Acquisition Of DNA Methylation Research Articles

Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline

How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline? Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells. The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L(+/-) males. This is a cross-sectional study of DNA methylation in Dnmt3L(+/+) versus Dnmt3L(+/-) male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2-20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L(+/+) and Dnmt3L(+/-) males following ≈ 2 months in culture. DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning. At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L(+/-) SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6-28. While the methylation of most loci was stable in culture over time, differences at ≈ 1% of sites were found between Dnmt3L(+/-) and Dnmt3L(+/+) cultures. Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment. The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a 'window' of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.

SINE Retrotransposons Cause Epigenetic Reprogramming of Adjacent Gene Promoters

Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2, and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.

DNA Methylation Defects in Prenatal and Postnatal Germ Cells of Dnmt3L Haploinsufficient Male Mice.

The enzyme DNA methyltransferase 3-like (DNMT3L) is essential for the acquisition of DNA methylation patterns in germ cells as well as normal germ cell development and has been reported as the first paternal effect gene in mammals. Although Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction, postmeiotic gene expression and sire an increased number of pups with XO monosomy. To examine the postulated epigenetic basis of the paternal effects, we examined DNA methylation acquisition at intergenic (non-CpG island, non-repeat) sites along chromosome 9 in male germ cells isolated during the prenatal and postnatal periods. At 16.5 dpc (days post coitum), 42% of intergenic loci examined were hypomethylated, by 20–30%, in Dnmt3L heterozygotes as compared to Dnmt3L wildtype mice. By day 1 after birth DNA methylation differences in spermatogonia between the two genotypes had resolved for 67% of the sites; in contrast, 33% of sites showed residual hypomethylation, in the range of 10%, associated with Dnmt3L haploinsufficiency. By postnatal days 4 and 6, germ cell DNA methylation levels were similar in the heterozygous and wildtype mice and remained similar later in spermatogenesis in spermatozoa. The findings suggest that germ cell DNA methylation differences between the genotypes resolve shortly after birth before the spermatogonia begin to divide. To confirm and extend these results, higher resolution studies using Reduced Representation Bisulphite Sequencing (RRBS) are being carried out to examine methylation differences at 3-5 million CpG sites across the genome. The DNA methylation defects described here in prenatal and early postnatal male germ cells of Dnmt3L heterozygous mice are the earliest epigenetic perturbation yet identified for a mammalian paternal effect gene and may influence downstream epigenetic and genetic events that lead to the previously observed abnormalities during spermatogenesis and paternal effects in the offspring. (Supported by CIHR).

Comparative Analysis of DNA Methylation Acquisition at the Imprinted Gtl2/Dlk1 Locus During Mouse Embryonic Development.

Comparative Analysis of DNA Methylation Acquisition at the Imprinted Gtl2/Dlk1 Locus During Mouse Embryonic Development.

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes.

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

Effect of postovulatory oocyte aging on DNA methylation imprinting acquisition in offspring oocytes

To investigate whether postovulatory aging of oocytes in the mother affects DNA methylation acquisition of imprinted genes in oocytes from the offspring. Randomized research experimental study. Academic basic research laboratory. Mice. Fresh oocytes and aged oocytes from mothers were artificially inseminated, and oocytes were collected from the resultant offspring. Methylation status was evaluated at differentially methylated regions (DMRs) in oocytes of maternally imprinted genes Peg3, Snrpn, and Peg1 and paternally imprinted gene H19. Our results showed that methylation patterns at DMRs of Peg3, Snrpn, Peg1, and H19 in oocytes from aged-oocyte offspring were mainly normal, with only a small number of oocytes showing aberrant methylation in the DMR of Peg3. Postovulatory oocyte aging causes a decline in reproductive outcomes but does not evidently lead to defects in DNA methylation imprinting acquisition in the oocytes from viable offspring.

Critical Period of Nonpromoter DNA Methylation Acquisition during Prenatal Male Germ Cell Development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.

