Imprinted chromatin around DIRAS3 regulates alternative splicing of GNG12-AS1, a long noncoding RNA.

Imprinted gene clusters are regulated by long noncoding RNAs (lncRNAs), CCCTC binding factor (CTCF)-mediated boundaries, and DNA methylation. DIRAS3 (also known as ARH1 or NOEY1) is an imprinted gene encoding a protein belonging to the RAS superfamily of GTPases and is located within an intron of a lncRNA called GNG12-AS1. In this study, we investigated whether GNG12-AS1 is imprinted and coregulated with DIRAS3. We report that GNG12-AS1 is coexpressed with DIRAS3 in several tissues and coordinately downregulated with DIRAS3 in breast cancers. GNG12-AS1 has several splice variants, all of which initiate from a single transcription start site. In placenta tissue and normal cell lines, GNG12-AS1 is biallelically expressed but some isoforms are allele-specifically spliced. Cohesin plays a role in allele-specific splicing of GNG12-AS1. In breast cancer cell lines with loss of DIRAS3 imprinting, DIRAS3 and GNG12-AS1 are silenced in cis and the remaining GNG12-AS1 transcripts are predominantly monoallelic. The GNG12-AS1 locus, which includes DIRAS3, provides an example of imprinted cotranscriptional splicing and a potential model system for studying the long-range effects of CTCF-cohesin binding on splicing and transcriptional interference.

Cohesin is required for higher-order chromatin conformation at the imprinted IGF2-H19 locus.

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.

Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling.

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.

Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer.

The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2-5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.

Network flux analysis: impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures.

The aim of this study was to test the assumption that (13)C-enrichment of respiratory substrate does not perturb metabolism. Cell suspension cultures of Arabidopsis thaliana were grown in MS medium containing unlabelled glucose (with (13)C at natural abundance), 100% [1-(13)C]glucose, 100% [U-(13)C(6)]glucose or 10% [U-(13)C(6)]glucose plus 90% unlabelled glucose. There was no significant difference in the metabolism of [U-(14)C]glucose between the cultures. Similarly, the pattern of (14)CO(2) release from specifically labelled [(14)C]-substrates was unaffected. Principal component analysis of (13)C-decoupled (1)H NMR metabolite fingerprints of cell extracts was unable to discriminate between the different culture conditions. It is concluded that (13)C-enrichment of the growth substrate has no effect on flux through the central pathways of carbon metabolism in higher plants. This conclusion supports the implicit assumption in metabolic flux analysis that steady-state (13)C-labelling does not perturb fluxes through the reactions of the metabolic network it seeks to quantify.

Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®.

Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression.

Predictors of insulin resistance in pediatric burn injury survivors 24 to 36 months postburn.

Burn injury is a dramatic event with acute and chronic consequences including insulin resistance. However, factors associated with insulin resistance have not been previously investigated. The purpose of this study was to identify factors associated with long-term insulin resistance in pediatric burn injury survivors. The study sample consisted of 61 pediatric burn injury survivors 24 to 36 months after the burn injury, who underwent an oral glucose tolerance test. To assess insulin resistance, the authors calculated the area under the curve for glucose and insulin. The diagnostic criteria of the American Diabetes Association were used to define individuals with impaired glucose metabolism. Additional data collected include body composition, anthropometric measurements, burn characteristics, and demographic information. The data were analyzed using multivariate linear regression analysis. Approximately 12% of the patients met the criteria for impaired glucose metabolism. After adjusting for possible confounders, burn size, age, and body fat percentage were associated with the area under the curve for glucose (P < .05 for all). Time postburn and lean mass were inversely associated with the area under the curve for glucose (P < .05 for both). Similarly, older age predicted higher insulin area under the curve. The results indicate that a significant proportion of pediatric injury survivors suffer from glucose abnormalities 24 to 36 months postburn. Burn size, time postburn, age, lean mass, and adiposity are significant predictors of insulin resistance in pediatric burn injury survivors. Clinical evaluation and screening for abnormal glucose metabolism should be emphasized in patients with large burns, older age, and survivors with high body fat.

Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue.

BACKGROUND: Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. RESULTS: Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs. CONCLUSIONS: Our validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.

Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing.

We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer.

BACKGROUND: Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis. CONCLUSIONS/SIGNIFICANCE: These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.