5-Formylcytosine can be a stable DNA modification in mammals.

5-Formylcytosine (5fC) is a rare base found in mammalian DNA and thought to be involved in active DNA demethylation. Here, we show that developmental dynamics of 5fC levels in mouse DNA differ from those of 5-hydroxymethylcytosine (5hmC), and using stable isotope labeling in vivo, we show that 5fC can be a stable DNA modification. These results suggest that 5fC has functional roles in DNA that go beyond being a demethylation intermediate.

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.

Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus.

Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2'deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes.

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

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.

Binding interactions between long noncoding RNA HOTAIR and PRC2 proteins.

Long noncoding RNAs (lncRNAs) play a key role in the epigenetic regulation of cells. Many of these lncRNAs function by interacting with histone repressive proteins of the Polycomb group (PcG) family, recruiting them to gene loci to facilitate silencing. Although there are now many RNAs known to interact with the PRC2 complex, little is known about the details of the molecular interactions. Here, we show that the PcG protein heterodimer EZH2-EED is necessary and sufficient for binding to the lncRNA HOTAIR. We also show that protein recognition occurs within a folded 89-mer domain of HOTAIR. This 89-mer represents a minimal binding motif, as further deletion of nucleotides results in substantial loss of affinity for PRC2. These findings provide molecular insights into an important system involved in epigenetic regulation.

Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith-Wiedemann syndrome and Silver-Russell syndrome.

Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF--cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF--cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF-cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF--cohesin binding sites surrounding the locus. In normal cells, lack of CTCF--cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF--cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively.

Epigenetic mechanisms in development and disease.

Our advances in technology allow us to sequence DNA to uncover genetic differences not only between individuals, but also between normal and diseased cells within an individual. However, there is still a lot we have yet to understand regarding the epigenetic mechanisms that also contribute to our individuality and to disease. The 80th Biochemical Society Annual Symposium entitled Epigenetic Mechanisms in Development and Disease brought together some leading researchers in the field who discussed their latest insights into epigenetic mechanisms. Methylation of DNA has been the focus of much study from both a developmental perspective and imprinting of genes to its contribution to diseases such as cancer. Recently, the modification of methylcytosine to hydoxymethylcytosine within cells was uncovered, which opened a host of potential new mechanisms, and a flurry of new studies are underway to uncover its significance. Epigenetics is not confined to a study of DNA, and the post-translational modifications on the histone proteins have a significant role to play in regulating gene expression. There are many different modifications and, as shown at the Symposium, new variations used by cells are still being uncovered. We are some way to identifying how these modifications are added and removed and the protein complexes responsible for these changes. A focus on the function of the complexes and the interactions between individual modifications to regulate gene expression is advancing our knowledge, as discussed in the accompanying papers, although there are clearly plenty of opportunities for new breakthroughs to be made.

Status of genomic imprinting in epigenetically distinct pluripotent stem cells.

Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.

Widespread allele-specific topological domains in the human genome are not confined to imprinted gene clusters.

BACKGROUND: There is widespread interest in the three-dimensional chromatin conformation of the genome and its impact on gene expression. However, these studies frequently do not consider parent-of-origin differences, such as genomic imprinting, which result in monoallelic expression. In addition, genome-wide allele-specific chromatin conformation associations have not been extensively explored. There are few accessible bioinformatic workflows for investigating allelic conformation differences and these require pre-phased haplotypes which are not widely available. RESULTS: We developed a bioinformatic pipeline, "HiCFlow," that performs haplotype assembly and visualization of parental chromatin architecture. We benchmarked the pipeline using prototype haplotype phased Hi-C data from GM12878 cells at three disease-associated imprinted gene clusters. Using Region Capture Hi-C and Hi-C data from human cell lines (1-7HB2, IMR-90, and H1-hESCs), we can robustly identify the known stable allele-specific interactions at the IGF2-H19 locus. Other imprinted loci (DLK1 and SNRPN) are more variable and there is no "canonical imprinted 3D structure," but we could detect allele-specific differences in A/B compartmentalization. Genome-wide, when topologically associating domains (TADs) are unbiasedly ranked according to their allele-specific contact frequencies, a set of allele-specific TADs could be defined. These occur in genomic regions of high sequence variation. In addition to imprinted genes, allele-specific TADs are also enriched for allele-specific expressed genes. We find loci that have not previously been identified as allele-specific expressed genes such as the bitter taste receptors (TAS2Rs). CONCLUSIONS: This study highlights the widespread differences in chromatin conformation between heterozygous loci and provides a new framework for understanding allele-specific expressed genes.

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.

Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression.

