FIGURE 4.

Direct TLR4 activation with LPS induces gene histone methylation and RE1 silencing transcription factor (REST) occupancy at cholinergic genes. Chromatin immunoprecipitation (ChIP) revealed that direct application of LPS (100 ng/mL; 24 h) to basal forebrain slice culture (FSC) media increased histone 3 lysine 9 dimethylation (H3K9me2) occupancy at the (A) CpG island in the Chat promoter by approximately 1.7-fold and (B) at the promoter of the tropomyosin receptor kinase A (Trka) gene by approximately 2-fold relative to vehicle-treated FSCs (n = 7 wells/group, two-tailed t-test). ChIP assessment revealed that direct application of LPS to FSC media significantly increased REST occupancy (C) approximately 10-fold at the Chat promoter CpG island and (D) approximately 3-fold at the promoter of the LIM/homeobox protein 8 (Lhx8) gene relative to vehicle-treated FSCs (n = 6 wells/group, two-tailed t-test). (E) Simplified schematics depicting locations of promoters and promoter CpG islands assessed for H3K9me2 and REST occupancy. Chromosome location and sequence regions for each promoter primer is presented. TSS: transcription start site. Data are presented as mean ± SEM. ChIP analyses were run in duplicate. *p < 0.05, ^**p < 0.01.