Fig 2: Silencing of Lsd1 and Prmt6 ameliorates the disease phenotype of SBMA mice.a Body weight, hanging wire, and rotarod analysis of male WT and AR100Q mice treated with either vehicle or amiR-Lsd1/Prmt6 (n = 8 WT CTR, n = 10 WT amiR-Lsd1/Prmt6, n = 9 AR100Q CTR, n = 8 AR100Q amiR-Lsd1/Prmt6 mice. b NADH staining of 8-week-old mice treated as indicated (n = 1 WT CTR, n = 4 AR100Q CTR and n = 4 AR100Q amiR-Lsd1/Prmt6; n = 11,000 counted fibers/group. Scale bar = 4 µm. c RT-PCR analysis of denervation markers in 13-week-old WT CTR, AR100Q CTR, and AR100Q amiR-Lsd1/Prmt6 mice (n = 4 WT CTR, n = 9 AR100Q CTR, n = 8 AR100Q amiR-Lsd1/Prmt6. d Western blots of AR in the skeletal muscle of AR100Q CTR and AR100Q amiR-Lsd1/Prmt6 mice (n = 3 mice/group). HMW, high molecular weight species. AR was detected with specific antibody and CNX was used as loading control. Graphs show mean ± SEM; two-way ANOVA followed by Fisher’s least significance difference test (a, Body weight: p = 0.008 week 9, p = 0.081 week 10, p = 0.045 week 11. Hanging wire: p = 0.058 week 12, p = 0.001 week 13. Rotarod: p = 0.036 week 9, p = 0.045 week 10, p = 0.057 week 11). *p < 0.05; **p < 0.01; ***p < 0.001, # marginally significant; two-tailed Student t-test (b); one-way ANOVA followed by Tukey HSD tests (c). Source data are provided as a Source data file.

Fig 3: Silencing of Lsd1 and Prmt6 modifies gene expression in SBMA muscle.a Venn diagrams showing intersection of differentially expressed genes (top, upregulated; bottom, downregulated; absolute fold-change >4 and adjusted p < 0.01) obtained by comparing control (AR100Q) versus amiR-treated SBMA mice; AR100Q versus WT mice; or amiR-treated versus WT mice. b Ring plot showing the number of differentially expressed genes in AR100Q versus WT mice from differential expression analysis and relative proportions of totally and partially rescued genes. c Heatmap of top 20 clusters obtained from functional enrichment analysis of rescued genes. Color is proportional to enrichment p-values. Each cluster name is based on the most statistically significant term within the cluster. d Similarity network of enriched terms. Each node represents an enriched term and is colored by cluster identifier. Node size is proportional to number of rescued genes in the term. Edge width is proportional with similarity score computed between pairs of nodes.

Fig 4: RelA coimmunoprecipitates with endogenous PRMT6 and they directly interact in vitro. (A) Primary MEFs were isolated from WT and ER*-PRMT6 Line A mice, cultured for 7 days and treated with OHT (2 μM) for 24 h. Cells were then lysed and an IP was performed using αFlag agarose. A WB using αRelA antibody reveals an interaction between the ER*-PRMT6 and the endogenous RelA in the lysate from the transgenic cells. (B) HEK 293 cells were grown to 90% confluency, then washed with PBS and lysed in co-IP buffer. Lysates were divided in equal volumes and incubated with αIgG or αPRMT6 antibodies overnight and with protein A/G agarose thereafter. A WB using αRelA antibody reveals an interaction between PRMT6 and RelA. (C) Immortalized WT and PRMT6-KO MEFs were grown to 90% confluency, and then washed with PBS and lysed in co-IP buffer. Lysates were incubated with αPRMT6 antibody (which recognized mouse PRMT6) overnight and with protein A/G agarose thereafter. A WB using αRelA antibody reveals an interaction between PRMT6 and RelA in the WT cells. (D) Direct binding between PRMT6 and RelA. GST and GST-RelA (1–431) were incubated with His-PRMT6 and a GST pull-down was performed. For an additional negative control, GST-RelA was also incubated with a His-Tudor-UHRF1. A WB using αHis antibody reveals direct binding between GST-RelA and His-PRMT6.

Fig 5: PRMT6 promotes RelA shuttling into the nucleus and ChIPs at a subset of NF-κB-regulated promoters. (A) Primary MEFs were isolated from WT and ER*-PRMT6 mice, cultured for 3 days and treated with OHT for 2 weeks. Cell fractionation was performed. Western analysis for the Flag-tag shows stabilization of ER-PRMT6 in the nuclear fraction, corresponding to increased nuclear RelA. WB for RelA and IκB-α on the cytoplasmic extracts shows no change in the levels of these two proteins. (B) Immunofluorescence using a αRelA antibody following transient transfection of GFP-PRMT6 in HeLa cells shows increased nuclear RelA in transfected cells. A GFP-expressing construct was used as negative control. (C) HeLa cells were transiently transfected with a GFP or GFP-PRMT6 expressing vector for 24 h, at which time cells were left untreated or treated with TNF-α (10 ng/ml, 30 min). ChIP analyses at the κB consensus region of IL-6 promoter were then performed using αRelA antibody. Values were normalized to an intergenic region 8000 bp upstream IL-6 consensus sequence. For each transfection, the mean value of the unstimulated group was arbitrarily set as 1. Error bars represent standard deviations calculated from triplicates. The P values of the TNF-α-stimulated groups compared to the unstimulated are 0.0024 and 0.0003 for the Mock and GFP-PRMT6-transfected cells, respectively. The P value of the fold increase difference for PRMT6-overexpressing cells versus the Mock is 0.023. (D) HeLa cells were grown to 90% confluency, at which point cells were left untreated or treated with TNF-α (10 ng/ml, 30 min). ChIP analyses at the κB consensus region of IL-6, TNF-α and IκB-α promoters were then performed using αPRMT6 antibody. Values were normalized to an intergenic region 8000 bp upstream IL-6 consensus sequence. Error bars represent standard deviations calculated from triplicates. The mean value of the unstimulated groups was arbitrarily set as 1. The P values of the TNF-α-stimulated groups compared to the unstimulated are 0.024, 0.0053 and 0.84 for IL-6, TNF-α and IκB-α genes, respectively. (E) The recruitment of PRMT6 and RelA to the IL-6 promoter consensus region was evaluated by ChIP/re-ChIP experiment. HeLa cells were either left unstimulated or treated with TNF-α (10 ng/ml, 30 min) before the ChIP/re-ChIP experiments were performed using indicated antibodies. The ChIP DNA was analyzed by qPCR with primers for the IL-6 consensus region. (F) ChIP experiment was performed using αPRMT6 antibody in control and RelA-knockdown HeLa cells, which were either left unstimulated or treated with TNF-α.