BACKGROUND AND AIMS: Although aberrant methylation of key genes in the progression of colorectal neoplasia has been reported, no model-based analysis of the incremental changes through the intermediate adenoma stage has been described. In addition, the biological drivers for these methylation changes have yet to be defined. Linear mixed-effects modelling was used in this study to understand the onset and patterns of the methylation changes of SFRP2, IGF2 DMR0, H19, LINE-1 and a CpG island methylator phenotype (CIMP) marker panel, and they were correlated with DNA methyltransferase 3B (DNMT3B) levels of expression in a sample set representative of colorectal neoplastic progression. METHODS: Methylation of the above CpG islands was measured using quantitative pyrosequencing assays in 261 tissue samples. This included a prospective collection of 44 colectomy specimens with concurrent normal mucosa, adenoma and invasive cancer tissues. Tissue microarrays from a subset of 64 cases were used for immunohistochemical analysis of DNMT3B expression. RESULTS: It is shown that the onset and pattern of methylation changes during colorectal neoplastic progression are locus dependent. The CIMP marker RUNX3 was the earliest CpG island showing significant change, followed by the CIMP markers NEUROG1 and CACNA1G at the hyperplastic polyp stage. SFRP2 and IGF2 DMR0 showed significant methylation changes at the adenomatous polyp stage, followed by the CIMP markers CDKN2A and hMLH1 at the adenocarcinoma stage. DNMT3B levels of immunohistochemical expression increased significantly (p < 0.001) from normal to hyperplastic and from adenomatous polyps to carcinoma samples. DNMT3B expression correlated positively with SFRP2 methylation (r = 0.42, p < 0.001, 95% CI 0.25 to 0.56), but correlated negatively with IGF2 DMR0 methylation (r = 0.26, p = 0.01, 95% CI -0.45 to -0.05). A subset of the CIMP panel (NEUROG1, CACNA1G and CDKN2A) positively correlated with DNMT3B levels of expression (p < 0.05). CONCLUSION: Hierarchical epigenetic alterations occur at transition points during colorectal neoplastic progression. These cumulative changes are closely correlated with a gain of DNMT3B expression, suggesting a causal relationship.

Generation of an epigenetic signature by chronic hypoxia in prostate cells.

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Setting up and maintaining differential insulators and boundaries for genomic imprinting.

It is becoming increasingly clear that gene expression is strongly regulated by the surrounding chromatin and nuclear environment. Gene regulatory elements can influence expression over long distances and the genome needs mechanisms whereby transcription can be contained. Our current understanding of the mechanisms whereby insulator/boundary elements organise the genome into active and silent domains is based on chromatin looping models that separate genes and regulatory elements. Imprinted genes have parent-of-origin specific chromatin conformation that seems to be maintained in somatic tissues and reprogrammed in the germline. This review focuses on the proteins found to be present at insulator/boundary sequences at imprinted genes and examines the experimental evidence at the IGF2-H19 locus for a model in which CTCF or other proteins determine primary looping scaffolds that are maintained in most cell lineages and speculates how these loops may enable dynamic secondary associations that can activate or silence genes.

Molecular mechanisms of genomic imprinting and clinical implications for cancer.

Genomic imprinting is an epigenetic marking of genes in the parental germline that ensures the stable transmission of monoallelic gene expression patterns in a parent-of-origin-specific manner. Epigenetic marking systems are thus able to regulate gene activity independently of the underlying DNA sequence. Several imprinted gene products regulate cell proliferation and fetal growth; loss of their imprinted state, which effectively alters their dosage, might promote or suppress tumourigenic processes. Conversely, global epigenetic changes that underlie tumourigenesis might affect imprinted gene expression. Here, we review imprinted genes with regard to their roles in epigenetic predisposition to cancer, and discuss acquired epigenetic changes (DNA methylation, histone modifications and chromatin conformation) either as a result of cancer or as an early event in neoplasia. We also address recent work showing the potential role of noncoding RNA in modifying chromatin and affecting imprinted gene expression, and summarise the effects of loss of imprinting in cancer with regard to the roles that imprinted genes play in regulating growth signalling cascades. Finally, we speculate on the clinical applications of epigenetic drugs in cancer.

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.

ApcMin/+ tumours and normal mouse small intestines show linear metabolite concentration and DNA cytosine hydroxymethylation gradients from pylorus to colon.

Topographical variations of metabolite concentrations have been reported in the duodenum, jejunum and ileum of the small intestine, and in human intestinal tumours from those regions, but there are no published metabolite concentrations measurements correlated with linear position in the mouse small intestine or intestinal tumours. Since DNA methylation dynamics are influenced by metabolite concentrations, they too could show linear anatomical variation. We measured metabolites by HR-MAS 1H NMR spectroscopy and DNA cytosine modifications by LC/MS, in normal small intestines of C57BL/6J wild-type mice, and in normal and tumour samples from ApcMin/+ mice. Wild-type mouse intestines showed approximately linear, negative concentration gradations from the pylorus (i.e. the junction with the stomach) of alanine, choline compounds, creatine, leucine and valine. ApcMin/+ mouse tumours showed negative choline and valine gradients, but a positive glycine gradient. 5-Hydroxymethylcytosine showed a positive gradient in the tumours. The linear gradients we found along the length of the mouse small intestine and in tumours contrast with previous reports of discrete concentration changes in the duodenum, jejunum and ileum. To our knowledge, this is also the first report of a systematic measurement of global levels of DNA cytosine modification in wild-type and ApcMin/+ mouse small intestine.

5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation.

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Being in a loop: how long non-coding RNAs organise genome architecture.

Chromatin architecture has a significant impact on gene expression. Evidence in the last two decades support RNA as an important component of chromatin structure [Genes Dev. (2005) 19, 1635-1655; PLoS ONE (2007) 2, e1182; Nat. Genet. (2002) 30, 329-334]. Long non-coding RNAs (lncRNAs) are able to control chromatin structure through nucleosome positioning, interaction with chromatin re-modellers and chromosome looping. These functions are carried out in cis at the site of lncRNAs transcription or in trans at distant loci. While the evidence for a role in lncRNAs in regulating gene expression through chromatin interactions is increasing, there is still very little conclusive evidence for a potential role in looping organisation. Here, we review models for the involvement of lncRNAs in genome architecture and the experimental evidence to support them.

Genomic imprinting: CTCF protects the boundaries.

The DNA-binding protein CTCF, which acts as a chromatin 'insulator', regulates imprinting of the mammalian Igf2 and H19 genes in a methylation-sensitive manner. It has now been shown that CTCF is also required for protection against de novo methylation of the differentially methylated domain of H19 in the female germline.