Fig. 7: Loss of XRN2 methylation underlies cGAS activation.
a,b, Phospho-CHK1, CHK2 and γH2AX levels after PRMT9 KD (a) or LD2 (b) in THP1 (n = 2 independent experiments). c, Comet assay of THP1 after PRMT9 KD for 48 and 72 h. Right: summary of each group (n = 50 cells). Scale bar, 50 μm. The P value was determined using an unpaired two-sided t-test. d, Luciferase activity of THP1-IRF cells after KO of the indicated genes. Data are the mean ± s.d. from three independent experiments. e, Methylation assay of KHDRBS1 (amino acids 326–339), XRN2 (amino acids 937–950) or DDX3X (amino acids 80–92) peptides. Methylation was analyzed using an anti-MMA antibody (n = 2 independent experiments). f, XRN2 and DDX3X levels after respective KO (n = 2 independent experiments). g, Luciferase activity of WT and cGAS KO THP1-IRF cells. gRNA-resistant XRN2 WT and R946K constructs were ectopically expressed in THP1-IRF cells (n = 5 independent experiments). A reporter assay was performed using the cells with KO endogenous XRN2. Data are the mean ± s.d. The P value was determined using a one-way ANOVA. h, Luciferase activity of THP1-IRF cells (n = 5 independent experiments). gRNA-resistant DDX3X WT or R88K constructs were ectopically expressed in THP1-IRF cells. A reporter assay was performed using the cells with KO endogenous DDX3X. Data are the mean ± s.d. The P value was determined using an unpaired two-sided t-test. i,j, In vitro methylation of XRN2 peptides with PRMT9 (i) or PRMT5 (j) with increased dose of LD2 (i) or EPZ015666 (j) (n = 1). k, XRN2-engineered THP1 cells were prepared for IP using anti-FLAG beads; interactors were detected as indicated (n = 2 independent experiments). l,m, R-loop signals by dot blots (l, n = 2) or immunostaining (m) in THP1 cells. Scale bar, 10 μm. ssDNA, single-stranded DNA. n,o, R-loop signals in RNASEH1-overexpressed THP1 cells treated with LD2 (2.5 μM) (n) or PRMT9 KD (o) (n = 2 independent experiments). p, Cell cycle of THP1 cells treated for 48 h with LD2 (2.5 μM), n = 5 independent experiments. Right: statistics. Data are the mean ± s.d. Right: P values were determined using a one-way ANOVA. q, Phospho-CHK1 in engineered THP1 cells treated with LD2 (2.5 μM) (n = 2 independent experiments). r, Luciferase activity of THP1-Lucia luciferase cells treated with (2.5 μM) LD2 (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA.
