Figure 5.
SIRT1 deficiency increases IL-1β transcription via downregulating DNMT activity and hypomethylating IL-1β proximal promoter. A, B, Nuclear extracts from SIRT1F/F or LysM-Cre/SIRT1F/F or 5 μm 5-aza-CdR-treated SIRT1F/F cells were incubated with DNMT substrate with methyl donor. Methylated DNA was recognized with anti-5-methylcytosine antibody and measured by optical density; n = 4–5 samples/condition from two experiments; *p < 0.05; **p < 0.01, unpaired Student's t test. C, Mouse IL-1β promoter. Vertical lines indicate CpG sites. CpG sites −1439 bp to translation start site in the IL-1β promoter were analyzed. D, E, Percentage of IL-1β methylation at 10 CpG sites with or without 5 μm 5-aza-CdR for 48 h;. n = 4–10/samples from two experiments; **p < 0.01, unpaired Student's t test. F, SIRT1F/F or LysM-Cre/SIRT1F/F cells were treated with or without 5 μm 5-aza-CdR for 48 h. Using qRT-PCR, IL-1β mRNA were measured; n = 4–5 samples/condition from two experiments; *p < 0.05; **p < 0.01, one-way ANOVA with Tukey's multiple-comparison test. G, Potential mechanism of microglial SIRT1-mediated regulation of IL-1β transcript. With SIRT1, DNMT1 is deacetylated and activated. Activated DNMT1 methylates CpG at −215bp, recruits corepressor complex, and prevents the transcription factors (TFs) from binding to the promoter, and suppresses IL-1β transcription. Without SIRT1, DNMT1 remains acetylated and its activity is inhibited, leading to hypomethylation of IL-1β promoter at −215 bp, allowing TF binding. Values are mean ± SEM (A, B, D–F).