Specific epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT). All normal semen samples presented the expected high global methylation level for all CpGs analysed. In the teratozoospermia group, 11 of 19 patients presented a loss of methylation at variable CpG positions either in the IGF2 DMR2 or in both the IGF2 DMR2 and the 6th CTCF of the H19 DMR. In the OAT group, 16 of 22 patients presented a severe loss of methylation of the 6th CTCF, closely correlated with sperm concentration. The methylation state of DMR0 and of the 3rd CTCF was never affected by the pathological status of sperm samples. This study demonstrates that epigenetic perturbations of the 6th CTCF site of the H19 DMR might be a relevant biomarker for quantitative defects of spermatogenesis in humans. Moreover, we defined a methylation threshold sustaining the classification of patients in two groups, unmethylated and methylated. Using this new classification of patients, the observed intrinsic imprinting defects of spermatozoa appear not to impair significantly the outcome of assisted reproductive technologies.     

Changes in genomic imprinting and gene expression associated with transformation in a model of human osteosarcoma

Genomic imprinting, a heritable form of epigenetic information, is thought to play an important role in tumor progression. DNA methylation is a common mechanism of genomic imprinting. To evaluate the genome-wide effects of malignant transformation on osteosarcoma progression, we examined multiple biological properties, including DNA methylation, in human osteoblast hFOB1.19 cells (ATCC Catalog No. CRL-11372) transformed by treatment with carcinogenic agent N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG, 1.0 μg/ml) and carcinogenic promoting agent 12-O-tetradecanoyl phorbol-13-acetate (TPA, 200 ng/ml). We also examined global changes in expression of imprinted genes during transformation using microarray analysis. Ten imprinted genes, including H19, MKRN3, NDN, CDKN1C, PHLDA2, MEST, CD81, GRB10, SLC22A18, and SLC22A3 were aberrantly regulated in transformed cells, suggesting roles in tumorigenesis. Moreover, we analyzed the methylation state of the promoter regions of H19, PHLDA2, and SLC22A18 genes by bisulfite sequencing array and observed a correlation between upregulated expression of H19 and PHLDA2 genes and hypomethylation of their promoter regions, although this was not observed for SLC22A18. Our results suggest that changes in expression of imprinted genes caused by changes in methylation are involved, and are among the earliest events, in neoplastic progression.     

Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse

We have examined the relative methylation levels of several dispersed repeated and low-copy-number gene sequences during gametogenesis and early embryogenesis. Southern blot analyses revealed that L1, intercisternal A particle (IAP), and major urinary protein (MUP) sequences were undermethylated extensively at MspI sites in DNA from diplotene oocytes. In contrast, the same sequences were highly methylated in DNA from pachytene spermatocytes, round spermatids, and epididymal sperm. These results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavage-stage embryos and inner cell masses (ICM) were methylated at intermediate levels, consistent with transient maintenance of gametic methylation levels during early embryogenesis. Gametic differences in DNA methylation observed here indicate that methylation could provide a mechanism for imprinting maternal and paternal genomes resulting in differential regulation of parental genomes during early development.     

The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development

BACKGROUND: Germline-specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the 'imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re-established during oocyte growth. RESULTS: We have examined the timing of the reprogramming of the paternal methylation imprint of the mouse H19 gene during germ cell development. In both male and female PGCs, the paternal allele is partially methylated whereas the maternal allele is unmethylated. This partial methylation is completely erased in the female germline by entry into meiosis, establishing the oocyte methylation pattern. In the male germline, both alleles become methylated, mainly during the gonocyte stage, establishing the sperm methylation pattern. CONCLUSION: The paternal methylation imprint of H19 is established in the male germline and erased in the female germline at specific developmental stages. The identification of the timings of the methylation and demethylation should help to identify and characterize the biochemical basis of the reprogramming of imprinting.     

