Fig. 3.

Neonatal alcohol effects on epigenetic mediators in hypothalamic microglia in vivo. Hypothalamic microglia from PND 6 AD, PF, and AF rat pups were measured for protein levels of epigenetic mediators of inflammatory gene transcription. In vivo, hypothalamic microglia from AF animals showed decreased levels of negative regulators of transcription including methyl CpG-binding protein 2 (MeCP2) (a) (n = 5–6/treatment group) and decreased global DNA methylation (d) (n = 4–6) compared to control animals. Levels of DNMT1 (b) (n = 7–9) and DNMT3a (c) (n = 4-11) were not significantly different in microglia from AF animals compared to AD and PF controls. In addition, compared to microglia from control pups, microglia from AF pups showed decreased protein levels of histone deacetylases HDAC1 (e) (n = 9–11) and sirtuin 1 (SIRT1) (f) (n = 8), while showing increased global acetylation of their target histone H3 lysine 9 (H3K9ac) (g) (n = 6–12), a positive regulator of inflammatory gene transcription. Finally, ChIP of H3K9ac from hypothalamic microglia showed increased enrichment of the IL-6 (h) and TNF-α (i) promoter gene regions in AF pups compared to PF and AD pups (n = 4). Data are mean ± SEM (n = 4–11), and were analyzed by one-way ANOVA followed post-tests, *p < 0.05; **p < 0.01; ***p < 0.001 AD compared to AF, #p < 0.05; ##p < 0.01; ###p < 0.001 PF compared to AF