The importance of altered gene promoter methylation and transcription factor binding in developmental programming of central appetitive drive

Emerging data from human epidemiological and well-controlled animal studies have stimulated interest in the central role of nutrition during plastic periods of development of an individual's phenotype and their long-term health. Developmental programming of postnatal function demonstrates several key features including persistence after the stimulus has been removed, and stage of development and sex specificity. Epigenetic changes resulting from both under- and overnutrition in fetal and early neonatal life can change the trajectory of development of key systems that predispose to adult obesity. Postnatal appetite control is central to the development of adiposity and animal studies have shown maternal diet-induced alterations in offspring expression of hypothalamic peptides such as the orexigenic neuropeptide AgRP that have the potential to increase food intake and result in obesity (Kirk et al. 2009). The molecular basis of epigenetic mechanisms that underlie developmental programming are rapidly being characterized (Aagaard-Tillery et al. 2008; Gluckman et al. 2009). In contrast to genetic changes such as spontaneous mutations where the DNA primary sequence is changed, epigenetic programming involves alterations such as covalent modification by methylation of cytosine rings found at the dinucleotide sequence CpG and histone methylation and acetylation in chromatin in response to environmental exposures. These modifications provide a dynamic mechanism that alters the intricate patterns of gene expression characteristic of complex organisms. The epigenome is a central factor that determines accessibility of DNA by the transcriptional machinery. It is generally accepted that DNA methylation plays an important role in regulating gene expression. DNA methylation in distinct regulatory regions is believed to silence gene expression whereas hypomethylated genes are accessible and transcribed (Gluckman et al. 2009). The developmental programming hypothesis proposes that environmental exposures alter phenotype through this mechanism. In this issue of The Journal of PhysiologyPlagemann et al. (2009) present data on the DNA methylation status of CpG dinucleotides in gene promoters for the orexigenic neuropeptide Y (NPY) and the anorexigenic neuropeptide proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus of rats at weaning. Together these two peptides are important factors that regulate the balance of food intake and energy output. Neonatal overfeeding was induced by rearing rats in small litters (three pups per mother while normal litters were reduced to twelve pups per mother). Overfeeding resulted in a metabolic syndrome phenotype characterized by obesity, hyperleptinaemia, hyperglycaemia, hyperinsulinaemia, and increased insulin/glucose ratio with greater weight gain up to weaning. In the offspring of the small litters POMC promoter analysis revealed hypermethylation of one CpG site (−152) within a target binding site for transcription factors Sp1 and NFκB. In addition, DNA methylation of the CpG dinucleotide within the Sp1/NFκB site was inversely correlated to the quotients of POMC expression/plasma leptin and POMC expression/plasma insulin suggesting functional epigenomic changes in the hypothalamus. POMC promoter changes were specific since the NPY promoter showed a hypomethylation status (< 5%) with no difference between overfed and normally fed offspring. These results demonstrate a decrease in DNA methylation that corresponds to overfeeding prior to weaning and suggest that early overfeeding may be an epigenetic risk factor for the programming of central nervous system mechanisms that predispose to augmented food intake, obesity and subsequent development of the metabolic syndrome. While several studies have now shown altered methylation and acetylation as a result of nutritional status during development, this report is the first to show alterations in mechanisms involving transcription factors that regulate gene expression and open the way for studies designed to demonstrate that the nutritional environment can influence epigenetic programming of key central nervous system and other organ systems using approaches that elucidate protein–DNA interactions and transactivational changes by binding and transcriptional assays, respectively. Results from such assays are needed to determine the specific mechanisms whereby nutrition can programme later adult obesity. In addition, it will be interesting to determine the methylation status of genes within the POMC signalling network, such as leptin, leptin receptor and AMP kinase, in response to the overfed nutritional environment. It will be important to identify the network of gene promoters within the central nervous system that are epigenetically programmed by the nutritional environment present during development. There are several questions that arise from this interesting study. What are the mechanisms responsible for the differential epigenetic effects on different genes in the presence of the same nutritional challenge as shown here by POMC and NPY? Do the differences reflect the need for plasticity in some gene pathways and stability in others? Are there differences according to offspring sex? What is the interplay between availability of various factors such as micro- and macronutrients, hormones and growth factors? Information is also needed on the precise change in availability of the various one-carbon cycle components in the different animal models of epigenetic programming. To date few measurements have been made of either the circulating components or the several enzymes that regulate the one-carbon cycle in affected tissues. Finally, in addition to knowledge of windows of susceptibility it will be important to determine windows of reversibility. Methyl donor-rich dietary supplements have been shown to both prevent the transgenerational predisposition to obesity observed in agouti mice (Waterland et al. 2008) and reverse some but not all of the epigenetic changes that are present in offspring of mothers fed a low protein diet indicating that timing of rescue will prove important (Burdge et al. 2009). This report provides a further step on our way to understanding the mechanisms responsible for the protracted memory of the original insult.