Peg3 regulates orexin expression in an independent manner from paternal Snord116. (A) Upper panel, the gene expression analysis of Peg3 in PWScr^m+/p− mice (red) versus controls (black). Peg3 mRNA assessed by qPCR was significantly increased in PWScr^m+/p− mice compared with PWScr^m+/p+ mice (unpaired t test; t(6) = 2.46, P = 0.04). Values expressed are relative to the WT control mean ± SEM. Gapdh was used as a housekeeping gene; see Supplemental Methods. Bottom panel, ChIP analysis of PEG3 binding to the Ppox promoter region in PWScr^m+/p− mice (red) versus controls (black). PEG3 binding was lower in PWScr^m+/p– mice than in PWScr^m+/p+ mice (unpaired t test; t(2) = 7.11, P = 0.01); see Supplemental Methods. (B) Peg3 gene expression (upper panel) and Snord116 gene expression (bottom panel) in Ppox-KO and orexin neuron–ablated (ataxin-3 [Atx] mice). One-way ANOVA indicated that Peg3 was significantly increased in Atx mice relative to KO mice, (F_[2,16] = 0.02, Bonferroni’s post hoc test, P = 0.03). Snord116 was increased in Atx mice relative to KO and control mice (WT) (1-way ANOVA; F_[2,16]= 3.50, Bonferroni’s post hoc test, P = 0.002). The following genotypes of narcoleptic mice were investigated: WT (n = 4), KO (n = 12), and Atx (n = 4). (C) Snord116 and Peg3 gene expression analysis in the Snord116 siRNA–treated immortalized hypothalamic rat cell line. Snord116 siRNA (green bars) reduced the expression of the Snord116 gene compared with untreated cells or scrambled siRNA–treated cells (white and orange bars, respectively) (1-way ANOVA; F_[2,6] = 11.36, Bonferroni’s post hoc test, P = 0.009). Peg3 mRNA levels were unchanged, similar to Snord116 siRNA, in untreated cells and scrambled siRNA–treated cells. The experiment was conducted in triplicate. Data are presented as the mean ± SEM. *P ≤ 0.05; **P ≤ 0.01.