DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.

Selective oestrogen receptor antagonists inhibit oesophageal cancer cell proliferation in vitro.

BACKGROUND: Oestrogen receptors (ER) have a well-established role to the initiation, progression and regulation of responses to treatment of breast, prostate, and lung cancers. Previous data indicates altered ER expression in oesophageal cancers (OC). However the role of ER subtypes and ER specific inhibitors in the regulation of OC progression remains unclear. This study sought to assess levels of ERα and ERß in OC. The effects of highly selective ER antagonists on cell proliferation and apoptosis in two OC adenocarcinoma cell lines was also studied. METHODS: ERα and ERß expression profiling in paired normal oesophageal mucosa and tumour tissues (n = 34; adenocarcinoma n = 28; squamous cell carcinoma n = 6) was performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Correlation between levels of ER with the clinico-pathological features for OC was determined. The effect of selective ER antagonists on proliferation of OE33 and OE19 OC cell lines was studied. RESULTS: ERα and ERß mRNA expression was significantly higher (p < 0.05) in tumour tissues relative to their paired normal mucosa and correlated inversely with survival outcome (p < 0.05). Upregulation of ERα mRNA correlated with higher pathological T-stage (p < 0.05) and lymph node metastasis (p < 0.05) while ERß mRNA upregulation correlated with positive vascular invasion (p < 0.05). A significant concentration-dependent inhibition of proliferation in OE33 and OE19 cell lines was induced by a highly-selective ERα antagonist (MPP) and an ERß specific antagonist (PHTPP) (p < 0.05). Moreover, anti-oestrogens induced cell death through stimulation of apoptotic caspase activity. CONCLUSION: These findings indicate that the ER system is involved in OC progression and thus may provide a novel target for the treatment of OC.

Expression of a SOX1 overlapping transcript in neural differentiation and cancer models.

SOX1 is a member of the SOXB1 subgroup of transcription factors involved in early embryogenesis, CNS development and maintenance of neural stem cells. The structure and regulation of the human SOX1 locus has been less studied than that of SOX2, another member of the SOXB1 subgroup for which an overlapping transcript has been reported. Here we report that the SOX1 locus harbours a SOX1 overlapping transcript (SOX1-OT), and describe expression, splicing variants and detection of SOX1-OT in different stem and cancer cells. RT-PCR and RACE experiments were performed to detect and characterise the structure of SOX1-OT in neuroprogenitor cultures and across different cancer cell lines. SOX1-OT was found to present a complex structure including several unannotated exons, different transcript variants and at least two potential transcription start sites. SOX1-OT was found to be highly expressed in differentiated neural stem cells across different time points of differentiation, and its expression correlated with SOX1 gene expression. Concomitant expression of SOX1 and SOX1-OT was further observed in several cancer cell models. While the function of this transcript is unknown, the regulatory role reported for other lncRNAs strongly suggests a possible role for SOX1-OT in regulating SOX1 expression, as previously observed for SOX2. The elucidation of the genetic and regulatory context governing SOX1 expression will contribute to clarifying its role in stem cell differentiation and tumorigenesis.

Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer.

LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that in cancer, aberrantly active LINE-1 promoters can drive transcription of flanking unique sequences giving rise to LINE-1 chimeric transcripts (LCTs). Here, we show that one such LCT, LCT13, is a large transcript (>300 kb) running antisense to the metastasis-suppressor gene TFPI-2. We have modelled antisense RNA expression at TFPI-2 in transgenic mouse embryonic stem (ES) cells and demonstrate that antisense RNA induces silencing and deposition of repressive histone modifications implying a causal link. Consistent with this, LCT13 expression in breast and colon cancer cell lines is associated with silencing and repressive chromatin at TFPI-2. Furthermore, we detected LCT13 transcripts in 56% of colorectal tumours exhibiting reduced TFPI-2 expression. Our findings implicate activation of LINE-1 elements in subsequent epigenetic remodelling of surrounding genes, thus hinting a novel retrotransposition-independent role for LINE-1 elements in malignancy.

Current understanding of the interplay between extracellular matrix remodelling and gut permeability in health and disease.

