Fig. 7: DNA hypermethylation underlies the repression of REV-ERBα in Psen2N141I/+ immune cells.
a Methylation analysis of CpG islands in the Nr1d1 promoter. Difference in the value of the sum of methylation rate (calculated as methylation depth / (methylation depth + non-methylation depth)) between KI/+ and WT at each methylated CpG site is shown as a graph with a blue bar for each position. b Comparison of overall DNA methylation ratios in the 2-kbp promoter region of clock genes between KI/+ and WT microglia (n = 4) (#p = 0.0383; unpaired t-test). c, d Pharmacological modulation with 5-Azacytidine (5-Aza, 10 μM) treatment for 24 h restores Nr1d1 transcript in KI/+ microglia and BMDM (c; n = 4, respectively) and protein levels in KI/+ microglia (d; n = 3) (untreated WT vs untreated KI/+: ###p = 0.0008, ##p = 0.0062, and #p = 0.03; untreated KI/+ vs 5-Aza-treated KI/+: ††p = 0.0084, †††p = 0.0009, and †p = 0.0118; one-way ANOVA). Signals on western blots were normalized to those of ACTB. e, f 5-Aza-induced demethylation reduces the mRNA level (e; n = 3 for Il-6; n = 4 for Tnf) and secretion (f; n = 4) of IL-6 but not TNF-α in KI/+ microglia (LPS-treated WT vs LPS-treated KI/+: ###p = 0.0008 and ###p < 0.0001; LPS-treated KI/+ vs LPS and 5-Aza-treated KI/+: †††p < 0.0001; one-way ANOVA). Cells were pre-treated with 5-Aza for 12 h, and mRNA levels and secretion were analyzed after co-treatment with LPS for 12 h. mRNA levels were normalized to Actb. Data are mean ± SEM. †p < 0.05, ††p < 0.01, and †††p < 0.001 vs untreated KI/+ control. #p < 0.05, ##p < 0.01, and ###p < 0.001 for the indicated comparisons. Source data are provided as a Source Data file.
