To determine whether PRMT1, -2, and -3 could affect NF-κB function, their potential to regulate effects on κB transcription was examined. Sequence comparison of the arginine methyltransferases has revealed several motifs shared by these proteins (Fig. , top). Transient cotransfections were performed using PRMT1, PRMT2, and PRMT3 expression plasmids with an HIV-1 reporter plasmid in the human renal epithelial cell line 293T. While PRMT1 stimulated κB transcription ~10 fold, PRMT2 inhibited transcription ~50 fold, and PRMT3 did not affect transcription of the reporter plasmid (Fig. , bottom left). These results suggest that PRMT2 is unique among the PRMTs in its ability to inhibit κB-dependent transcription.
To map the domains responsible for inhibition of this transcription, truncation and point mutations were made in PRMT2 (Fig. , top) and cotransfected with an NF-κB reporter in 293T cells. PRMT2-A represents an alternatively spliced form of PRMT2 found in the expressed sequence tag database. This isoform contains the first 218 amino acids of PRMT2 and differs from full-length PRMT2 by the absence of the less-conserved COOH-terminal domain. PRMT2-N was generated by introducing a stop codon after amino acid 95 before the putative Ado-Met domain of PRMT2. To analyze the role of the Ado-Met domain further, another mutant, PRMT2-4A, was prepared in which the sequence 141ILDV144 was altered to four consecutive alanines to compare the effects of point mutations in this highly conserved region. Under conditions in which PRMT2, PRMT2-A, and PRMT2-4A inhibited NF-κB activity, PRMT2-N did not (Fig. , bottom left), suggesting that a structural, but not necessarily a functional, methyltransferase domain is required for transcriptional inhibition.
The HIV-1 long terminal repeat contains two highly conserved κB-binding sites that play an important regulatory role in HIV-1 gene expression (37
). To study the effect of PRMT2 on transcription, PRMT2 was cotransfected with HIV-1, HIV-2, human T-cell lymphotropic virus type 1 (HTLV-1), or HTLV-2 reporter plasmids into 293 cells. Despite the presence of a single κB site in HIV-2, its expression shows greater dependency on Ets family transcription factors (30
). No significant reduction was seen with either HIV-2 or HTLV reporter plasmids, while HIV-1 chloramphenicol acetyltransferase (CAT) expression was substantially inhibited, documenting the specificity of PRMT2 (Fig. ). To determine its dependence on NF-κB, HIV-1 reporter plasmids with WT or mutant (ΔκB) sites were cotransfected transiently with control or PRMT2 expression plasmids. PRMT2 significantly inhibited both basal and TNF-α-dependent HIV-1 transcription from the wild type but not the κB-mutant reporter in 293 renal epithelial cell lines (Fig. , left and middle). The κB effect was dose dependent and was also observed with other inducers of NF-κB, including phorbol myristic acid (PMA) (Fig. , right). These results suggested that PRMT2 could block NF-κB activation from various stimuli. PRMT2 was also able to modulate the expression of endogenous κB-regulated genes. PRMT2 transfection of 293T cells decreased endogenous major histocompatibility complex class I (MHC-I) cell surface expression by flow cytometry, in contrast to CD9, which is an NF-κB-independent gene (Fig. ).
FIG. 2. Transcriptional inhibition by PRMT2 is κB dependent, and IKK-2- or p65-induced NF-κB activation is blocked by PRMT2. (A) Transcriptional inhibition by PRMT2 is promoter specific; the effect of PRMT2 on HIV and other enhancers by a CAT (more ...)
The mechanism and site of action of PRMT2 in the NF-κB signaling pathway was further defined by cotransfection of PRMT2 and its mutants with different regulators in this pathway with an NF-κB reporter in 293T cells (Fig. ). PRMT2 and PRMT2-A inhibited both IKK2- and p65-induced NF-κB activity (Fig. ; see Fig. S1 in the supplemental material), while PRMT2N was unable to block this effect (Fig. ), suggesting that PRMT2 exerted its inhibitory action on nuclear NF-κB rather than by modulation of cytoplasmic IκB or the IκB kinase complex.