The intestinal wall represents an interactive network regulated by the intestinal epithelium, extracellular matrix (ECM) and mesenchymal compartment. Under healthy physiological conditions, the epithelium undergoes constant renewal and forms an integral and selective barrier. Following damage, the healthy epithelium is restored via a series of signalling pathways that result in remodelling of the scaffolding tissue through finely-regulated proteolysis of the ECM by proteases such as matrix metalloproteinases (MMPs). However, chronic inflammation of the gastrointestinal tract, as occurs in Inflammatory Bowel Disease (IBD), is associated with prolonged disruption of the epithelial barrier and persistent damage to the intestinal mucosa. Increased barrier permeability exhibits distinctive signatures of inflammatory, immunological and ECM components, accompanied by increased ECM proteolytic activity. This narrative review aims to bring together the current knowledge of the interplay between gut barrier, immune and ECM features in health and disease, discussing the role of barrier permeability as a discriminant between homoeostasis and IBD.

Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease.

Nearly all human genetic disorders result from a limited repertoire of mutations in an associated gene or its regulatory elements. We recently described an individual with an inherited form of anemia (alpha-thalassemia) who has a deletion that results in a truncated, widely expressed gene (LUC7L) becoming juxtaposed to a structurally normal alpha-globin gene (HBA2). Although it retains all of its local and remote cis-regulatory elements, expression of HBA2 is silenced and its CpG island becomes completely methylated early during development. Here we show that in the affected individual, in a transgenic model and in differentiating embryonic stem cells, transcription of antisense RNA mediates silencing and methylation of the associated CpG island. These findings identify a new mechanism underlying human genetic disease.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Differences in the pattern and regulation of mineral deposition in human cell lines of osteogenic and non-osteogenic origin.

Bone marrow-derived mesenchymal stem cells (MSCs) are widely used as a cellular model of bone formation, and can mineralize in vitro in response to osteogenic medium (OM). It is unclear, however, whether this property is specific to cells of mesenchymal origin. We analysed the OM response in 3 non-osteogenic lines, HEK293, HeLa and NTera, compared to MSCs. Whereas HEK293 cells failed to respond to OM conditions, the 2 carcinoma-derived lines NTera and HeLa deposited a calcium phosphate mineral comparable to that present in MSC cultures. However, unlike MSCs, HeLa and NTera cultures did so in the absence of dexamethasone. This discrepancy was confirmed, as bone morphogenetic protein inhibition obliterated the OM response in MSCs but not in HeLa or NTera, indicating that these 2 models can deposit mineral through a mechanism independent of established dexamethasone or bone morphogenetic protein signalling.

MicroRNAs: relevant tools for a colorectal surgeon?

Colorectal cancer is the third most common malignancy and cause of cancer-related deaths worldwide. Approximately half of the patients diagnosed with colorectal cancer ultimately die of the condition. Death from colorectal cancer can be prevented by early detection, but unfortunately presentation is often late, with a worse prognosis. Screening by fecal occult blood testing reduces disease-specific mortality, but there is a need for sensitive and specific noninvasive biomarkers to facilitate detecting the disease, staging it, and predicting the best therapeutic options. MicroRNAs (miRNAs) are short noncoding RNA sequences that have a crucial role in the regulation of gene expression. They have significant regulatory functions in basic cellular processes, such as cell differentiation, proliferation, and apoptosis. Evidence suggests that miRNAs may function as both tumor suppressors and oncogenes. The main mechanism for changes in the function of miRNAs in cancer cells is due to aberrant gene expression. Accurate discrimination of miRNA profiles between tumor and normal mucosa in colorectal cancer allows definition of specific expression patterns of miRNAs, giving good potential as diagnostic and therapeutic targets. MiRNAs expressed in colorectal cancers are also abundantly present and stable in stool and plasma samples. Their extraction from these three sources is feasible and reproducible. The ease and reliability of determining miRNA profiles in plasma or stool makes them potential molecular markers for colorectal cancer screening. This review summarizes the role miRNAs have in colorectal cancer, highlighting particularly the potential diagnostic, prognostic, and therapeutic implications in the future treatment of the disease.

Increased mitochondrial proline metabolism sustains proliferation and survival of colorectal cancer cells.

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells' proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells' survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.

Isolation of cancer-specific chimeric transcripts induced by hypomethylation of the LINE-1 antisense promoter.

The antisense promoter of human LINE-1 (L1) retroelements can direct transcription of adjacent unique genomic sequences generating chimeric RNAs, which can perturb transcription of neighbouring genes. As L1 elements constitute 17% of the human genome, chimeric transcription is potentially widespread, but the extent to which this occurs is largely unknown. Using a genome-wide screen we have isolated novel chimeric transcripts that are unique to breast cancer cell lines, primary tumours and colon cancer cells. Expression of the cancer-specific chimeric transcripts can be induced in non-malignant breast epithelial cells by the demethylating drug 5-azacytidine. These findings indicate that loss of L1 methylation in cancer cells is linked to the expression of L1-chimeric transcripts which may therefore constitute a useful set of markers of malignancy.

