Lower DNA Methylation Research Articles

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.

Epigenetic Regulation of Glucose Transporter 4 by Estrogen Receptor β

Glucose transporter 4 (Glut4) is an important regulator of cellular glucose uptake in adipose tissue and skeletal muscle. The estrogen receptors α and β (ERα and ERβ) have been shown to regulate Glut4. However, the regulatory mechanisms are unclear, and there are conflicting results about the effects of the two ER isoforms on Glut4 activity. In this study we investigated how the lack of either ER isoform affects Glut4 expression in differentiated mouse embryonic fibroblasts. Our results demonstrate that Glut4 transcription is markedly reduced in cells lacking ERβ, both basally and upon induction by liver X receptor. These changes in Glut4 expression could not be explained by the lack of ERβ as ligand-activated transcription factor. They were rather brought about by hypermethylation of one single CpG in the Glut4 promoter in the ERβ-deficient cells. This CpG is part of an Sp1-binding site, and Sp1 binding was reduced by its methylation. Treatment with Sp1 inhibitor diminished Glut4 expression in wild-type, but not in ERβ-deficient cells, suggesting that reduced recruitment of Sp1 to the Glut4 promoter is responsible for the differences in Glut4 expression. Reintroduction of ERβ into ERβ-deficient cells partly restored Glut4 transcription and stabilized low DNA methylation after treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine. Our findings demonstrate the involvement of DNA methylation in Glut4 regulation and imply a novel function for ERβ in mediating epigenetic events and thereby regulating gene expression.

Genome-wide combination profiling of DNA copy number and methylation for deciphering biomarkers in non-small cell lung cancer patients

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease’s high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31–3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients ( p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients.

A multi-tissue analysis identifies HLA complex group 9 gene methylation differences in bipolar disorder

Epigenetic studies of DNA and histone modifications represent a new and important activity in molecular investigations of human disease. Our previous epigenome-wide scan identified numerous DNA methylation differences in post-mortem brain samples from individuals affected with major psychosis. In this article, we present the results of fine mapping DNA methylation differences at the human leukocyte antigen (HLA) complex group 9 gene (HCG9) in bipolar disorder (BPD). Sodium bisulfite conversion coupled with pyrosequencing was used to interrogate 28 CpGs spanning ∼700 bp region of HCG9 in 1402 DNA samples from post-mortem brains, peripheral blood cells and germline (sperm) of bipolar disease patients and controls. The analysis of nearly 40 000 CpGs revealed complex relationships between DNA methylation and age, medication as well as DNA sequence variation (rs1128306). Two brain tissue cohorts exhibited lower DNA methylation in bipolar disease patients compared with controls at an extended HCG9 region (P=0.026). Logistic regression modeling of BPD as a function of rs1128306 genotype, age and DNA methylation uncovered an independent effect of DNA methylation in white blood cells (odds ratio (OR)=1.08, P=0.0077) and the overall sample (OR=1.24, P=0.0011). Receiver operating characteristic curve A prime statistics estimated a 69-72% probability of correct BPD prediction from a case vs control pool. Finally, sperm DNA demonstrated a significant association (P=0.018) with BPD at one of the regions demonstrating epigenetic changes in the post-mortem brain and peripheral blood samples. The consistent multi-tissue epigenetic differences at HCG9 argue for a causal association with BPD.

Abstract 101: Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

Abstract The tight junction protein claudin-4 is aberrantly up-regulated in gastric cancer, but its clinical significance and the molecular mechanism underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanism regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression didn't show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it didn't affect cell growth. Claudin-4 expression also increased the barrier function of tight junctions. Claudin-4 up-regulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression as well as it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 101. doi:10.1158/1538-7445.AM2011-101

Androgen Receptor Mutations Are Associated with Altered Epigenomic Programming as Evidenced by HOXA5 Methylation

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.

DNA Methylation Status of Bovine Blastocyst Embryos Obtained from Various Procedures

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoter-associated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P=0.005) and p15 methylation was associated with better overall (HR 0.4, P=0.001) and disease-free survival (HR 0.4, P=0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients.

