Epigenetic modifications of gene promoter DNA in the liver of adult female mice masculinized by testosterone.

Testosterone (T) is known to masculinize the female phenotype of the liver, evidenced as up- and down-regulated expressions of male- and female-predominant genes, respectively, involved in hepatic metabolism. This study is aimed at identifying epigenetic modifications of promoters of these differently expressed genes in the liver after masculinization by T of adult female C57BL/6 mice using methylated DNA immunoprecipitation and NimbleGen microarrays. Among the 17,354 promoters examined, 82 promoters in the liver have been identified to be significantly changed by T (p<0.05), with 47 and 35 promoters exhibiting increased and decreased DNA methylation, respectively. Most of these promoters display the changes of DNA methylation in their Ups-regions, which are between +500 and +2000 bp upstream from the transcription start site (TSS) of the genes. Less T-induced modifications have been detected in the Cor-regions of the promoters, i.e., +500 to -500 bp around the TSS. Only 13 and 7 Cor-promoters are hyper- and hypo-methylated, respectively, among which are 10 hyper- and 5 hypo-methylated promoters of genes with annotated functions. Surprisingly, the promoters are largely unmethylated in those genes whose expression has been previously found to be permanently deregulated by T in the liver, as e.g. the T-upregulated male-predominant genes Cyp7b1, Cyp2d9, Cyp4a10, Ugt2b1, Ugt2b38, Hsd3b5, Slco1a1 as well as the T-downregulated female-predominant genes Cyp2b9, Cyp2b13, Cyp3a41, Cyp3a44, Fmo3, Sult2a2, respectively. Though methylatable, the promoter DNA of Ar, Esr1, and Esr2 remained unaffected by T. However, T decreases DNA-methylation of the Cor-promoter region of Ddc encoding the AR-coactivator dopa decarboxylase. Among the identified 15 Cor-promoters of genes with annotated functions are also those of Defb43, Cst11, and Sele involved in innate immunity. Our data support the view that T may exert long-lasting epigenetic effects on functions of the liver-inherent immune system.