Exploration of epigenetic marks at the Mc4r promoter ruled out DNA 5-methylcytosine methylation but showed increased H3K27 acetylation. (A) DNA methylation at the Mc4r promoter. Methylation was evaluated using sequencing of bisulfite-treated DNA from PVN punches. The total number of methylations in each position was calculated by averaging the total methylations for all the rats in the same group (n = 6 per group, 5 to 6 colonies per rat; P = NS). (B) Evaluation of H3K27ac binding. PVN samples were immunoprecipitated with antibody against H3K27ac and subjected to qPCR with specific primers to the TRE area at position −570 to −490 bp upstream of the Mc4R coding sequence and primers to a control area, −3091 to −2996 bp upstream of the Mc4R coding sequence. Control group PCR abundance was set to 1; IgG was used as a negative control. Chow (control [C]) pups, n = 13; HFD pups, n = 9; IgG, n = 6. Data presented as mean ± standard error of mean; *statistically significant change from chow group at P < 0.05.