Loss of Tet1 associated 5-hydroxymethylcytosine is concomitant with aberrant promoter hypermethylation in liver cancer

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations which occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, CGI underwent hypo-hydroxymethylation prior to hypermethylation, whilst retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation observed during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprogramming of DNA methylation at silent CGI during progression

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4(+) T cells tolerized by peptide immunotherapy

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. - See more at: http://elifesciences.org/content/3/e03416#sthash.n6isQlkn.dpu

Chronic constipation diagnosis and treatment evaluation: The "CHRO.CO.DI.T.E." study

Background: According to Rome criteria, chronic constipation (CC) includes functional constipation (FC) and irritable bowel syndrome with constipation (IBS-C). Some patients do not meet these criteria (No Rome Constipation, NRC). The aim of the study was is to evaluate the various clinical presentation and management of FC, IBS-C and NRC in Italy. Methods: During a 2-month period, 52 Italian gastroenterologists recorded clinical data of FC, IBS-C and NRC patients, using Bristol scale, PAC-SYM and PAC-QoL questionnaires. In addition, gastroenterologists were also asked to record whether the patients were clinically assessed for CC for the first time or were in follow up. Diagnostic tests and prescribed therapies were also recorded. Results: Eight hundred seventy-eight consecutive CC patients (706 F) were enrolled (FC 62.5%, IBS-C 31.3%, NRC 6.2%). PAC-SYM and PAC-QoL scores were higher in IBS-C than in FC and NRC. 49.5% were at their first gastroenterological evaluation for CC. In 48.5% CC duration was longer than 10 years. A specialist consultation was requested in 31.6%, more frequently in IBS-C than in NRC. Digital rectal examination was performed in only 56.4%. Diagnostic tests were prescribed to 80.0%. Faecal calprotectin, thyroid tests, celiac serology, breath tests were more frequently suggested in IBS-C and anorectal manometry in FC. More than 90% had at least one treatment suggested on chronic constipation, most frequently dietary changes, macrogol and fibers. Antispasmodics and psychotherapy were more frequently prescribed in IBS-C, prucalopride and pelvic floor rehabilitation in FC. Conclusions: Patients with IBS-C reported more severe symptoms and worse quality of life than FC and NRC. Digital rectal examination was often not performed but at least one diagnostic test was prescribed to most patients. Colonoscopy and blood tests were the "first line" diagnostic tools. Macrogol was the most prescribed laxative, and prucalopride and pelvic floor rehabilitation represented a "second line" approach. Diagnostic tests and prescribed therapies increased by increasing CC severity

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

BackgroundThe DNA methylation profile of mammalian cell lines differs from the primary tissue from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblast, MEFS, to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of Vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T-cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs; suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.We thank Professors Adrian Bird and Nicholas Hastie for their comments on our manuscript. JT and RO are funded by IMI-MARCAR (under grant agreement number (115001) (MARCAR project)). Work in RM's lab is supported by the MRC, IMI-MARCAR and the BBSRC. This work in RM's lab was also initially funded by the Breakthrough Breast Cancer charity. Work in MB's lab was supported by Linkoping University strategic research funding and the Ake Wibergs fund (3772738). Work in SP's lab is supported by the BBSRC.</p

Functional analysis of 5-hydroxymethylcytosine

Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture

RunPHI: Enabling Mixed-criticality Containers via Partitioning Hypervisors in Industry 4.0

Orchestration systems are becoming a key component to automatically manage distributed computing resources in many fields with criticality requirements like Industry 4.0 (I4.0). However, they are mainly linked to OS-level virtualization, which is known to suffer from reduced isolation. In this paper, we propose RunPHI with the aim of integrating partitioning hypervisors, as a solution for assuring strong isolation, with OS-level orchestration systems. The purpose is to enable container orchestration in mixed-criticality systems with isolation requirements through partitioned containers

Non-alcoholic fatty liver disease (NAFLD) is associated with dynamic changes in DNA hydroxymethylation

Non-alcoholic fatty liver disease (NAFLD) is now the commonest cause of liver disease in developed countries affecting 25–33% of the general population and up to 75% of those with obesity. Recent data suggest that alterations in DNA methylation may be related to NAFLD pathogenesis and progression and we have previously shown that dynamic changes in the cell lineage identifier 5-hydroxymethylcytosine (5hmC) may be important in the pathogenesis of liver disease. We used a model of diet-induced obesity, maintaining male mice on a high-fat diet (HFD) to generate hepatic steatosis. We profiled hepatic gene expression, global and locus-specific 5hmC and additionally investigated the effects of weight loss on the phenotype. HFD led to increased weight gain, fasting hyperglycaemia, glucose intolerance, insulin resistance and hepatic periportal macrovesicular steatosis. Diet-induced hepatic steatosis associated with reversible 5hmC changes at a discrete number of functionally important genes. We propose that 5hmC profiles are a useful signature of gene transcription and a marker of cell state in NAFLD and suggest that 5hmC profiles hold potential as a biomarker of abnormal liver physiology