To investigate this mechanism further, p65 expression levels and cellular localization of RelA and IκB were examined. Immunoblotting for RelA in cytoplasmic and nuclear extracts from 293 cells transfected with PRMT2 revealed no effect on RelA protein levels or on its subcellular localization (Fig. ). Thus, PRMT2 appeared to affect RelA function without altering its nuclear accumulation, for example, by interfering with its DNA-binding activity. To determine whether PRMT2 could affect nuclear NF-κB DNA-binding activity, PRMT2 was cotransfected into 293 cells with the NF-κB1 (p50) and RelA (p65) expression vectors. Analysis of nuclear extracts from transfected cells by mobility shift assays, using a consensus κB-binding site double-stranded oligonucleotide, showed that PRMT2 inhibited DNA binding of the p50/p65 complex in a dose-dependent manner (Fig. , lanes 2, 5, and 6). In contrast, the inactive PRMT2-N mutant did not affect NF-κB DNA binding (Fig. , lane 3). The nature of these complexes was confirmed by supershifts with antibodies directed against p50 and p65 (Fig. , lanes 8 and 9).
FIG. 3. PRMT2 does not interfere with p50/p65 dimerization or DNA binding. (A) PRMT2 does not alter p65 expression or localization. A total of 10 μg of cytoplasmic (CE) or nuclear (NE) extract from 293 cells transfected with vector and PRMT2 expression (more ...)
To examine whether PRMT2 directly affected NF-κB DNA binding, a recombinant GST PRMT2 fusion protein, GST-PRMT2, was added to the gel shift reaction mixture. No decrease in DNA binding over GST control was observed (Fig. , lanes 14 to 16), suggesting that the inhibition of NF-κB DNA binding in PRMT2-transfected extracts was indirect. Because p50/p65 dimerization is important for efficient NF-κB DNA binding (44
), PRMT2 might inhibit DNA binding by antagonizing p50/p65 complex formation. To test this possibility, p50/p65 complexes were immunoprecipitated from PRMT2-transfected 293 cell nuclear extracts with an anti-p65 antibody. Western blotting for p50 showed that equal amounts of p50 coimmunoprecipitated with p65 from cells transfected with PRMT2 or PRMT2-N (Fig. , lanes 17 and 18), suggesting that decreased NF-κB DNA binding in PRMT2-transfected cell extracts was not due to interference with p50/p65 dimerization. In this assay, PRMT2 also did not affect interactions of p65 with p300 and the general transcriptional machinery (see Fig. S2A in the supplemental material), nor did it catalyze the methylation of histones (see Fig. S2B in the supplemental material), p65, p50, IκB, hnRNPU, and CRM1 in both bacterially purified and cell extract-immunoprecipitated PRMT2 by an in vitro methyltransferase assay (data not shown). Whole-cell hypomethylated extracts from PRMT2-transfected cells showed minimal changes in methylation when incubated in vitro with [methyl
-methionine over control, while PRMT1-transfected extracts were hypermethylated (see Fig. S2C in the supplemental material). Taken together, these data suggest that the methyltransferase function of PRMT2 is not necessary for inhibiting NF-κB activity.