Embryonic imprinting perturbations do not originate from superovulation-induced defects in DNA methylation acquisition

To investigate whether superovulation disrupts maternal imprint acquisition in oocytes. Animal model. Academic institute. Spontaneously ovulated and superovulated mice. Low and high hormone dosage treatments were administered to females, and ovulated metaphase II oocytes were collected. Imprinted DNA methylation was analyzed at Snrpn, Kcnq1ot1, Peg3, and H19 in individual oocytes. Examination of 125 individual oocytes derived from females subjected to low and high hormone treatments revealed normal imprinted methylation patterns that were comparable to oocytes derived from spontaneously ovulated females. Maternal imprint acquisition was not affected by superovulation. Given its aberrant effects during preimplantation development, superovulation must instead disrupt maternal-effect gene products that are required after fertilization for imprint maintenance. These results eliminate imprint acquisition per se as the initial stage of imprint loss and point to the importance of analyses on early embryos after procedures involving oocyte manipulation.

DNA methylation: a new twist in the tail

DNA methylation is the epigenetic mark with the longest history and that we probably understand best, yet we still have no adequate account for why specific DNA sequences are selected to become methylated. Gene-specific DNA methylation is fundamental to processes such as developmental silencing of genes, classical epigenetic phenomena such as genomic imprinting, and occurs pathologically in the silencing of tumor suppressor genes in cancer. Fully understanding the mechanisms of methylation is thus of huge importance. In mammals, the acquisition of DNA methylation is determined by one of two de novo DNA methyltransferase enzymes, Dnmt3a and Dnmt3b. These activities are assisted by the related, but catalytically inactive protein Dnmt3L. Dnmt3a and Dnmt3b have similar structures, comprising a PWWP domain, PHD-like or ADD domain and a carboxy-terminal catalytic domain 1. The PWWP domains are required for binding of Dnmt3a and Dnmt3b to chromatin in vivo, and the PHD domain is thought to be dispensable for this 2. Dnmt3L, which is a truncated protein lacking the PWWP and a functional catalytic domain, forms a heterotetramer with Dnmt3a or Dnmt3b 3 and is described as stimulating the activity of its de novo partners, or guiding the recognition of DNA targets with a particular periodicity of CpG sites 3, or attenuating the inherent sequence specificities of Dnmt3a or Dnmt3b so that they methylate targets more homogenously 4.

Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis.

The gene for the atypical Notch ligand Delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms functioning in multiple developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally-inherited chromosome1,2. Here we show that mice deficient in Dlk1 exhibit defects in postnatal neurogenesis within the subventricular zone (SVZ), a developmental continuum resulting in depletion of mature neurons in the olfactory bulb. We show that DLK1 is a factor secreted by niche-astrocytes, while its membrane-bound isoform is present in neural stem cells (NSCs) being required for the inductive effect of secreted DLK1 on self-renewal. Surprisingly, we find a requirement for Dlk1 expressed from both maternal and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ line-derived imprinting control region (IG-DMR). The results emphasize molecular relationships between NSCs and niche-astrocytes identifying a signalling system coded by a single gene functioning co-ordinately in both cell types. The modulation of genomic imprinting in a stem cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche raising wider questions about the adaptability, function, and evolution of imprinting within specific developmental contexts.

Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development

The Dlk1- Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1- Dio3 cluster.

Abstract 2791: SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters

Abstract Retrotransposons of SINE and LINE classes have been extremely successful in colonizing mammalian genomes. Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The comparison of human and mouse genomes revealed a spatial concordance in positioning of SINE but not LINE repeats, indicating evolutionary pressure to maintain and/or exclude these repeats from orthologous regions. We hypothesized that this phenomenon is due to a negative influence of SINE repeats on gene expression and counter selection over evolution, resulting in exclusion of these repeats from vulnerable genomic environments. To test the capacity of SINE B1 elements in promoting gene silencing, we generated a system where the luciferase gene is under the control of three different mouse gene promoters (cdkn2a, p14arf and mlh1), and we inserted two and four copies of SINE B1 elements upstream to these promoters. Using this in vitro system, we found that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. Interestingly, SINE sequences themselves do not immediately acquired DNA methylation, but became marked by H3K9me2 and H3K27me3. Moreover, gain of DNA methylation depended on cryptic genomic methylation centers rather than spreading from SINE B1 repeats. Since mouse SINE B1 elements caused transcriptional repression, we sought to investigate whether human SINE Alu repeats could have the same effect. The human E-cadherin gene has three SINE Alus adjacent to the 3’ region of its CpG island, and in reporter assays the removal of these repeats resulted in higher, stable promoter activity. Genome-wide analysis of SINE repeats distribution in the human and mouse genomes showed that they are excluded from proximal promoters and their enrichment is directly correlated with the presence of USF1, USF2 and CTCF binding, proteins with insulator function. In conclusion, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is dependent on, and co-evolved with the presence of insulator elements. 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 2791. doi:10.1158/1538-7445.AM2011-2791

