DNA Methylation In Gene Regulation Research Articles

DNMT3A moderates cognitive decline in subjects with mild cognitive impairment: replicated evidence from two mild cognitive impairment cohorts.

Epigenetic dysregulation has been associated with cognitive decline and Alzheimer's disease. The present study investigated associations between common SNPs in genes regulating DNA methylation and age-related changes in cognitive decline in two independent prospective cohorts of patients suffering from mild cognitive impairment. An association between the rs1187120 SNP in DNMT3A and annual decline in cognitive functioning was discovered and replicated, suggesting that DNMT3A moderates cognitive decline in subjects with mild cognitive impairment.

The expression patterns of DNA methylation reprogramming related genes are associated with the developmental competence of cloned embryos after zygotic genome activation in pigs

DNA methylation reprogramming, regulated by DNA methylation and demethylation related genes, is essential for early embryo development; however, it is incomplete in cloned embryos, leading to poor cloning efficiency. Previous studies have shown that DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), could enhance the development of cloned embryos, thus, the genes regulating DNA methylation reprogramming should appropriately express in these embryos. To examine whether there is a correlation between embryo development and the expression patterns of DNA methylation reprogramming related genes, we investigated the developmental progress and transcription levels of candidate genes containing DNA methyltransferases (Dnmt1 and Dnmt3a), ten eleven translocation (Tet) dioxygenases (Tet1, Tet2 and Tet3) and base excision repair related genes including activation induced deamination (Aid), thymine DNA glycosylase (Tdg) and AP endonuclease 1 (Apex1) in porcine early embryos. In this study, our results demonstrated that compared with in vitro fertilized embryos, delayed and reduced development and downregulated transcripts of DNA methylation reprogramming related genes after the 4-cell stage were observed in cloned embryos, showing the significantly (P < 0.05) lower proportions of embryos at the 8-cell, morula and blastocyst stages (19.69% vs 32.64% at 72 h, 16.67% vs 25.49% at 120 h and 19.82% vs 26.29% at 156 h, respectively) and transcription levels of Dnmt3a, Tet1, Tet2, Tet3, Aid, Tdg and Apex1. When cloned embryos were treated with 5-aza-dC, the developmental progress and transcription levels of DNA methylation reprogramming related genes were improved, more similar to those detected in fertilized counterparts. Furthermore, we found that the transcripts of zygotic genome activation and blastocyst quality related genes were also effectively promoted in porcine cloned embryos after 5-aza-dC treatment. In conclusion, our results demonstrated that the disturbed transcripts of DNA methylation reprogramming related genes were observed in porcine cloned embryos, while the enhanced development of porcine cloned embryos induced by 5-aza-dC was accompanied with the improved expression of DNA methylation reprogramming related genes after the 4-cell stage, providing a positive correlation between the expression patterns of DNA methylation reprogramming related genes and the developmental competence of porcine cloned embryos after zygotic genome activation.

Shall we crosstalk? – The relationship between DNA methylation and histone H3 lysine 27 trimethylation (comment on DOI 10.1002/bies.201300130)