Epigenetics Online: Multimedia Teaching Resources

The Human Genome Project, declared complete in 2003, elucidated the sequence of our genome. Researchers are now learning that another level of information—the epigenome—controls gene expression in part by controlling access to DNA. The gene-reading machinery is blocked when methyl molecules bind to DNA or histones. Conversely, when acetyl molecules bind to histones, DNA becomes less tightly wound and genes become available for transcription. The pattern of these chemical tags can be copied across cell divisions and may even be passed on to an individual's offspring. However, because acetylation and methylation are driven by cell signals, the epigenome is dynamic, enabling it to respond to the environment. Thus, epigenetic tags in a cell lineage can change over the course of an individual's lifetime, even leading to differences between identical twins. This feature focuses on multimedia resources that can be used in teaching epigenetics at the undergraduate and secondary level; several hands-on activities also are included.

Effect of neonatal exposure to environmental pollutants on the DNA methylation of rat testis

To investigate the effects of neonatal exposure of DNA methylation inhibitor, Cadmium and PCB153 on DNA methylation, apoptosis and spermatogenesis in SD rats. Neonatal SD rats were randomly divided into 10 groups and received oral administrations of PCB153 (0.025, 0. 250, 2.500 mg/kg), or Cadmium (1, 2, 4 mg/kg), or positive control 5-Aza-CdR (0.025, 0.250 mg/kg), or vehicle control for five days from PND3. Half of the rats were killed 24 h after the last administration. The remains were fed until 12 weeks. Sperm numbers, apoptosis and DNA methylation levels in testis were investigated. The daily sperm production was significantly decreased in each neonatal exposed group (P < 0.05). Neonatal rats exposed to 5-Aza-CdR and Cadmium reduced the global DNA methylation level, increased apoptosis, while PCB153 exposure did not significantly change DNA methylation and apoptosis. Neonatal rats exposed to chemicals could reduce spermatogenesis via multiple pathways. Lower DNA methylation and increased neonatal apoptosis were suggested as one of the causes.

Functional analysis of 5-lipoxygenase promoter repeat variants

Variants of a hexanucleotide repeat polymorphism in the promoter of the 5-lipoxygenase (5-LO) gene have been associated with cardiovascular disease traits in humans, which may be due, at least in part, to differential expression of the at-risk alleles. To more fully characterize these variants, we carried out gene expression and DNA methylation studies in primary leukocytes from healthy individuals carrying various 5-LO promoter alleles. Regardless of genotype, 5-LO and 5-LO-activating protein (FLAP) gene expression was higher in granulocytes compared with monocytes and lymphocytes, whereas leukotriene A4 hydrolase (LTA4H) expression was higher in monocytes. In all three leukocyte populations, 5-LO mRNA levels were positively correlated with those of FLAP and LTA4H, with the highest correlation observed in granulocytes. In lymphocytes, individuals homozygous for the shorter 3 and 4 repeat alleles had between 20-35% higher 5-LO, FLAP and LTA4H expression compared with homozygous carriers of the wild-type 5 repeat allele (P = 0.03-0.0001). DNA methylation analysis of four CpG islands in a 1500 bp region encompassing the 5-LO promoter and the first approximately 100 bp of intron 1 revealed relatively low overall DNA methylation across all genotypes and leukocyte populations. However, analysis of the promoter repeats themselves demonstrated that, regardless of cell population, the 4 allele was methylated approximately twice as much as the 3 allele (P < 0.0001). Our results demonstrate that, in lymphocytes, the shorter repeat alleles of the 5-LO promoter lead to higher gene expression, which may be regulated through differential DNA methylation of the CpGs located within these repeats.

DNA methylation patterns of mouse tetraploid embryos

In the present study, the DNA methylation patterns of in vitro-derived mouse tetraploid embryos were investigated by immunofluorescence staining with an antibody against 5-methylcytosine (5MeC). Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass (ICM) than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts. So the DNA methylation patterns of mouse tetraploid embryos are aberrant, which may lead to subsequent developmental failure and embryo death. This is the first report on the methylation patterns of in vitro-derived mouse tetraploid embryos.