Sex difference in methylation of single-copy genes in human meiotic germ cells: Implications for X chromosome inactivation,parental imprinting,and origin of CpG mutations

To determine the methylation status of female germ cells in reference to the programmed reversal of X chromosome inactivation in these cells, we examined human fetal ovaries at developmental stages from the time germ cells initiate meiosis to when they cease to synthesize DNA (8–21 weeks gestation). Using methylation-sensitive restriction enzymes, we analyzed 57 MspI sites (32 sites in the CpG islands, and 25 nonclustered sites) from five X-linked housekeeping genes (HPRT, G6PD, P3, PGK, and GLA) and two tissue specific genes (X-linked F9 and autosomal EPO). Methylation patterns were compared to those of male germ cells, sperm, and somatic tissues of both sexes. All 32 MspI sites in CpG islands were unmethylated in germ-cell fractions of fetal ovary and adult testes, which could explain the reversibility of X inactivation in these tissues. However, whereas male meiotic germ cells were extensively methylated outside the islands (in the body of genes) and the methylation patterns resembled those of most somatic tissues, none of the 25 nonclustered CpGs was methylated in DNA contributed by the germ-cell component of fetal ovaries. The presence of faint MspI-like fragments in HpaII digests of fetal testes as well as fetal ovary prior to the onset of meiosis suggests that DNA of primordial germ cells is unmethylated in both sexes. Our observations of meiotic germ cells suggest that the female germ cells remain unmethylated, but that methylation in male germ cells occurs postnatally, prior to or during the early stages of spermatogenesis. In any event, the striking sex difference in methylation status of endogenous single-copy genes in meiotic germ cells could provide a molecular basis for parental imprinting of the mammalian genome.     

Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region

Mouse chromosome 7F4/F5, where the imprinting domain is located, is syntenic to human 11p15.5, the locus for Beckwith-Wiedemann syndrome. The domain is thought to consist of the two subdomains Kip2 (p57(kip2))/Lit1 and Igf2/H19. Because DNA methylation is believed to be a key factor in genomic imprinting, we performed large-scale DNA methylation analysis to identify the cis-element crucial for the regulation of the Kip2/Lit1 subdomain. Ten CpG islands (CGIs) were found, and these were located at the promoter sites, upstream of genes, and within intergenic regions. Bisulphite sequencing revealed that CGIs 4, 5, 8, and 10 were differentially methylated regions (DMRs). CGIs 4, 5, and 10 were methylated paternally in somatic tissues but not in germ cells. CGI8 was methylated in oocyte and maternally in somatic tissues during development. Parental-specific DNase I hypersensitive sites (HSSs) were found near CGI8. These data indicate that CGI8, called DMR-Lit1, is not only the region for gametic methylation but might also be the imprinting control region (ICR) of the subdomain.     

Parental allele specific methylation of the human insulin-like growth factor II gene and Beckwith-Wiedemann syndrome.

In an attempt to elucidate the role of methylation in parental imprinting at the IGF-II gene locus, for which imprinting has already been described in the mouse, we undertook an allele specific methylation study of the human IGF-II gene (mapped to 11p15.5) in a control population and in patients with Beckwith-Wiedemann syndrome. In control leucocyte DNA (16 unrelated adults and eight families), the maternal allele of the IGF-II gene was specifically hypomethylated, whereas no such allele specific methylation was found for either the insulin or the calcitonin genes which are located in 11p15.5 and 11p15.1, respectively. Furthermore, the IGF-II gene specific hypomethylation was localised on the 5' portion of exon 9. In the patients with Beckwith-Wiedemann syndrome in which the IGF-II gene is thought to be involved and where paternal isodisomy has been described, hypomethylation of the maternal allele was conserved in leucocyte DNA, but abnormal methylation was detected in malformed tissues where the paternal allele was also demethylated. Some specific mechanism linked to methylation therefore seems to be involved in the pathogenesis of Beckwith-Wiedemann syndrome.     

DNA甲基化与印记基因

[摘要] 遗传外修饰指不影响DNA遗传编码而调控基因组活性的过程,甲基化转移酶在该过程中发挥着重要的作用。配子的遗传外编程是印记建立的必要条件,与印记的形成机制有关,对基因组功能有重要影响。DNA甲基化、组蛋白乙酰化在印记基因表达过程中相互作用。印记基因可以解释肿瘤的发生以及一些遗传性疾病。有逐渐增多的证据表明,辅助生殖技术 (assisted reproductive technologies,ART)与印记基因存在联系,探索ART影响印记基因的机制将有助于ART技术的完善,防止ART相关并发症的发生。本文就DNA甲基化与印记基因研究进展进行综述。     

Paternal imprints can be established on the maternal Igf2-H19 locus without altering replication timing of DNA

Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.     

Epigenetic marks by DNA methylation specific to stem,germ and somatic cells in mice

BACKGROUND: DNA methylation is involved in many gene functions such as gene-silencing, X-inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue-dependent and differentially methylated region (T-DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation. RESULTS: We investigated the genome-wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T-DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation. CONCLUSIONS: Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei.     

Mammalian DNA (cytosine-5) methyltransferases and their expression

Two classes of functional DNA (cytosine-5) methyltransferases have been discovered in mammals to date. One class methylates the unmodified DNA and is designated as the de novo enzyme, whereas the other maintains the methylation status of the daughter strand during DNA replication and thus is referred to as a maintenance DNA methyltransferase. Each enzyme catalyzes methyl group transfer from S-adenosyl-L-methionine to cytosine bases in DNA. During methylation the enzyme flips its target base out of the DNA duplex into a typically concave catalytic pocket. This flipped cytosine base is then a substrate for the enzyme-catalyzed reaction. The newly formed 5-methylcytosine confers epigenetic information on the parental genome without altering nucleotide sequences. This epigenetic information is inherited during DNA replication and cell division. In mammals, DNA methylation participates in gene expression, protection of the genome against selfish DNA, parental imprinting, mammalian X chromosome inactivation, developmental regulation, T cell development, and various diseases.     

Reduced expression of DNMT3B in the germ cells of patients with bilateral spermatogenic arrest does not lead to changes in the global methylation status

DNA methylation events during spermatogenesis have important implications for gamete integrity and transmission of epigenetic information to the next generation. However, the role of DNA methyltransferases in the disorders of human spermatogenesis has not been elucidated. The aim of the present study was to evaluate the expression of DNMT3B, crucial for full germ cell methylation, in testicular germ cells of patients with spermatogenic arrest and to determine whether or not there is an association with the global methylation status. In order to determine the DNMTs expression status at various stages of spermatogenesis, immunohistochemical localization was performed on 16 fertile controls having normal spermatogenesis and 11 patients with bilateral spermatogenic arrest. DNMT3B was expressed in most of the germ cell types in both controls and patients with bilateral spermatogenic arrest. The number of DNMT3B positive preleptotene/zygotene cells and pachytene spermatocytes was significantly lower in patients with bilateral arrest. However, evaluation of 5-methylcytosine, a global methylation marker, in the few matured germ cells of these patients did not reveal altered methylation. In conclusion, the global methylation status of germ cells is not affected by spermatogenic defects in spite of aberrant DNMT3B expression indicating the necessity of proper methylation for full spermatogenesis.     

A study of DNA methylation in myotonic dystrophy.

We have examined the hypothesis that the severe congenital form of myotonic dystrophy is caused by genomic imprinting at the level of differential DNA methylation of maternal and paternal alleles. Probes encompassing the 5', central, and 3' regions of the myotonic dystrophy protein kinase gene were used on blots of blood DNA from congenital and adult onset patients, digested with combinations of methylation sensitive and insensitive restriction enzymes. We observed similar patterns of methylation in each of the different classes of patient, and found no methylation differences between paternally and maternally derived alleles. Within the limitations of the experiment, our results provide no evidence for a role for genomic imprinting in congenital myotonic dystrophy and suggest that the explanation for this form of the disease will be found elsewhere.     

