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BackgroundThe DNA modification 5-hydroxymethylcytosine (5hmC) is now referred to as the sixth base of DNA with evidence of tissue-specific patterns and correlation with gene regulation and expression. This epigenetic mark was recently reported as a potential biomarker for multiple types of cancer, but its application in the clinic is limited by the utility of recent 5hmC quantification assays. We use a recently developed, ultra-sensitive, fluorescence-based single-molecule method for global quantification of 5hmC in genomic DNA. The high sensitivity of the method gives access to precise quantification of extremely low 5hmC levels common in many cancers.MethodsWe assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach.ResultsUsing our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker.ConclusionsSingle-molecule detection of 5hmC allows distinguishing between malignant and healthy tissue in clinically relevant and accessible tissue such as blood and colon. The presented method outperforms current commercially available quantification kits and may potentially be developed into a widely used, 5hmC quantification assay for research and clinical diagnostics. Furthermore, using this method, we confirm that 5hmC is a good molecular biomarker for diagnosing colon and various types of blood cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0368-9) contains supplementary material, which is available to authorized users.

This study aimed to determine whether epigenetic malprogramming induced by high-fat diet (HFD) has an obesogenic effect on nonmated and mated female rats and their offspring. Further, it aimed to reprogram offspring's epigenetic malprogramming and phenotype by providing normal diet after weaning. Body weight (BW) was measured, and plasma and hypothalamic arcuate nuclei were collected for analysis of hormones, mRNA, and DNA CpG methylation of the promoter of Pomc, a key factor in control of food intake. In nonmated females, HFD decreased Pomc/leptin ratio by ∼38%. This finding was associated with Pomc promoter hypermethylation. While heavier during pregnancy, during lactation HFD dams showed sharper BW decrease (2.5-fold) and loss of Pomc promoter hypermethylation. Moreover, their weight loss was correlated with demethylation (r=-0.707) and with gadd45b mRNA expression levels (r=0.905). Even though offspring of HFD dams ate standard chow from weaning, they displayed increased BW, Pomc promoter hypermethylation, and vulnerability to HFD challenge (3-fold kilocalorie intake increase). These findings demonstrate a long-term effect of maternal HFD on CpG methylation of the Pomc promoter in the offspring, which was not reprogrammed by standard chow from weaning. Further, the results suggest a possible mechanism of demethylation of the Pomc promoter following pregnancy and lactation.

A maternal high-fat diet (HFD) alters the offspring's feeding regulation, leading to obesity. This phenomenon is partially mediated by aberrant expression of the hypothalamic anorexigenic neuropeptide proopiomelanocortin (POMC). Nevertheless, although some individual offspring suffer from morbid obesity, others escape the malprogramming. It is suggested that this difference is due to epigenetic programming. In this study, we report that in lean offspring of non-HFD–fed dams, essential promoter regions for Pomc expression were enriched with 5-hydroxymethylcytosine (5hmC) together with a reduction in the level of 5-methylcytosine (5mC). Moreover, 5hmC was negatively correlated whereas 5mC was positively correlated with body weight in offspring from both HFD- and control-fed dams. We further found that Pomc expression in obese offspring is determined by a two-step epigenetic inhibitory mechanism in which CpG methylation is linked with histone posttranslational modifications. An increase in CpG methylation at the Poxmc promoter enables binding of methyl-binding domain 1 (MBD1) to 5mC, but not to its derivative 5hmC. MBD1 then interacts with SET domain bifurcated 1 methyltransferase to promote bimethylation on the histone 3 lysine 9 residue, reducing Pomc mRNA expression. These results suggest an epigenetic regulatory mechanism that affects obesity-prone or resilient traits.

Author Contributions: Drs Fallon and Laird had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

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Maternal obesity is a risk factor for offspring obesity. The melanocortin 4 receptor (Mc4r) is one of the mediators of food intake and energy balance. The present study examined the epigenetic mechanisms underlying altered Mc4r levels in the hypothalamic paraventricular nucleus in the offspring of high-fat diet (HFD)-induced obese dams and sought to elucidate the role of thyroid hormones in epigenetic regulation and tagging of their nucleosome at the Mc4r promoter. Female Wistar rats were fed an HFD or standard chow from weaning through gestation and lactation. Epigenetic alterations were analyzed in the offspring on postnatal day 21 at the Mc4r promoter using chromatin immunoprecipitation and bisulfite sequencing. To study the role of triiodothyronine (T3) in Mc4r downregulation, dams received methimazole (MMI), an inhibitor of thyroid hormone production. Offspring of HFD-fed dams had a greater body weight, elevated plasma T3 concentrations, and lower Mc4r messenger RNA levels than controls. At the Mc4r promoter, offspring of HFD-fed mothers demonstrated increased histone 3 lysine 27 acetylation (H3K27ac) with a greater association to thyroid hormone receptor-β (TRβ), an inhibitor of Mc4r transcription. Moreover, TRβ coimmunoprecipitated with H3K27ac, supporting their presence in the same complex. Maternal MMI administration prevented the HFD reduction in Mc4r levels, the increase in TRβ, and the increase in the TRβ-H3K27ac association, providing further support for the role of T3 in downregulating Mc4r levels. These findings demonstrate that a perinatal HFD environment affects Mc4r regulation through a T3 metabolic pathway involving histone acetylation of its promoter.

5015 Background: Androgen-deprivation therapy (ADT) used in prostate-cancer may increase risk of cardiovascular disease (CVD). Limited preclinical and retrospective clinical data suggest that use of gonadotrophin-releasing hormone (GnRH)-antagonist may be associated with lower risk of CVD compared to GnRH-agonist. Methods: We conducted a randomized open-label study comparing the one year incidence of major cardiovascular and cerebrovascular event (MACCE) in prostate-cancer patients with pre-existing CVD commencing on GnRH-agonists or antagonists. Patients were followed every 3 months for the development of MACCE defined as either death, myocardial infarction (MI), cerebrovascular event (CVA), or percutaneous-coronary intervention (PCI). Serum levels of N-terminal pro-B-type natriuretic peptide (NTproBNP) were analyzed at baseline, 3, 6 and 12-months. Results: Eighty patients were enrolled (41 randomized to GnRH-antagonist, 39 to GnRH-agonist). Patients in both arms had similar age, baseline cardiovascular and prostate-cancer characteristics. During follow-up 15 patients developed a new cardiovascular event. Of these, nine patients developed MACCE (two deaths, one MI, two CVAs, and four PCI). Twenty percent (n = 8) of patients randomized to GnRH-agonists had a MACCE compared to 3% (n = 1) randomized to antagonists (log-rank p = 0.013). The absolute risk reduction for MACCE at 12 months using GnRH-antagonist was 18% (95%CI 5-31). Baseline levels of NTproBNP predicted events (AUC = 0.73 95%CI 0.54-0.91 p = 0.03) and increased over time only among patients with CV events. Conclusions: This is the first prospective study to test cardiovascular outcome among prostate-cancer patients receiving ADT. We demonstrated that in patients with pre-existing CVD, GnRH-antagonists was associated with development of fewer cardiovascular events compared to GnRH-agonists. Clinical trial information: NCT02475057.