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCE(high) tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCE(high) SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

38 EFFECTS OF 5-AZACYTIDINE ON DNA HYPERMETHYLATION STATUS OF CLONED MINIATURE PIG EMBRYO

During somatic cell nuclear transfer, somatic cells should undergo epigenetic reprogramming in order to attain successful totipotency. Although there are many reasons of low efficiency of cloning, the aberrant DNA methylation status in donor cells is thought to reduce efficiency and increase abnormalities in cloned embryos. The methylation level of cloned embryos is higher than that of in vivo-produced or in vitro-fertilized embryos before occurring de novo methylation. This hyper DNA methylation status has been considered as a reason for the abnormal development of cloned embryos and the related decrease in term number. The aim of this research is to validate the DNA methylation pattern in cloned porcine embryos and to confirm whether reduction of hyper DNA methylation levels results in an improvement in cloning efficiency. First, immunostaining was used to evaluate the stage-specific changing pattern of DNA methylation of cloned embryos. In this result, we demonstrated that the 1-cell stage embryos and blastocyst had the highest level of methylation compared to 2- or 4-cell stage embryos. Second, this methylation level was higher in SCNT-derived embryos compared to parthenogenic embryos, suggesting that the methylation level was associated with the nucleus of donor cells. Third, cloned embryos were treated with various concentration of 5-azacytidine, which is a demethylating agent, to find adequate concentration. In results, 500 nm of 5-azacytidin group from 0 to 24 h after activation produced significantly more blastocysts compared to control group (control; 5.4 ± 2.3, 500 nm/0 24-h treatment; 12.7 ± 2.1, P &lt; 0.05) and also showed low DNA methylation level of inner cell mass different from that of control group. In conclusion, our results suggest that cloned porcine embryos show typical demethylation. But, they are generally on a hyper level of DNA methylation. This high DNA methylation level is associated with somatic cells and affects on blastocyst development. We also suggest that 5-azacytidine could improve blastocyst development rate of cloned porcine embryos by aiding weak and abnormal demethylation process.

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

In somatic cell nuclear transfer (SCNT) technologies, the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of genome function, including establishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05), and three tested CpGs sites (1, 2, 3) exhibited unmethylation in one cloned bovine (9C3); however, Xist showed similar DNA methylation levels between clones and controls, and both showed hypermethylation (96.11% and 86.67%).

Deoxyribonucleic Acid Methyltransferase 3B Promotes Epigenetic Silencing through Histone 3 Chromatin Modifications in Pituitary Cells

Epigenetic dysregulation is implicated in pituitary neoplasia as the cause of silencing of several tumor suppressor genes. However, the upstream mediators of such events remain unknown. We examined the three members of the DNA methyltransferase (DNMT) enzyme family in normal and neoplastic human and mouse pituitary cells. This study was performed at a university-affiliated cancer research institute. Gene expression, promoter DNA methylation, histone modifications, and cell proliferation were determined. In contrast to DNMT1 and DNMT3a, DNMT3b was expressed at relatively higher levels in neoplastic pituitary cells. However, examination of the human DNMT3b 5' region showed uniformly low DNA methylation levels with little difference between normal and tumor samples. Through pharmacological methylation inhibition or histone deacetylation inhibition, we identified that DNMT3b gene expression is subject to histone modifications. Down-regulation of DNMT3b resulted in induction of retinoblastoma, p21, and p27, and reduction in cell proliferation. These targeted effects were associated with enhanced histone 3 acetylation and diminished histone methylation. Our findings identify DNMT3b as a putative mediator of epigenetic control through histone modifications of gene expression in pituitary cells.

Regulation of the CLDN3 gene in ovarian cancer cells

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.