Size-Dependent Acquisition of Global DNA Methylation in Oocytes Is Altered by Hormonal Stimulation.

The normal progression of acquisition of global DNA methylation in oocytes of adult female mice is not known. Assisted reproductive technologies (ART) are commonly used for the treatment of infertility problems. Several studies have shown that ART procedures alter the expression of genes, causing delayed embryonic development, increased abnormal blastocyst formation, pronounced fetal growth retardation and is associated with increased incidence of loss-of-imprinting syndromes. A previous study from our laboratory showed that the maternal pronuclei of one-cell mouse embryos formed after superovulation (SO) had 50% less methylation when compared to those formed after natural ovulation (NO). We hypothesize that oocytes in ovaries of females undergoing a SO scheme will have different levels of global DNA methylation than oocytes from naturally cycling animals. First, we determined the progression of global DNA methylation during oocyte growth in naturally cycling animals and then compared this to the levels of DNA methylation in oocytes from females undergoing a SO scheme. A group of 14 cyclic females were selected at diestrus and divided into seven groups. Two females were sacrificed and collected ovaries at diestrus (0h). Six females underwent a SO scheme while the remaining six did not. In brief, SO females were injected with 5 IU of eCG followed 44 h later by 5 IU of hCG. Ovaries were collected at 24- and 44 h post-eCG and 10 h post-hCG (data not discussed). Ovaries from their NO counterparts were collected at the same time as the SO females. Ovarian sections were stained with an antibody that recognizes methylated cytosines (5MeC). All oocytes in a section were photographed and the largest cross section was used for analysis. Measurements related to oocyte growth were; 1) oocyte size in micrometers, 2) area of germinal vesicle, 3) size and type of enclosing follicle. Global DNA methylation was determined using the background corrected density macro of ImageJ. The 5MeC staining intensity level of the GV was normalized to the staining of the surrounding granulosa cells. For analyses, correlations were made between oocyte size and level of methylation and GV size and level of methylation. These numbers were then compared between similar size oocytes from SO and NO females. Results show that at diestrus the level of methylation increases in a linear manner form 10 to 80 µm diameter. The curve becomes exponential at 24 and 44 h in the NO group but is sigmoid in both SO groups. Analysis of full-grown oocytes (>70 µm) show that global methylation increases from diestrus (0 h) to 24 h and again to 44 h in the NO group but not in the SO groups. Interestingly, the levels of methylation remain low until ~ 40 µm diameter and start increasing exponentially in oocytes > 41 µm. This is coincident with the initiation of the formation of the second layer of granulosa cells. At 44 h, levels of DNA methylation were different in full-grown oocytes of NO vs. SO groups (P < 0.02). These full-grown oocytes are expected to ovulate upon the LH surge. Our results show that acquisition of DNA methylation continues in full grown oocytes until the presumed LH surge and we speculated that this is necessary to attain full developmental competence. On the other hand, SO full-grown oocytes do not attained full methylation before ovulation and this may in part be responsible for the lower developmental competence observed in embryos produced from SO oocytes. (poster)

Developmental changes in DNA methylation of pollen mother cells of David lily during meiotic prophase I

