Histone deacetylase 3 (HDAC3) participates in the transcriptional repression of the p16^INK4a gene in mammary gland of the female rat offspring exposed to an early-life high-fat diet

Abstract

Maternal exposure to environmental agents throughout pregnancy and lactation may affect offspring’s mammary gland growth and alter the epigenome. This may predispose the offspring’s mammary glands to be more susceptible to carcinogenesis. The purpose of this study was to examine the effect of a maternal high-fat diet on the regulation of p16^INK4a gene expression in the mammary gland of rat offspring. Timed-pregnant Sprague-Dawley rats were fed one of the two diets, a control (C, 16% of fat) or a high fat (HF, 45% of fat) diet, throughout gestation and lactation and sacrificed at 12 weeks of age. Compared with C, HF offspring showed a decrease of p16^INK4a gene expression in the mammary gland at both mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assay demonstrated that the downregulation of p16^INK4a transcription in HF offspring was associated with reduced acetylation of histone H4 and increased recruitment of histone deacetylase 3 (HDAC3) within the p16^INK4a promoter region, but was not associated with acetylation of histone H3 or HDAC1. Methylated DNA immunoprecipitation (MeDIP) did not detect differences in methylation at different regions of the p16^INK4a gene between C and HF offspring. We conclude that maternal high fat exposure represses p16^INK4a gene expression in the mammary gland of offspring through changes of histone modifications and HDAC3 binding activity within the regulatory regions of the p16^INK4a gene.

Key words:

• pregnancy
• epigenetics
• developmental programming
• thrifty phenotype hypothesis

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This project was supported, in whole or in part, by the USDA Cooperative State Research, Education and Extension Service, Hatch project number # ILLU-698-374 and by National Institutes of Health Grants CA-139557 (to Y.X.P.).

Figures and Tables

Figure 1 Maternal food intake and offspring body weight. (A) Gestational growth curve for (C) and high-fat (HF) fed dams. (B) Food intake for (C) and high-fat (HF) fed dams. (C) Growth curve after weaning for offspring of (C) and high-fat (HF) fed dams. The values are presented as the mean ± SEM.

Figure 2 p16^INK4a mRNA and protein levels in offspring mammary glands. (A) Expression of p16^INK4a mRNA in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 6) presented as the ratio to L7a housekeeping gene. The values are presented as the mean ± SEM; *p < 0.05 when compared with C group. (B) Expression of protein in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 4). The right part is a representative image of p16^INK4a protein as measured by immunoblotting, and values in the left part are presented as the mean ± SEM *p < 0.05 when compared with C group.

Figure 3 Cell cycle analysis in offspring mammary glands. Cell cycle analysis was performed in cells from mammary glands of offspring of control (C) and high-fat (HF) fed dams using flow cytometry (n = 3). Data are shown as the percentages of total cells corresponding to G₀/G₁, S and G₂/M phases. The values are presented as the relative mean ± SEM, *p < 0.05 when compared with C group.

Figure 4 DNA methylation at the p16^INK4a gene in offspring mammary glands. (A) CpG islands at the rat p16^INK4a gene. (B) Relative methylation levels relative to input corresponding to the negative 3 kb region, promoter region and CpG islands 1 to 4 of the p16^INK4a gene in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). (C) Non-specific IgG levels relative to input corresponding to the negative 3 kb region, promoter region and CpG islands 1 to 4 of the p16^INK4a gene in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). The values presented as the mean ± SEM.

Figure 5 Histone modifications within the p16^INK4a gene in offspring mammary glands. (A) Histone modifications at the p16^INK4a promoter region in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). (B) Histone modifications at the p16^INK4a gene body (around + 5 kb region) in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). (C) Histone modifications at CpG-rich regions on the p16^INK4a gene in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). Data are shown as a ratio to the input DNA. H4Ac: acetylated histone 4; H3Ac: acetylated histone 3; H3K4me2: di-methylated histone 3 at lysine 4 residues; H3K9me3: tri-methylated histone 3 at lysine 9 residues. The values are presented as the relative mean ± SEM, *p < 0.05 when compared with C group for each modification.

Figure 6 The binding of HDCA3 and HDAC1 within the p16^INK4a promoter. (A and B) Expression of HDAC3 and HDAC1 protein levels in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 4). The lower parts are a representative image of HDAC3 (left) and HDAC1 (right) protein as measured by immunoblotting. (C) Recruitment of HDAC3 at the p16^INK4a promoter in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). (D) Recruitment of HDAC1 at the p16^INK4a promoter in mammary glands of offspring of control (C) and high-fat (HF) fed dams (n = 5). The values present the mean ± SEM, *p < 0.05.

Table 1 Maternal diet composition

Table 2 p16 primers (ENSRNOT00000066011)