Fig 2: Inhibition of TET activity by SMCHD1.(A) Reduction in 5hmC levels by coexpression of SMCHD1 with TET3 in 293T cells. 5hmC and 5mC contents were assessed using antibody-based dot blots. One-way analysis of variance (ANOVA) was performed comparing the mean of each group with the mean of the second group (**P < 0.01 and ***P < 0.001; mean ± SEM). ns, not significant. (B) Inhibition of TET3S-induced reactivation of a methylation-silenced luciferase construct by SMCHD1 in 293T cells (top). One-way ANOVA was performed (**P < 0.01 and ****P < 0.0001). Data are for means ± SEM of three independent experiments. An unmethylated luciferase vector was used as a control (bottom). (C) FLAG purification of TET2-CD and SMCHD1 full length (SMCHD1-FL) from Sf9 insect cells. Coomassie blue staining. (D) Inhibition of TET2-CD activity on fully methylated DNA in the presence of SMCHD1 as shown by combined bisulfite restriction analysis (COBRA) assay (BstU I cleavage indicates methylation). P.C., positive control with excess TET protein (18 µg); N.C., negative control without TET treatment. Different molar ratios of SMCHD1 and TET protein (1.15 µg) are shown. The H19 imprinting control region was analyzed. (E) Bisulfite sequencing analysis of H19 methylation analyzed in duplicates. Solid black circles indicate modified CpGs; open circles indicate TET-oxidized mCpGs. The purple arrows indicate BstU I sites. (F) Percentages of modified cytosines (%Me) of the different samples. P values were determined by Fisher’s exact test (two sided).

Fig 3: GABA acts through the aKG/Tet2/ PPAR? axis to mediate metabolic-epigenetic regulation of AMs. (A) Schematic diagram of the TCA cycle, with the key enzymes regulated upon stress labeled. Red arrow, down-regulated, and Green arrow, up-regulated in stressed AMs versus non-stressed AMs post IAV infection. (B) Serum levels of aKG and succinate analyzed by GC–MS, with the ratio of aKG to succinate provided. (C-F) Stressed and Non-stressed (NS) mice were infected or uninfected with IAV for 3 days. CD45+CD11b-CD64+CD11c+SiglecF+ AMs were sorted for subsequent analysis. (C) Immunoblotting of Tet2; (D) Dot-blot analysis of 5hmc or 5mc level. MB, methylene blue staining (as loading control). (E) ChIP test of 5hmc or 5mc enrichment at the ppar? locus; (F) Immunoblotting of PPAR?; (G) Immunoblotting of Tet2 and PPAR? in MH-S cells treated with GABA at the indicated doses; (H) Immunoblotting of Tet2 or PPAR? in GABA-treated macrophages with or without bicuculline (Bic; 40 µM). (I-K) MH-S cells were transfected with Tet2 siRNA (siTet2) or siRNA (siNC), and then treated with GABA (100 µM) or 0.01% DMSO prior to IAV infection. (I) PPAR? luciferase activity; (J) Immunoblotting of Tet2 and PPAR?; (K) Heatmapping of AM-associated genes. Shown are representative images. The bar graph is the densitometric quantification of western blot bands. Data from three individual experiments are shown as means ± SD. *P < 0.05, **P < 0.01, with one-way ANOVA (three or more groups) followed by Bonferroni post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)