Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets

Abstract

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter-enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

Fig. 1.

DNA methylation and histone modification patterns at the Hnf4a locus in rat islets. (A) Schematic representation of the gene. Enh, enhancer region; 1D–10, coding exons. (B) mRNA expression analysis by qRT-PCR. Data were normalized against cyclophilin A (Ppia) and presented relative to the mean level of P2 transcripts, arbitrarily set to 1 (n = 5). **P < 0.01. (C) Bisulphite sequencing analysis of DNA methylation (n = 2). Filled squares represent methylated CpG dinucleotides. TSS, transcription start site. (D) Native ChIP analysis of histone marks. Actb and Hbb promoter regions were used as controls (n = 7).

Fig. 2.

Role of histone modifications in controlling Hnf4a P2 expression. (A) mRNA analysis by qRT-PCR in adult rat islets, BRIN cells, and INS-1 cells, normalized against Ppia (n = 3 per group). Data are shown relative to islets, arbitrarily normalized to 1. Native ChIP analysis of histone marks in BRIN (B) and INS-1 (D) cells (n = 4 per group). qRT-PCR analysis in BRIN- (C) and INS-1–treated (E) cells. Data were normalized against Ppia and are shown relative to levels in untreated cells, arbitrarily set to 1 (n = 4 per group). Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Early diet- and aging-associated effects on transcriptional activity and epigenetic regulation at the Hnf4a locus in rat islets. (A) mRNA analysis by qRT-PCR in 3M and 15M C and LP samples (3M, n = 10 per group; 15M, n = 8 per group). Data were normalized against Ppia and are shown relative to 3M C, arbitrarily set to 1. (B) MassArray analysis of DNA methylation at the P2 promoter in 3M and 15M C and LP samples (n = 6 per group). Native ChIP analysis of histone marks at the enhancer (C) and P2 promoter (D) in 3M and 15M C and LP samples (3M, n = 7 per group; 15M, n = 6 per group). Note that the levels of H3K4me3 were not compared between C and LP islets at the enhancer because of its very low levels at this region (Fig. 1D). H3K4me1 was not compared at the P2 promoter because it is a mark for active enhancers (23). Data are shown relative to the 3M C samples, arbitrarily normalized to 1. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Long-range interactions at the rat Hnf4a locus. (A) Schematic representation of the 300-kb region on rat chromosome 3 containing the Hnf4a locus, analyzed by q3C. Green arrows indicate the transcriptional orientation of the genes within this region. Blue vertical bars indicate CTCF/cohesin-binding sites validated by ChIP in rat liver. (B) Comparative P2-enhancer association frequency in adult liver (n = 4), INS-1 cells (n = 5), and adult islets (n = 3). (C) Proposed looping models that summarize the long-range interactions in islets and liver. Red circles indicate CTCF/cohesin-binding sites, and blue circles indicate sites not bound by CTCF/cohesin. (D) P2-enhancer association frequency in 3M C (n = 4) and LP samples (n = 5). Data are shown relative to the 3M C samples, arbitrarily normalized to 1. 3M C products after 33 PCR cycles are shown. Error bars represent SEM. *P < 0.05; **P < 0.01.