Newly synthesized IκB-α can be detected in the cytoplasm but also in the nucleus, where it associates with NF-κB/RelA complexes. As newly synthesized IκB-α accumulates in the nucleus, there is a progressive reduction of both NF-κB DNA binding and NF-κB-dependent transcription (4
), presumably by export of NF-κB-IκB-α complexes from the nucleus (3
). PRMT2 could therefore potentially affect nuclear IκB-α levels, resulting in decreased NF-κB DNA binding. To examine whether PRMT2 increased nuclear IκB-α levels, nuclear and cytoplasmic extracts were prepared from PRMT2 or inactive, PRMT2-N-transfected 293 cells. Immunoblotting for IκB-α and RelA proteins in the two fractions revealed no significant changes in the levels of cytoplasmic IκB-α (Fig. , left, lane 1 versus lane 2) or nuclear p50 and RelA (p65) levels (Fig. , right, lane 3 versus lane 4), but a distinct increase in the amount of nuclear IκB-α was observed with PRMT2-transfected cells compared to the functionally inactive PRMT2-N mutant control (Fig. , right, lane 3 versus lane 4, and Fig. ; P
< 0.01, PRMT2 compared to the mutant PRMT2-N using Student's t
test) with cells that had been stimulated with TNF-α. This increase in the nuclear accumulation of IκB-α therefore appeared to be responsible for the PRMT2-mediated inhibition of NF-κB DNA binding and NF-κB-dependent transcription.
FIG. 4. PRMT2 promotes nuclear accumulation of IκB-α. (A) Cells were stimulated with TNF-α (200 U/ml) 24 h after transfection and harvested at 36 h. A total of 10 μg of cytoplasmic extracts was resolved by 4 to 15% SDS-PAGE and (more ...)
A polyclonal antibody to recombinant PRMT2 was used to examine the association between endogenous PRMT2 and IκB-α in vivo. Immunoprecipitation of IκB-α from NIH 3T3 cell extracts with a control or anti-IκB-α antibody, followed by immunoblotting with antibody to PRMT2, revealed that PRMT2 interacted with endogenous IκB-α (Fig. ) but not IκB-β (data not shown). The domain of IκB-α required for association with PRMT2 was mapped by in vivo immunoprecipitation assays where HA-tagged PRMT2 was coexpressed with truncation mutants of His-tagged IκB-α (Fig. , top) in 293 cells. The ankyrin domain was necessary for this association (Fig. , bottom, lanes 1 and 2). The domain of PRMT2 that interacted with endogenous IκB-α was mapped by immunoprecipitation, following expression of HA-tagged PRMT2 truncation mutants (Fig. , top). IκB-α interacted with PRMT2 and PRMT2-A (Fig. , bottom, lanes 10 and 11) but did not associate with PRMT2-N (Fig. , bottom, lane 12), indicating that the Ado-Met domain is necessary to promote IκB-α binding. When the ratios of PRMT2 or PRMT2-A binding to IκB-α were compared, both interacted with IκB-α with similar affinity. PRMT2 or the mutants did not interact with endogenous p65 (Fig. , bottom, lanes 7, 8, and 9).
FIG. 5. PRMT2 associates with the endogenous IκB-α complex. (A) Immunoprecipitation of endogenous PRMT2-IκB-α complex. NIH 3T3 cell extracts (2 mg) were immunoprecipitated with agarose-conjugated control IgG or IκB-α (more ...)
To determine whether similar effects would be observed in nontransformed cell lines with physiological levels of protein, NF-κB inducibility was analyzed in MEFs derived from PRMT2 null mice (T. Yoshimoto et al., unpublished data). A κB-luciferase reporter was transfected with control or PRMT2 expression plasmid into WT and prmt2−/− MEFs and incubated in the presence or absence of TNF-α. Compared to wild-type cells and consistent with the transfection results in 293 cells, prmt2−/− MEFs were more responsive to NF-κB induction by TNF-α (Fig. ). Complementation of prmt2−/− MEFs with PRMT2 completely abolished NF-κB induction by TNF-α (Fig. ). IκB-α and p65 levels in cytoplasmic and nuclear extracts from control and prmt2−/− MEFs were examined after TNF-α stimulation for 0, 15, 30, and 60 min. Immunoblotting for p65, p50, and IκB-α in the two fractions revealed no significant changes in the levels of cytoplasmic IκB-α at a representative 30-min time point (Fig. , bottom left) or p50 (Fig. , middle), but a moderate increase in RelA (p65) levels (Fig. , top) and a distinct decrease in the amount of nuclear IκB-α was observed with prmt2−/− compared to control MEFs (Fig. , bottom right).