Cancer cells usurp silencing mechanisms to extinguish functional pathways by epigenetic processes. There are two important epigenetic silencing mechanisms, polycomb repressive complex 2 (PRC2)-based histone H3 lysine 27 trimethylation (H3K27me3), and DNA methylation, which are associated with a variety of cellular functions. Studies have shown that these two epigenetic marks seem to be dependent on one another, although the precise mechanistic link between them remains to be fully characterized 1. This crosstalk between DNA methylation and PRC2-H3K27me3 has implications for understanding stem cell differentiation, somatic cell reprogramming, and tumorigenesis as well: dysregulation of these two epigenetic mechanisms has frequently been observed in many types of cancers. It is well appreciated that stem cell PRC2 targets – which are generally associated with embryonic development – are more likely to have cancer-specific promoter DNA methylation than non-targets 1. During tumorigenesis, these vulnerable genes undergo replacement of dynamic H3K27me3 modification with static DNA methylation. By contrast, DNA methylation in CpG sequences interferes with PRC2 recruitment 2. H3K27me3 and DNA methylation are mutually exclusive in CpG islands (CGIs), as ascertained via precise genome-wide analyses 3. These data lead to an idea that during tumorigenesis, aberrant DNA methylation patterns affect the affinity of PRC2 binding and drive redistribution of the PRC2 pattern. Recently, Meehan and coworkers 4, 5 reported that DNA methylation could be a mark for appropriate polycomb-mediated gene repression by constraining PRC2 targeting. Since dense DNA methylation in CGIs interferes with the binding of polycomb protein components, altered DNA methylation patterns in cancer cells influence the PRC2 binding landscape. Meehan et al. uncovered an unappreciated plausible role of DNA methylation in gene regulation. Consistently, studies have shown that artificial removal of DNA methylation induces the accumulation of PRC2 in those genomic loci 3. Fascinatingly, data from these studies evoke another plausible feature of PRC2. PRC2 is mostly enriched in CGIs, although precise mechanisms of its recruitment are not fully understood so far. It is possible that PRC2 stochastically and preferentially modifies DNA at certain loci with low transcription activity but lack of DNA methylation. Since DNA methylated CGIs generally confer low transcription activity, removal of DNA methylation in the CGIs may provide preferential targets for PRC2 binding. Consequently, high levels of DNA methylation and H3K27me3 are rarely found at the same silenced loci, as was reported in whole epigenome analyses. Hence, a direct mechanistic link between PRC2 and DNA methylation may not be required in this regard. An aberrant epigenome is a hallmark of cancer. Understanding such complex epigenetic abnormalities may open the door for developing better therapeutic strategies for cancer treatment.

Genetic and Epigenetic Regulation of Human lincRNA Gene Expression

Large intergenic noncoding RNAs (lincRNAs) are still poorly functionally characterized. We analyzed the genetic and epigenetic regulation of human lincRNA expression in the GenCord collection by using three cell types from 195 unrelated European individuals. We detected a considerable number of cis expression quantitative trait loci (cis-eQTLs) and demonstrated that the genetic regulation of lincRNA expression is independent of the regulation of neighboring protein-coding genes. lincRNAs have relatively more cis-eQTLs than do equally expressed protein-coding genes with the same exon number. lincRNA cis-eQTLs are located closer to transcription startsites (TSSs) and their effect sizes are higher than cis-eQTLs found for protein-coding genes, suggesting that lincRNA expression levelsare less constrained than that of protein-coding genes. Additionally, lincRNA cis-eQTLs can influence the expression level of nearby protein-coding genes and thus could be considered as QTLs for enhancer activity. Enrichment of expressed lincRNA promoters in enhancer marks provides an additional argument for the involvement of lincRNAs in the regulation of transcription in cis. By investigating the epigenetic regulation of lincRNAs, we observed both positive and negative correlations between DNA methylation and gene expression (expression quantitative trait methylation [eQTMs]), as expected, and found that the landscapes of passive and active roles ofDNA methylation in gene regulation are similar to protein-coding genes. However, lincRNA eQTMs are located closer to TSSs thanareprotein-coding gene eQTMs. These similarities and differences in genetic and epigenetic regulation between lincRNAs and protein-coding genes contribute to the elucidation of potential functions of lincRNAs.

Mutations Of Genes Regulating DNA Methylation Are Common In MLL Partial Tandem Duplication AML and DNMT3A Mutations Are Associated With Adverse Outcome