SOMATIC CELL NUCLEAR TRANSFER USING DONOR CELLS SELECTED FOR DNA METHYLTRANSFERASE 1 (DNMT1) EXPRESSION

Bovine nuclear transfer (NT) embryos and fetuses often display abnormalities that are not observed in their in vivo produced counterparts, and are attributed to large offspring syndrome. Gene expression is altered in NT embryos and is thought to be due to inadequate reprogramming of the somatic cell nucleus. It is known that the embryonic genome must be rapidly demethylated, but often does not occur normally in NT embryos. This may be due to the introduction of Dnmt1s into the NT embryo, the enzyme responsible for maintaining DNA methylation (DM) in somatic cells, but which is never found in early embryos. Chemicals, such as 5azaC, have been used to artificially reduce DM, but have proven problematic in NT. We previously showed that culture without chemicals can be used, with cell type and time in culture affecting expression of Dnmt1 in donor cells. Here we tested the effects of using donor cells selected for Dnmt1 expression (low vs. high) as a means to reduce DM in NT embryos. Cell lines were grown to passage 4, 8 and 12, diluted out and frozen in individual use aliquots for later use. An aliquot of each cell line was taken for RNA isolation, and Dnmt1 expression analysis using Real-time RTPCR. Two cell lines with the same genotype, but at different passages were selected as having higher (HD) and lower Dnmt1 (LD) expression for use in NT experiments. Cumulus oocyte complexes were retrieved by slashing 2–8mm follicles on ovaries obtained from the abattoir, and were matured for 17–19h. Metaphase II oocytes (n=874) were selected for polar body extrusion and were enucleated for use in NT. Cells were transferred from either HD or LD groups, and fused. Fused couplets were activated with ionomycin and DMAP using standard protocols, and cultured within treatment in KSOM to d7 (activation=0). Global DM was evaluated in NT (LD and HD) and in vitro produced (IVP) control embryos (2-cell, 8-cell and blastocyst stages, 10/treatment and stage) by immunocytochemistry using 5MeC antibody (Calbiochem) and a FITC-conjugated secondary antibody (Santa Cruz). IgG and PBS served as negative controls to show antibody specificity. Pixel data were collected using AxioVision software (Zeiss) and were analyzed by LSMeans ANOVA using SAS, and significance was deemed p<0.05. There was significantly less DM in 2-cell IVP when compared to either LD or HD NT embryos (p<0.05). By the 8-cell stage, NT produced with LD had lower DM than 8- cell HD NT (p<0.05), but was still higher than 8-cell IVP (p<0.05). NT blastocysts produced with LD had DM similar to IVP (p>0.05), and both were lower than HD NT blastocysts (p<0.05). These data indicate that cell lines selected for reduced Dnmt1 expression can reduce global DM in the developing NT embryo. However, DM was not reduced to the levels found in IVP embryos at either the 2- or the 8-cell stages. Future efforts will need to determine if these reductions are adequate and allow for normal development in vivo, or if additional efforts will need to be made to further mimic normal embryonic reprogramming events. This project was supported by National Research Initiative Competitive Grant no. 2006- 35203-16620 from the USDA Cooperative State Research, Education, and Extension Service. (platform) (platform)

Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL): molecular cloning, expression distribution, splice variants and DNA methylation profile

BackgroundThe genetic basis of telomere length heterogeneity among mammalian species is still not well understood. Recently, a gene named regulator of telomere length elongation helicase (RTEL) was identified and predicted to be an essential participant in species-specific telomere length regulation in two murine species. To obtain broader insights into its structure and biological functions and to ascertain whether RTEL is also a candidate gene in the regulation of telomere length diversity in other mammalian species, data from other mammals may be helpful.ResultsHere we report the cDNA cloning, genomic structure, chromosomal location, alternative splicing pattern, expression distribution and DNA methylation profile of the bovine homolog of RTEL. The longest transcript of bovine RTEL is 4440 nt, encompassing 24.8 kb of genomic sequence that was mapped to chromosome 13q2.2. It encodes a conserved helicase-like protein containing seven characterized helicase motifs in the first 750 aa and a PIP box in the C-terminus. Four splice variants were identified within the transcripts in both the coding and 5'-untranslated regions; Western blot revealed that the most abundant splice variant SV-1 was translated to a truncated isoform of RTEL. The different 5'UTRs imply alternative transcription start sites in the promoter; Bovine RTEL was transcribed at the blastocyst stage, and expression levels were highest in adult testis, liver and ovary. DNA methylation analysis of tissues that differed significantly in expression level indicated that relatively low DNA methylation is associated with higher expression.ConclusionIn this study, we have identified and characterized a bovine RTEL homolog and obtained basic information about it, including gene structure, expression distribution, splice variants and profile of DNA methylation around two putative transcription start sites. These data may be helpful for further comparative and functional analysis of RTEL in mammals.

Detailed DNA methylation profiles of the E-cadherin promoter in the NCI-60 cancer cells

E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad's distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad's promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of approximately 20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy.