Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation

Differential modifications of the genome during gametogenesis result in a functional difference between the paternal and maternal genomes at the moment of fertilization. A possible cause of this imprinting is the methylation of DNA. The insertion of foreign DNA into transgenic mice allows the tagging of regions that are differentially methylated during gametogenesis. We describe here a transgenic mouse strain in which the expression of the hepatitis B surface antigen gene is irreversibly repressed following its passage through the female germ line. This inhibition is accompanied by the methylation of all the HpaII and HhaI sites within the foreign gene, which we have shown to be integrated into a site on chromosome 13. The irreversibility reported here contrasts with what is found with other transgenic mice sequences which are reversibly methylated after passage through the male or female germ line, though in both cases methylation appears to be important in the imprinting process.     

Cell lineage-specific undermethylation of mouse repetitive DNA

Several distinct cell lineages are established during mouse embryogenesis. The trophectoderm and primitive endoderm give rise to extraembryonic structures alone, while the primitive ectoderm becomes the fetus proper. Recent studies suggest that the levels of DNA modification are lower in inactive X chromosomes from extraembryonic tissues than in embryonic and adult somatic tissues. Using HpaII/MspI isoschizomers, Southern blots and cloned probes, we show here that repetitive DNA sequences from all derivatives of the two extraembryonic lineages, trophectoderm and primitive endoderm, are substantially undermethylated compared with primitive ectoderm derivatives. This contrasts with the highly methylated state of these repetitive elements observed in adult somatic tissues. Specific demethylation or inhibition of de novo methylation, or a combination of both mechanisms, may be involved. These findings suggest that elements of gene regulation dependent on DNA modification may be different in extraembryonic cell lineages.     

Differential activity of maternally and paternally derived chromosome regions in mice

Although both parental sexes contribute equivalent genetic information to the zygote, in mammals this information is not necessarily functionally equivalent. Diploid parthenotes possessing two maternal genomes are generally inviable, embryos possessing two paternal genomes in man may form hydatidiform moles, and nuclear transplantation experiments in mice have shown that both parental genomes are necessary for complete embryogenesis. Not all of the genome is involved in these parental effects, however, because zygotes with maternal or paternal disomy for chromosomes 1, 4, 5, 9, 13, 14 and 15 of the mouse survive normally. On the other hand, only the maternal X chromosome is active in mouse extraembryonic membranes, maternal disomy 6 is lethal, while non-complementation of maternal duplication/paternal deficiency or its reciprocal for regions of chromosome 2, 8 and 17 has been recognized. We report that animals with maternal duplication/paternal deficiency and its reciprocal for each of two particular chromosome regions show anomalous phenotypes which depart from normal in opposite directions, suggesting a differential functioning of gene loci within these regions. A further example of non-complementation lethality is also reported.     

Non-invasive prenatal measurement of the fetal genome

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.     

Seed development: Early paternal gene activity in Arabidopsis.

Both parental genomes are expressed during embryogenesis, although the time of activation of the paternally inherited genes varies between organisms. Results reported by Vielle-Calzada et al. have suggested that delayed activation of the paternal genome seems to be the rule in plant development. We find, however, that during early embryogenesis in Arabidopsis, paternal genes are expressed and are sufficient for normal development. Our findings indicate that there is no overall maternal control of early embryogenesis, and that the contribution of the parental alleles needs to be assessed for each gene individually.     

Genome-scale DNA methylation maps of pluripotent and differentiated cells

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.     

Predicted methylation landscape of all CpG islands on the human genome

CpG island methylation plays important role in various biological processes. To investigate methylation landscape of all CpG islands on the human genome, we develop a model for predicting the CpG island methylation status. This model outperforms other existing methods. We apply the model on the whole human genome and predict the landscape of DNA methylation of all CpG islands. Based on the methylation profile, we find that about 31% of CpG islands are methylation-prone and CpG islands located in promoter regions are seldom methylated. There is no significant difference in the CpG island methylation level between R and G bands among the chromosomes. The occupancy of RNA polymerase II is significantly higher in methylation-resistant promoter CpG islands, indicating that genes with such promoter CpG islands tend to be more active.     

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.     

High frequency of unequal recombination in pseudoautosomal region shown by proviral insertion in transgenic mouse

The mammalian X and Y chromosomes, in contrast to the autosomes, pair during male meiosis only near the telomeres. Alleles localized in this region can undergo reciprocal exchange during meiosis. Because such sequences do not show strict sex-linked inheritance, they have been termed pseudoautosomal. In man, several DNA sequences have been described which show pseudoautosomal transmission and which are localized in the pairing region at the ends of the short arms of both the X and Y chromosomes (refs 6-9, and D. Page, unpublished results). We now show that the transgenic mouse strain, Mov-15, contains a single Moloney murine leukaemia virus (M-MuLV) genome in its germline, and genetic evidence indicates that the provirus is integrated into the pseudoautosomal region of the sex chromosome. Proviral copies are lost or gained in 7% of male meioses in this strain, and mouse sequences flanking the provirus are tandemly repeated and highly variable. We conclude that unequal recombination events occur with high frequency in the pairing region, possibly because of the presence of repeated sequences.     

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 that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally 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. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately 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 in specific developmental contexts.     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

Influence of parental chromosomes on spatial specificity in androgenetic----parthenogenetic chimaeras in the mouse

The presence of both parental genomes is essential for development to term in the mouse embryo probably because of germline-specific modifications of homologous chromosomes. Neither androgenetic nor parthenogenetic embryos can by themselves develop to term; any post-implantation embryos they produce have opposite phenotypes, which reflects the presence of complementary information in parental chromosomes. The development of androgenetic----parthenogenetic chimaeras is of considerable interest because in this case both parental chromosomes are available even though they are in separate cells. We demonstrate here that in post-implantation chimaeric fetuses, the expression of parental information results in spatial specificity so that parthenogenetic cells are confined to the embryo but the trophoblast consists almost entirely of androgenetic cells. The yolk sac contains both cell types. However, there is incomplete functional complementation because the chimaeras do not reach term. Although failure to reach term may occur partly because of inadequate intermingling and interactions between embryonic cells, it is more likely that further control of mouse development depends on the presence of both sets of chromosomes within the same cells.     

Degree of methylation of transgenes is dependent on gamete of origin

Data derived from both pronuclear transplantation experiments and classical genetic experiments indicate that the maternal and paternal genetic contributions to the mammalian zygote nucleus do not function equivalently during subsequent development. These observations have been interpreted as resulting from differential 'genome imprinting' during male and female gametogenesis. The molecular mechanism responsible for genome imprinting is unknown, but data gathered to date require that the mechanism fulfill at least four criteria: (1) the imprint must be physically linked to the pronucleus; (2) the imprint must persist through DNA replication and cell division; (3) the mechanism must be capable of affecting gene expression; and (4) the mechanism must be capable of switching the identity of the imprint from one sex to the other in successive generations. One molecular mechanism which could satisfy the first three criteria is differential DNA methylation during gametogenesis itself, or before formation of the zygote nucleus during embryogenesis. We present data indicating that the methylation patterns of exogenous DNA sequences in transgenic mice can be changed by switching their gamete of origin in successive generations. These data suggest that DNA methylation can also satisfy the fourth criterion for an imprinting mechanism.     

高活力水稻种子萌发过程中DNA甲基化变化的MSAP分析

DNA甲基化是基因组DNA的一种主要表观遗传修饰形式,是调节基因组功能的重要手段。本实验采用MSAP方法分析,旨在研究高活力水稻种子萌发过程中的甲基化与去甲基化变化的规律,为研究种子的老化机理和种子的长期保存提供依据。结果表明:水稻种子萌发过程中,同时发生了甲基化与去甲基化作用,且去甲基化作用先于甲基化作用发生。发生去甲基化可能与基因活化有关,发生甲基化可能与组织特异性有关。     

Speciation- induced heritable cytosine methylation changes in polyploid wheat

To study possible epigenetic changes accompanying polyploid speciation, genomic DNA from natural polyploid wheats and their putative diploid progenitors were digested with a pair of isoschizomers Hpa II / Msp I and hybridized to 21 different types of low-copy DNA sequences. It was found that cytosine methylation changes were abundant in natural polyploid wheats after their speciation. The hybridization of the same set of sequences to a synthetic hexaploid wheat along with its parental lines indicated that the extensive DNA methylation changes already existed in the early generations (S5, S6 and Sy) of this plant. Moreover, the high similarity of the changed restriction fragment length polymorphism (RFLP) patterns among three randomly chosen individual plants suggested that the methylation changes occurred even earlier, and/or were of a nonrandom nature. The changed patterns were stably inherited in the three successive selfed generations. Though methylation changes are probably a genome-wide occurrence, they appeared to be confined to the specific types of DNA sequences. The possible implications of the rapid and extensive cytosine methylation changes for several attributes of allopolyploid genome evolution, such as genetic diploidization and gene diversification, are discussed .     

小麦异源多倍体中亲本基因组相互作用产生快速基因组变异的ISSR标记分析

通过ISSR标记分析,研究了异源多倍体基因组的进化现象.结果表明,基因组组成和普通小麦相同的人工合成异源六倍体小麦,在形成早期发生了迅速、广泛、以非随机性为主的基因组变化,包括遗传变异--主要表现为序列变异和表观遗传变异--主要表现为DNA甲基化变异.而且异源六倍体小麦中来自父本的基因组比来自母本的基因组发生了更多的遗...