Abstract Background and purpose Abnormalities of genes regulating DNA methylation have been described in acute myeloid leukemia (AML). MLL protein is a transcriptional regulator and governs proper hematopoiesis through its histone methyltransferase activity. AML with partial tandem duplication of MLL (MLL-PTD) was associated with an unfavorable prognosis. The cooperating roles of MLL-PTD with other mutated genes regulating DNA methylation have not been comprehensively studied in AML. We aimed to determine the prevalence and clinical impact of mutations of DNA methylation regulators in AML with MLL-PTD. Materials and methods Bone marrow samples from 98 AML patients with MLL-PTD were analyzed for gene mutations of TET2, DNMT3A, IDH1 and IDH2. MLL-PTD was screened by RT-PCR and confirmed by real-time quantitative PCR assays. The mutational analysis was performed with PCR assays followed by direct sequencing for TET2 (whole coding exons 3–11) and IDH1/2 (hotspots exon 4). For the detection of DNMT3A mutations, the PCR products amplified for entire coding exons 2 to 23 were first screened with denaturing high-performance liquid chromatography followed by direct sequencing for the abnormal profiles. Results The frequency of TET2, IDH1, IDH2 and DNMT3A mutations in AML patients with MLL-PTD was 17.0% (16/94), 10.2% (10/98), 18.4% (18/98), and 31.6% (31/98), respectively. Taken together, 61.1% of patients with MLL-PTD had at least one mutated gene of DNA methylation regulators. TET2, IDH1 and IDH2 mutations were mutually exclusive with each other whereas DNMT3A mutations frequently co-existed with other DNA methylation modifiers:TET2 (n=8), IDH1 (n=5) and IDH2 (n=4). No differences were observed between the mutation status of the DNA methylation modifiers and clinico-hematologic features of patients with MLL-PTD except that TET2 (P=0.012) and DNMT3A (P=0.024) mutations were associated with older age. Of the 55 MLL-PTD patients who received standard chemotherapy, IDH2 mutation was associated with a lower complete remission rate (25.0% vs 67.8%, P=0.018), while DNMT3A mutations conferred an inferior event-free survival (0.0 vs 6.8 months, P=0.027) and overall survival (6.0 vs 11.5 months, P=0.032). In multivariate analysis, older age (P=0.008) and DNMT3A mutations (P=0.049) were independent adverse factors for overall survival. The crosstalk between MLL-PTD and genes involving DNA methylation in the leukemogenesis of AML warrants further investigation. Conclusions Gene mutations involving DNA methylation frequently co-existed in AML patients with MLL-PTD, especially DNMT3A mutations which conferred a poor outcome. Our study demonstrated the importance of genetic alterations involving DNA methylation in the pathogenesis of MLL-PTD AML and provided potential epigenetic-targeted therapy. Grant support The work was supported by NHRI-EX93-9011SL, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3 and MMH-E-101-09. Disclosures: No relevant conflicts of interest to declare.

Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues

Mammalian development requires cytosine methylation, a heritable epigenetic mark of cellular memory believed to maintain a cell’s unique gene expression pattern. However, it remains unclear how dynamic DNA methylation relates to cell-type specific gene expression and animal development. Here, by mapping base resolution methylomes in 17 adult mouse tissues at shallow coverage, we identify 302,864 tissue-specific differentially methylated regions (tsDMRs) and estimate that >6.7% of the mouse genome is variably methylated. Supporting a prominent role for DNA methylation in gene regulation, most tsDMRs occur at distal cis-regulatory elements. Surprisingly, some tsDMRs mark enhancers dormant in adult tissues but active in embryonic development. These “vestigial” enhancers are hypomethylated and lack active histone modifications in adult tissue, but nevertheless exhibit activity during embryonic development. Our results provide new insights into the role of DNA methylation at tissue-specific enhancers and suggest that epigenetic memory of embryonic development may be retained in adult tissues.

Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes.

BackgroundDNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear.ResultsBy genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression.ConclusionsAn intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease.

Post-implantation mortality of in vitro produced embryos is associated with DNA methyltransferase 1 dysfunction in sheep placenta

Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)? DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss. DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos. Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation. Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured. Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the developing embryos. The studied genes represent only a small fraction of genes regulating DNA methylation. Further studies are needed to evaluate changes in the expression and methylation status of other genes that may lead to developmental arrest of IVP embryos. As this is the only study evaluating the functionality of DNMT1 machinery in placentae from ART embryos, studies on other species are needed to confirm if our observation may be applicable to all mammalian embryos produced in vitro. The knowledge about compromised activity of DNMT1 in placentae obtained from IVP embryos should stimulate detailed studies on the metabolic requirements of oocytes and embryos in order to adequately enrich the culture media.

Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors

DNA methylation plays an essential role in maintenance of cellular function. A growing number of human diseases have been found to be associated with aberrant DNA methylation, especially cancer. However, current technologies used in DNA methylation detection are complicated and time consuming. A promotor of the Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was used as the detection target DNA sequence. The double recognition mechanism was realized with oligonucleotide probe hybridization and specific protein binding. First, complementary target DNA was captured by the probe immobilized onto a surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG binding domain (MBD) protein was passed over the surface to recognize and bind to methylated CpG sites. Binding resulted in an increase in the refractive index, and a detectable optical signal was generated. Five picomoles of methylated APC promotor DNA could be easily detected with this method. The entire detection could be completed within 1 h. This work represents the first SPR based biosensor technology, which achieves simple and specific DNA methylation detection and avoids complicated bisulfite treatment and methylation-sensitive restriction digestion. It will improve our ability to detect DNA methylation specifically and rapidly, and promote our understanding of the role of DNA methylation in gene regulation and diseases.

Role of Dnmt3a in cytokine gene regulation during Th1/Th2 differentiation (88.23)

Abstract Helper T cell polarization is accompanied by DNA methylation-mediated silencing of specific genes. Dnmt3a/3b, de novo DNA methyltransferases, have been shown to associate with cytokine gene promoters during Th1/Th2 differentiation. To elucidate the functional significance of Dnmt3a/3b-mediated DNA methylation in gene regulation of CD4 cell development, we generated conditional knockout mice by crossing Dnmt3aloxP/loxP and Dnmt3bloxP/loxP mice (a gift from Dr. En Li) with CD4-CRE mice. The conditional knockout mice exhibited normal lymphoid organ cellularity. Analysis of gene expression revealed that Dnmt3a-deficient Th1/Th2 cells secreted higher levels of effector cytokines in comparison with littermate controls, whereas absence of Dnmt3b did not show significant change, compared to controls. Using a DNA methylation-sensitive restriction method, we found that although absence of Dnmt3a did not alter global methylation levels of CD4 cells, Dnmt3a deficiency led to reduced levels of DNA methylation at specific gene loci, indicating that Dnmt3a participated in the establishment of local DNA methylation. Meanwhile, Chromatin IP data showed histone modification alterations in the absence of Dnmt3a. Taken together, the results indicated that Dnmt3a had an inhibitory effect on gene expression in Th1/Th2 development.

Integrating Epigenomics into Pharmacogenomic Studies.

The goal of personalized medicine is to recommend drug treatment based on an individual's genetic makeup. Pharmacogenomic studies utilize two main approaches: candidate gene and whole-genome. Both approaches analyze genetic variants such as single nucleotide polymorphisms (SNPs) to identify associations with drug response. In addition to DNA sequence variations, non-genetic but heritable epigenetic systems have also been implicated in regulating gene expression that could influence drug response. The International HapMap Project lymphoblastoid cell lines (LCLs) have been used to study genetic determinants responsible for expression variation and drug response. Recent studies have demonstrated that common genetic variants, including both SNPs and copy number variants (CNVs) account for a substantial fraction of natural variation in gene expression. Given the critical role played by DNA methylation in gene regulation and the fact that DNA methylation is currently the most studied epigenetic system, we suggest that profiling the variation in DNA methylation in the HapMap samples will provide new insights into the regulation of gene expression as well as the mechanisms of individual drug response at a new level of complexity. Epigenomics will substantially add to our knowledge of how genetics explains gene expression and pharmacogenomics.

Cyclical DNA methylation of a transcriptionally active promoter

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

Functional Reassembly of Split Enzymes On‐Site: A Novel Approach for Highly Sequence‐Specific Targeted DNA Methylation

