL4^DBD-BCL6 and PRMT5/MEP50. Western blots demonstrate representative expression of GAL4^DBD-BCL6, PRMT5, and MEP50 following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. ***P < .001. (C) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6 and PRMT5/MEP50. Western blots demonstrate representative expression of BCL6, PRMT5, and MEP50 following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. ***P < .001. (D) GAL4 luciferase reporter assays in 293T cells transfected with full-length GAL4^DBD-BCL6, GAL4^DBD-BTB/POZ, or GAL4^DBD-RD2 domains of BCL6 in the presence or absence of cotransfected PRMT5/MEP50 siRNAs from GE Dharmacon (Lafayette, CO). Western blots demonstrate representative expression of PRMT5 and BCL6 or MEP50 and BCL6 following transfection of increasing concentrations of PRMT5 siRNA-1 and MEP50 siRNA-1, respectively. All the experiments were repeated 3 times in triplicate. ***P < .001. Independent siRNA to PRMT5 and MEP50 shown in supplemental Figure 3. (E) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6 alone or in the presence of GSK591 (*P < .05; **P < .01; ***P < .001). (F) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with plasmids encoding BCL6, PRMT5, or catalytically inactive PRMT5 mutant (G367A/R368A, muPRMT5). Western blots demonstrate representative expression of BCL6, PRMT5, and muPRMT5 (G367A/R368A) following expression of corresponding expression vectors. All the experiments were repeated 3 times in triplicate. *P < .05; **P < .01.