In Silico Identification of SOX1 Post-Translational Modifications Highlights a Shared Protein Motif.

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer.

The Janus-like role of proline metabolism in cancer.

The metabolism of the non-essential amino acid L-proline is emerging as a key pathway in the metabolic rewiring that sustains cancer cells proliferation, survival and metastatic spread. Pyrroline-5-carboxylate reductase (PYCR) and proline dehydrogenase (PRODH) enzymes, which catalyze the last step in proline biosynthesis and the first step of its catabolism, respectively, have been extensively associated with the progression of several malignancies, and have been exposed as potential targets for anticancer drug development. As investigations into the links between proline metabolism and cancer accumulate, the complexity, and sometimes contradictory nature of this interaction emerge. It is clear that the role of proline metabolism enzymes in cancer depends on tumor type, with different cancers and cancer-related phenotypes displaying different dependencies on these enzymes. Unexpectedly, the outcome of rewiring proline metabolism also differs between conditions of nutrient and oxygen limitation. Here, we provide a comprehensive review of proline metabolism in cancer; we collate the experimental evidence that links proline metabolism with the different aspects of cancer progression and critically discuss the potential mechanisms involved.

Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer.

Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5'RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation.

The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.

The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation.

AIMS: The protective effects of cannabidiol (CBD) have been widely shown in preclinical models and have translated into medicines for the treatment of multiple sclerosis and epilepsy. However, the direct vascular effects of CBD in humans are unknown. METHODS AND RESULTS: Using wire myography, the vascular effects of CBD were assessed in human mesenteric arteries, and the mechanisms of action probed pharmacologically. CBD-induced intracellular signalling was characterized using human aortic endothelial cells (HAECs). CBD caused acute, non-recoverable vasorelaxation of human mesenteric arteries with an Rmax of ∼ 40%. This was inhibited by cannabinoid receptor 1 (CB1) receptor antagonists, desensitization of transient receptor potential channels using capsaicin, removal of the endothelium, and inhibition of potassium efflux. There was no role for cannabinoid receptor-2 (CB2) receptor, peroxisome proliferator activated receptor (PPAR)γ, the novel endothelial cannabinoid receptor (CBe), or cyclooxygenase. CBD-induced vasorelaxation was blunted in males, and in patients with type 2 diabetes or hypercholesterolemia. In HAECs, CBD significantly reduced phosphorylated JNK, NFκB, p70s6 K and STAT5, and significantly increased phosphorylated CREB, ERK1/2, and Akt levels. CBD also increased phosphorylated eNOS (ser1177), which was correlated with increased levels of ERK1/2 and Akt levels. CB1 receptor antagonism prevented the increase in eNOS phosphorylation. CONCLUSION: This study shows, for the first time, that CBD causes vasorelaxation of human mesenteric arteries via activation of CB1 and TRP channels, and is endothelium- and nitric oxide-dependent.

Endocannabinoids modulate human blood-brain barrier permeability in vitro.

BACKGROUND AND PURPOSE: Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood-brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model. EXPERIMENTAL APPROACH: Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR. KEY RESULTS: Anandamide (10 µM) and oleoylethanolamide (OEA, 10 µM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 µM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB1 receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB2 receptors was only present in astrocytes. CONCLUSION AND IMPLICATION: The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.

Inflammation and MiR-21 pathways functionally interact to downregulate PDCD4 in colorectal cancer.

Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE2), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE2 up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE2 and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.

LINE-1 activation and epigenetic silencing of suppressor genes in cancer: Causally related events?

The ability of active retrotransposon elements to move within the host genome and alter gene expression with subsequent phenotypic variation led to their initial discovery. In recent years it has become apparent that these elements can also modulate host gene expression independently of their transposition activity. Many retrotransposons maintain endogenous promoter motifs that can potentially drive expression of adjacent DNA modules. Similarly to transposition dependent dysregulation, these proto-promoters can progress disease states when active. Indeed aberrant activation of retrotransposon derived promoters in cancer can lead to transcription of oncogenic isoforms of cellular genes. Here we propose that activation of promoters of transposable elements in cancer can also drive transcription of long non-coding RNAs whose expression leads to silencing of linked tumor suppressor genes. Such transcription driven by aberrantly active transposable elements in cancer can lead to a characteristic reprogramming of epigenetic profiles, thus extending the potential molecular mechanisms whereby retrotransposons can directly contribute to cancer development and subsequent progression.

DNMTs are required for delayed genome instability caused by radiation.

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.