In mammalian genomes, a significant fraction of the cytosine residues is methylated at the 5-position (5-methylcytosine), and this modified nucleobase is found in 5’-CG-3’ sequences (CpG sites). The methylation pattern of the genome changes during ontogenesis, depends on the tissue and can substantially differ in several diseases, notably cancer. We are only at the beginning of understanding the biological role of DNA methylation in higher organisms, but the emerging view is that methylation of the promoter region of a substantial fraction of genes leads to transcriptional inactivation (gene silencing). Recognition of the importance of DNA methylation in gene regulation raised the possibility of silencing selected genes by exogenous, targeted methylation of their promoters. Directing DNA methylation to predetermined sites, besides being a promising research tool for silencing genes of interest and studying DNA methylation in higher eukaryotes, could lead to therapeutic applications in diseases characterised by aberrant expression of one or a small number of genes. A potential advantage of DNA methylation-mediated gene silencing is that the de novo established methylation pattern is stably propagated by maintenance methylation through cycles of semiconservative replication. The silencing mechanism is likely to involve other epigenetic factors (e.g. , histone methyltransferases, histone deacetylases) recruited by the DNA methylation marks. DNA methylation is catalysed by DNA methyltransferases (MTases), which transfer the activated methyl group from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet or SAM) to their target nucleobase within short DNA recognition sequences ranging from two to eight base pairs (bps). Directing DNA MTases to a preselected recognition sequence (targeted DNA methylation) was pioneered by Xu and Bestor, who genetically fused the bacterial DNA (cytosine-C5) MTase M.SssI (recognition sequence 5’CG-3’) to a zinc finger protein (ZFP) that recognises a 9 bp DNA sequence with high specificity and serves as targeting domain. The idea was that the chimeric protein would bind to the DNA sequence specific for the targeting domain, and the DNA MTase would selectively methylate CpG sequences located close to the binding site of the fusion partner. Indeed, preferential methylation of a CpG sequence located in the vicinity of the ZFP binding site was observed in in vitro experiments. Later research demonstrated that this approach can, in principle, also be applied in vivo for targeting chromosomal as well as mitochondrial DNA. In the most extensive study so far, the catalytic domains of the murine DNA MTases Dnmt3a and Dnmt3b were fused to sequence-specific DNA-binding proteins. In transient cotransfection experiments on human cells, dense methylation of the targeted promoter regions and silencing of the targeted genes was demonstrated. In almost all such studies, ZFPs were used as targeting devices because customised ZFPs can now be engineered to bind with high affinity and sequence specificity to almost any DNA sequence. A key issue of targeted DNA methylation is specificity, that is, the difference between levels of exogenous methylation at the targeted site and nontargeted sites. Analysis of the methylation status of nontargeted sites revealed either methylation far from the target site, 8] or extensive methylation of regions flanking the binding site of the targeting domain. Although efficient gene silencing might require methylation of many CpG sites in a promoter, and thus the latter phenomenon could be an advantage in many cases, high-resolution analysis of the effect of DNA methylation would require a method suitable for the methylation of single CpG sites. Methylation of nontargeted sites is not surprising owing to the inherent affinity of DNA MTases for their recognition sequences (Scheme 1A). Nontargeted methylation limits the use of simple DNA MTase fusions as a research tool and would be a serious obstacle to therapeutic application. Therefore, novel strategies that allow highly sequence-specific targeted DNA methylation are of prime importance. One approach to improving targeting specificity employs DNA MTase variants with reduced DNA-binding affinity. Here, DNA binding of the mutant DNA MTase-ZFP fusions is dominated by the targeting ZFP domain’s improving the specificity. A recent paper by Nomura and Barbas describes another approach that appears to be a significant step towards the goal of methylating single CpG sites within whole genomes. The authors capitalised on previous observations of protein fragment complementa[a] Prof. Dr. E. Weinhold Institut f r Organische Chemie RWTH Aachen University Landoltweg 1, 52056 Aachen (Germany) Fax: (+49)241-80-92528 E-mail : elmar.weinhold@oc.rwth-aachen.de [b] Prof. A. Kiss Institute of Biochemistry Biological Research Centre of the Hungarian Academy of Sciences Temesv7ri krt. 62, 6726 Szeged (Hungary) Fax: (+36)62-433-506 E-mail : kissa@brc.hu

Role of histone and DNA methylation in gene regulation

Transcription is known to be regulated by given chromatin states, distinguished as transcriptionally active euchromatin and silent heterochromatin. In plants, silencing in heterochromatin is associated with hypermethylation of DNA and specific covalent modifications of histone H3. Several lines of evidence have suggested that maintenance of DNA methylation patterns at CG sequences is responsible for the formation of stable and thus heritable activity states termed epialleles. By contrast, histone modification and DNA methylation outside CGs confer the flexibility of transcriptional regulation necessary for plant development and adaptive responses to the environment. Recent studies have refined our understanding of the biological significance of and the molecular mechanisms involved in the interplay between DNA and histone H3 methylation.