Epigenetic marks in the form of DNA methylation are involved in the development of germ cells and are important in the maintenance of fertility. However, the controlling system of the on-off switch for DNA methylation largely remains unclear. In this study, the extent of cytosine methylation during the meiotic prophase I in David lily is assessed using high pressure liquid chromatography to evaluate the DNA methylation rates. Comparing the degree of DNA methylation before, during, and after synizesis, both de novo methylation and demethylation occurred. Mainly the methylation level decreased by 21.3% (from 54.8 to 33.5%) during synizesis in the pollen mother cells. The developmental timing of genome-wide DNA methylation acquisition during pollen mother cell development is clarified in this paper. The relative amounts of 5-methyl-deoxycytidine of global methylation in leaf DNA in David lily were also higher than in other species reported.

Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping

The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This "omics" approach uncovered 11 large repressive zones (range, 0.35 approximately 5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.

Effects of Low Methyl Donor Levels in Culture Medium During Mouse Follicle Culture on Oocyte Imprinting Establishment1

Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. We previously demonstrated establishment of normal imprinting at four key imprinted genes in mouse metaphase II oocytes after in vitro follicle culture. Commercially available culture media feature a wide range of methyl donor levels. The aim of the present study was to examine the effect of low methyl donor (methionine, vitamin B(12), folic acid, choline, and vitamin B(6)) levels during follicle culture on acquisition of DNA methylation at imprinted genes in mouse oocytes. Follicle culture performed under low methyl donor levels led to decreased antral follicle development (mean [SD] antral follicle rate, 87.5% [12.6%] vs. 97.7% [4.3%] in control conditions; P < 0.05) and to a dramatic decrease in polar body (PB) oocyte rate (mean [SD] PB oocyte rate, 38.7% [25.5%] vs. 96.1% [7.1%]; P < 0.001). The methylation status of differentially methylated regions (DMRs) of four key imprinted genes was studied (by bisulphite sequencing) in normal-looking PB and germinal vesicle breakdown-arrested oocytes obtained from follicle culture under low methyl donor levels. DMRs of Snrpn, Igf2r, and H19 showed no alteration in DNA methylation, but at Mest DMR in PB oocytes, we found a significant reduction in DNA methylation compared to that in control follicle culture (DNA methylation, 89.9% and 98.2%, respectively; P = 0.0014). In conclusion, restriction of methyl donors during follicle culture led to a dramatic decrease in PB oocyte rate but induced no or only minor DNA methylation alterations at the studied regulatory sequences of key imprinted genes in oocytes.

Setting Epigenetic Programs in the Early Germline--Role of DNA Methyltransferases and Folate Pathway Enzymes.Jacquetta Trasler, M.D., Ph.D.

DNA methylation is an epigenetic modification of cytosine residues within CpG dinucleotides at about 20-30 million sites in the mammalian genome and is involved in gene regulation, X inactivation and genomic imprinting (variation in the expression of a subset of genes according to their maternal or paternal origin). Abnormalities in DNA methylation have been associated with perturbations in growth, placental function, neurobehavioral processes, and carcinogenesis. Methylation of DNA is catalyzed by DNA (cytosine-5)-methyltransferases (DNMTs) and is acquired in a sex-specific manner in the germline. In the male germline, the bulk of gamete methylation is initially acquired during germ cell development in the fetal testis; DNA methylation pattern acquisition is then completed postnatally during the transition from spermatogonia to pachytene spermatocytes. Data from mouse and human studies indicate that genome-wide methylation patterns are unique in sperm as compared to the patterns in somatic tissues. Cellular expression studies and gene-targeting experiments have identified critical roles for DNMT3a and DNMT3L in establishing methylation patterns on imprinted as well as single-copy, repeat and intergenic sequences during male germ cell development. Methyl donor (e.g. folate) supply to developing germ cells is essential during the prenatal and postnatal phases of DNA methylation acquisition. Deficiency in the key folate pathway enzyme methylenetetrahydrofolate reductase (MTHFR) is associated with DNA methylation defects in sperm and infertility that can be partially alleviated by provision of dietary methyl donors.

Methylation of Polycomb Target Genes in Intestinal Cancer Is Mediated by Inflammation

Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.