Epigenomics: maternal high-fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates

Abstract

The effect of in utero exposure to a maternal high-fat diet on the peripheral circadian system of the fetus is unknown. Using mRNA copy number analysis, we report that the components of the peripheral circadian machinery are transcribed in the nonhuman primate fetal liver in an intact phase-antiphase fashion and that Npas2, a paralog of the Clock transcription factor, serves as the rate-limiting transcript by virtue of its relative low abundance (10- to 1000-fold lower). We show that exposure to a maternal high-fat diet in utero significantly alters the expression of fetal hepatic Npas2 (up to 7.1-fold, P<0.001) compared with that in control diet-exposed animals and is reversible in fetal offspring from obese dams reversed to a control diet (1.3-fold, P>0.05). Although the Npas2 promoter remains largely unmethylated, differential Npas2 promoter occupancy of acetylation of fetal histone H3 at lysine 14 (H3K14ac) occurs in response to maternal high-fat diet exposure compared with control diet-exposed animals. Furthermore, we find that disruption of Npas2 is consistent with high-fat diet exposure in juvenile animals, regardless of in utero diet exposure. In summary, the data suggest that peripheral Npas2 expression is uniquely vulnerable to diet exposure.

Figure 1.

Expression of circadian genes in fetal liver. A) Schematic diagram showing the feedback loop of the circadian genes. Expression of circadian-regulated genes is driven by the binding of an NPAS2:BMAL1 dimer to the E-box within the circadian gene promoters. The dimer drives transcription of the Per and Cry genes as well as Rev-erb-α and RORα. Increased levels of Per, Cry, and Rev-erb-α inhibit expression of Bmal1 and Npas2, providing a feedback mechanism of transcriptional regulation. B) Relative mRNA copy number in fetal liver from control diet-fed mothers shows that low copy number of Npas2 is associated with increased Per2 and Rev-erb-α. C) mRNA copy number in fetal liver from HFD-fed mothers does not follow the tight pattern of expression as seen in the control diet animals. D) Although Npas2 has a very low copy number in fetal liver, it is readily amplified by PCR.

Figure 2.

Circadian gene expression is disrupted in fetal liver exposed to a maternal HFD. A) Npas2 expression level for each individual fetal animal in the control diet (n=9), HFD (n=10), and reversed diet (n=7) groups. Although the overall difference in average fold change does not differ among groups, the range of expression fold change is much greater in the HFD-exposed group. B) Npas2 expression level of 3 animals exposed to the HFD was greatly increased compared with those exposed to the control diet. Difference in expression between these two groups is significantly increased. C) Npas2 expression level of 4 animals was greatly decreased (<0.5-fold) compared with those fed the control diet. Change in expression was significantly decreased in these animals. D) Fetal hepatic Per1 is up-regulated 4.23-fold in HFD-exposed animals (P<0.001). Expression in reversed diet animals trends toward that of animals fed the control diet but is still significantly changed (1.26-fold) compared with that for the control diet (P<0.01). E) Expression of Rev-erb-α is up-regulated 2.7-fold with HFD exposure (P=0.011) and returns to control diet levels with diet reversion.

Figure 3.

Changes in fetal Npas2 expression due to maternal HFD exposure are not associated with changes in methylation of the proximal promoter region. A) Schematic diagram of the Npas2 proximal promoter region analyzed for differential methylation. Pink lines indicate location of individual CpG sites analyzed. RORE lies within region I. B) Methylation status of each CpG analyzed. ○, unmethylated cytosines; ● methylated cytosine. Additional animals were used to analyze the region around the RORE. Each horizontal line represents 1 clone.

Figure 4.

Exposure to a maternal HFD in utero is associated with changes in occupancy of the Npas2 promoter RORE of H3K14ac but not H3K9me3. A) ChIP of extracts from fetal hepatic tissue with an antibody specific for H3K14 acetylation shows enrichment in the RORE region of the Npas2 promoter in animals exposed to a maternal HFD in utero. This enrichment does not persist in animals exposed to a reversed diet. B) ChIP with an antibody specific for trimethylation of lysine 9 of histone H3 does not show enrichment of the RORE of Npas2 due to maternal diet.

Figure 5.

Disruption of hepatic circadian gene expression in juvenile animals is consistent with postweaning exposure to an HFD. A) Npas2 expression level is significantly increased in juvenile animals exposed to an HFD diet during the postweaning phase (CTR/HF, 2.7-fold, P=0.044; HF/HF, 2.3-fold, P=0.028). Animals exposed to the HFD in utero and weaned to a control diet do not show disrupted Npas2 expression (HF/CTR, 1.5-fold, P=0.182). B) Expression of Per1 differed from the CTR/CTR group only in the animals exposed in utero to a control diet and weaned to an HFD (CTR/HF, 0.4-fold, P=0.024).