DNA Methylation-dependent Epigenetic Regulation of Dimethylarginine Dimethylaminohydrolase 2 Gene in Trophoblast Cell Lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6.

We investigated the role of DNA methylation in gene regulation of the rat T-cell differentiation marker RT6. Analysis of the methylation status of various tissues revealed that the RT6 promoter was hypomethylated in RT6-expressing tissues, and methylated in nonexpressing ones. Remarkably, among RT6-nonexpressing tissues, the extent of methylated regions varied greatly between lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb region surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subpopulation of T cells. We now show that the RT6 alleles in these cells differed in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5-azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cells, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the control of monoallelic and tissue-specific RT6 expression.

Inhibition of internode growth due to mechanical stress in Bryonia dioica: relationship between changes in DNA methylation and ethylene metabolism

Changes in the level of cytosine methylation were assessed by HPLC in mechanically stressed Bryonia dioica. Rubbing young internodes. which results in growth inhibition and ethylene production, induced a rapid and transient decrease of cytosine methylation in DNA. The level of cytosine methylation was about 25% in young internodes and dropped to nearly 0% in less than 1 h before increasing to the normal level within 3 h. A decreasing gradient of DNA methylation occurred naturally along the plant, from the apex to the base. The pool of S‐adenosylmethionine was also measured in order to establish a possible relationship between ethylene metabolism and DNA methylation. The role of DNA methylation in gene regulation is discussed and different mechanisms for methylation are considered.

Extensive Changes in DNA Methylation Patterns Accompany Activation of a Silent T-DNA ipt Gene in Agrobacterium tumefaciens-Transformed Plant Cells

We crossed a male-sterile, Agrobacterium-transformed Nicotiana tabacum plant that contains a silent, hypermethylated T-DNA ipt oncogene with a normal tobacco plant and found that the methylated state of the ipt gene was stably inherited through meiosis in the offspring. However, when tissues of these plants were placed in cell culture, the ipt gene was spontaneously reactivated in a very small fraction of the cells; if 5-azacytidine was added to the culture medium, ipt gene reactivation occurred at high frequency. We analyzed the pattern of DNA methylation in a region spanning the ipt gene in a line that does not express the ipt gene, in five derivatives of this line that reexpressed the ipt gene either spontaneously or after 5-azacytidine treatment, and in tissues of a sibling of this line that reexpressed ipt spontaneously. We found that the ipt locus was highly methylated in the unexpressed state but that spontaneous or 5-azacytidine-induced reexpression always resulted in extensive demethylation of a region including 5' upstream, coding, and 3' downstream regions of the ipt gene. The role of DNA methylation in gene regulation in this system is discussed.

Extensive changes in DNA methylation patterns accompany activation of a silent T-DNA ipt gene in Agrobacterium tumefaciens-transformed plant cells.

We crossed a male-sterile, Agrobacterium-transformed Nicotiana tabacum plant that contains a silent, hypermethylated T-DNA ipt oncogene with a normal tobacco plant and found that the methylated state of the ipt gene was stably inherited through meiosis in the offspring. However, when tissues of these plants were placed in cell culture, the ipt gene was spontaneously reactivated in a very small fraction of the cells; if 5-azacytidine was added to the culture medium, ipt gene reactivation occurred at high frequency. We analyzed the pattern of DNA methylation in a region spanning the ipt gene in a line that does not express the ipt gene, in five derivatives of this line that reexpressed the ipt gene either spontaneously or after 5-azacytidine treatment, and in tissues of a sibling of this line that reexpressed ipt spontaneously. We found that the ipt locus was highly methylated in the unexpressed state but that spontaneous or 5-azacytidine-induced reexpression always resulted in extensive demethylation of a region including 5' upstream, coding, and 3' downstream regions of the ipt gene. The role of DNA methylation in gene regulation in this system is discussed.