Histone modifications, not DNA methylation, cause transcriptional repression of p16 (CDKN2A) in the mammary glands of offspring of protein-restricted rats

Maternal nutrition during pregnancy is an important intrauterine factor that results in persistent alteration of the offspring epigenome and that associates with health outcome in later life. This study examined the effect of maternal low-protein diet on the regulation of the p16 cell-cycle gene expression in the mammary gland of offspring rats. Timed-pregnant Sprague-Dawley rats were fed during gestation one of two isocaloric diets, control (18% casein) or low protein (LP, 9% casein). The expression of p16 mRNA in the mammary gland of the LP offspring was decreased by 75% vs. control. We also detected decreased p16 protein content in the mammary glands of pups gestated under the LP diet. Analysis of transcriptional and epigenetic regulation in offspring rats with maternal LP diet revealed the regulatory mechanisms underlying decreased p16 expression. Chromatin immunoprecipitation (ChIP) assay demonstrated that the altered p16 mRNA level and transcription rate in LP offspring resulted from histone code changes, including the reduced acetylation of histone H4 and the dimethylation of histone H3 at lysine 4 residues within the p16 promoter region. These results supported the hypothesis that maternal protein restriction during pregnancy programs p16 expression through histone code alterations in offspring mammary gland.