FIG. 6. Lack of NF-κB inhibition in prmt2−/− fibroblasts, reversal by complementation through transfection of PRMT2, and dependence on LMB-sensitive nuclear export. (A) NF-κB response in prmt2−/− fibroblasts or (more ...)
NF-κB DNA-binding and NF-κB-dependent transcriptional activation is reduced by accumulation of newly synthesized IκB-α in the nucleus (4
). NF-κB-IκBα complexes are exported from the nucleus to the cytoplasm by CRM1 (3
), and this nuclear export can be blocked by LMB (23
). To understand the role of PRMT2 in promoting nuclear IκBα accumulation, prmt2−/−
fibroblasts were transfected with an HA-tagged PRMT2 expression vector. At 36 h after transfection, the cells were treated with TNF-α for 30 min. The medium was then removed, and cells were incubated for an additional 30 min in the presence or absence of LMB. Cells were fixed, permeabilized, and stained for IκB-α (Fig. , left) and HA (PRMT2) (Fig. , middle). Confocal microscopy performed on the cells showed IκB-α accumulation in the nucleus in the presence of PRMT2 (Fig. , top left and right), which did not change in the presence of LMB (Fig. , bottom left and right).
To demonstrate the effect of PRMT2 further, nuclear IκB-α (Fig. ) was quantified in prmt2−/− fibroblasts and prmt2−/− fibroblasts complemented with PRMT2 in the presence or absence of LMB. Briefly, to quantitate the effect of PRMT2 on nuclear IκB-α, prmt2−/− fibroblasts were transfected with a control or HA-tagged PRMT2. At 36 h after transfection, cells were first treated with TNF-α for 30 min, washed, and then treated with LMB or vehicle for 30 min. Cells were fixed, permeabilized, and stained with IκB-α (green), PRMT2 (red), and DAPI (blue). Outlines of nuclei of cells with or without PRMT2 from each field of vehicle or LMB-treated cells were drawn with Leica confocal software. IκB-α pixel intensity from the nucleus of each individual cell in the field was measured using this software. In each field, the cells with PRMT2 were identified by the presence of the HA tag (red). The nuclear outline was defined by DAPI staining (blue). For each condition, the data from 10 fields were compiled (approximately five to six cells per field; 30% of the cells expressed PRMT2) and presented graphically, with P values as indicated. Vehicle- and LMB-treated cells are shown (Fig. , left and right, respectively). LMB promoted nuclear accumulation of IκB-α in the absence of PRMT2, and transfection of PRMT2 exerted the same effect. Together, these data are consistent with the hypothesis that PRMT2 inhibits the nuclear export of IκB-α through an LMB-sensitive CRM1 pathway. At the same time, we cannot exclude the possibility that these effects are coincidental and induced by an alternative mechanism yet to be defined.
Since PRMT2 inhibits NF-κB activity, which can regulate apoptosis in some cell types (7
), the ability of PRMT2 to independently regulate programmed cell death was next examined. Transfection of PRMT2 into 293 cells increased their susceptibility to TNF-induced cell death, comparable to levels observed with a mutant, stabilized, or superrepressor IκB (designated SR-IκB) (Fig. ) (7
). To evaluate the effect of PRMT2 on programmed cell death, wild-type, knockout PRMT2 MEF or knockout MEF cells complemented with PRMT2 were exposed to etoposide, a DNA-damaging agent with proapoptotic activity. Wild-type and PRMT2-complemented MEFs displayed a substantial increase in etoposide-induced cell death and annexin V staining compared to PRMT2-deficient cells (Fig. ).
FIG. 7. prmt2−/− cells are resistant to apoptosis, and this effect can be reversed by complementing prmt2−/− cells with PRMT2. (A) PRMT2 promotes TNF-α-induced apoptosis. Empty vector, mutant IκB-α (S32A/S36A (more ...)