Chloroplast DNA diversity within and among populations of Trifolium pratense

Summary In contrast to animal mitochondrial DNA, intraspecific variation in chloroplast DNA is thought to be very rare. This presumption has prevented the application to plant population biology of the diversity of molecular genetic techniques now well established for animal mitochondrial DNA. In Trifolium pratense, however, extensive intrapopulational variation does exist. In two paper I report a characterization of unprecedented restriction fragment profile variation within single populations. Populations typically contain a common genotype and many rare ones; often the rare genotypes differ from population to population. While both nucleon and nucleotide diversity, as well as estimates of Wright's F _ST, indicate a large within-population component and relatively little diversity among populations, the distribution of plastid genotype frequencies in each population is not homogeneous. Estimates of migration rate based on chloroplast DNA genotypes suggest a moderate number of migrants per generation. The unusually high level of genetic variation in Trifolium chloroplast DNA provides the first opportunity to use the plastid genome of plants to study population differentiation. Furthermore, it suggests that the plastid genome may not be as invariant as previously believed, but may instead exhibit high levels of genetic diversity at the population level.     

Alteration of methylation status in archival DNA samples: A qualitative assessment by methylation specific polymerase chain reaction

DNA methylation is an epigenetic mechanism that regulates gene expression, which also facilitates genomic imprinting. Genomic imprinting is responsible for differential expression of genes based on parent of origin. Altered methylation of parental alleles results in imprinting disorders diagnosed by methylation specific polymerase chain reaction (MS-PCR) technique. With increasing evidence of genes under epigenetic influence, methylation studies are extensively performed on archival samples. To evaluate effect of storage and storage conditions on DNA methylation, a systematic MS-PCR based analysis was planned on an imprinted gene, SNRPN, located on chromosome 15q11.2. It was assessed by MS-PCR on fresh, 4 −20, and −80°C stored DNA samples for different time periods for systematic evaluation of methylation status. Technical factors like type of sample processing, method of DNA isolation, primer region polymorphism, sample heterogeneity were also evaluated. DNA methylation was observed to be altered for SNRPN gene after storage at −80°C from 2 months onwards. Long-term storage of DNA at −80°C results in altered DNA methylation status. This may lead to false MS-PCR diagnosis of imprinting disorders. Our proof of concept study should be followed with quantitative validation since the findings have critical implications in the present era of biobanking.     

Increased cell proliferation and R.Msp1 fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism.

DNA methylation is one of the crucial mechanisms for cellular and tissue differentiation during developmental stages in mammals. 5-aza-2'-deoxycytidine, a specific cytosine DNA Methyltransferase inhibitor, is known to cause DNA hypomethylation in CpG, CpNpG and CCGG sequences. Therefore the present study was designed to determine the effects of 5-aza-2'-deoxycytidine on the germinal cells of the adult rat testicular tissue. Rat testicular tissues from the 5-aza-2'-deoxycytidine treated experimental and non-treated control groups were processed for light microscopy and also for genomic DNA isolation assays. The isolated genomic DNAs were digested with R.Msp1 in order to determine the methyl pattern differences in the enzyme cognate CCGG sequence. Testicular tissues from treated rats showed increased cell proliferation when investigated at the light microscopical level. On the other hand, genomic DNA of these proliferative tissue showed high fragmentation sizes of R.Msp1 digestion when compared to controls. While the R.Msp1 digested control group DNA fragmentation condensed at approximately 4700-5100 bps size, the experimental group DNA fragmentation was condensed at 700-900 bps size. In addition, 5-aza-2'-deoxycytidine has effects on increased cell proliferation via the loss of somatic de novo gene imprinting. These results imply that abnormally imprinted normal somatic cells in mammals are susceptible to epigenetic modification. These results also suggest that the genomic DNA of testicular tissues from control rats is resistant to R.Msp1 while DNA from the experimental group testicular cells demonstrating high proliferation rate could not resist to R.Msp1 digestion due to DNA hypomethylation in CCGG sequence. In conclusion, it could be suggested that the reversal of gene imprinting in germinal cells may cause an increased cellular proliferation and R.Msp1 fragmentation when induced by 5-aza-2'-deoxycytidine.