Figure 4.

Elevated androgen levels disrupt the circadian timing system in the liver through epigenetic modulation. (A) Representative traces of Period2::Luciferase (PER2::LUC) expression in liver explants from dihydrotestosterone (DHT) and placebo pellet mice. The peak of the tissue from DHT-pellet animals peaks slightly earlier than the placebo pellet-treated mice, indicating that DHT reduces the peak-to-peak time, thus advancing the overall peak of PER2 expression over 5 days. (B) The amplitude of the first 2 peaks of PER2::Luc luminescence was calculated by LM fit (damped sine) method (LumiCycle analysis, Actimetrics) from liver tissues isolated from placebo and DHT pellet-treated mice. Data are displayed as mean ± SE of the mean (n = 6 mice/ treatment). *P ≤ 0.05 vs placebo using Student’s t-test. (C) The time of day of the first PER2::LUC peak was used to establish the phase of the studied tissue. Phase relationship is represented in radians with circular mean deviation. The mean of the first peak is indicated by the vector lines and symbols indicate individual data points. Rayleigh test of uniformity revealed clustering of the studied tissues, indicated by arrowheads crossing the dotted circle (P = 0.05). (D) Schematic showing androgen-induced regulation of H3K27me3 mark. (E) Representative immunoblots showing total protein levels of EZH2, JMJD3, and H3K27me3 in liver samples isolated from wild-type mice treated with placebo pellet, DHT pellet, and hepatocyte-specific androgen receptor knockout (hepARKO) mice treated with DHT pellet. RPL19 and H3 protein levels are used as internal controls. (F) Relative expression of miR-101 by quantitative real-time polymerase chain reaction in livers isolated from wild-type mice treated with placebo pellet, DHT pellet, and hepARKO mice treated with DHT pellet. Data are displayed as means ± SE of the mean (n = 3 mice/ treatment) and normalized to snoRNA202. *P ≤ 0.01 vs placebo using Student’s t-test. (G) Anti-H3K27me3 chromatin immunoprecipitation assay in livers isolated from wild-type placebo and DHT pellet-treated mice as well as hepARKO mice treated with DHT pellet, showing H3K27me3 enrichment on Arntl (Bmal1) promoter region (within 500 bp from transcription start site). Immunoglobin G (IgG) represents a nonspecific antibody. Values represent percentage input. Data are displayed as mean ± SE of the mean (n = 6 mice/treatment). *P ≤ 0.05 vs placebo using 1-way analysis of variance followed by Dunnett’s multiple comparison test.