Differential histone modifications mark mouse imprinting control regions during spermatogenesis

Only some imprinting control regions (ICRs) acquire their DNA methylation in the male germ line. These imprints are protected against the global demethylation of the sperm genome following fertilisation, and are maintained throughout development. We find that in somatic cells and tissues, DNA methylation at these ICRs is associated with histone H4-lysine-20 and H3-lysine-9 trimethylation. The unmethylated allele, in contrast, has H3-lysine-4 dimethylation and H3 acetylation. These differential modifications are also detected at maternally methylated ICRs, and could be involved in the somatic maintenance of imprints. To explore whether the post-fertilisation protection of imprints relates to events during spermatogenesis, we assayed chromatin at stages preceding the global histone-to-protamine exchange. At these stages, H3-lysine-4 methylation and H3 acetylation are enriched at maternally methylated ICRs, but are absent at paternally methylated ICRs. H4 acetylation is enriched at all regions analysed. Thus, paternally and maternally methylated ICRs carry different histone modifications during the stages preceding the global histone-to-protamine exchange. These differences could influence the way ICRs are assembled into specific structures in late spermatogenesis, and may thus influence events after fertilisation.     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes

In different eukaryotic model systems, chromatin and gene expression are modulated by post-translational modification of histone tails. In this in vivo study, histone methylation and acetylation are investigated along the imprinted mouse genes Snrpn, Igf2r and U2af1-rs1. These imprinted genes all have a CpG-rich regulatory element at which methylation is present on the maternal allele, and originates from the female germ line. At these 'differentially methylated regions' (DMRs), histone H3 on the paternal allele has lysine-4 methylation and is acetylated. On the maternally inherited allele, in contrast, chromatin is marked by hypermethylation on lysine-9 of H3. Allele-specific patterns of lysine-4 and lysine-9 methylation are also detected at other regions of the imprinted loci. For the DMR at the U2af1-rs1 gene, we establish that the methyl-CpG-binding-domain (MBD) proteins MeCP2, MBD1 and MBD3 are associated with the maternal allele. These data support the hypothesis that MBD protein-associated histone deacetylase/chromatin-remodelling complexes are recruited to the parental allele that has methylated DNA and H3-K9 methylation, and are prevented from binding to the opposite allele by H3 lysine-4 methylation.     

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

Agglomerates of aberrant DNA methylation are associated with toxicant-induced malignant transformation

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals or cadmium. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. The agglomerative DNA methylation changes induced by arsenicals appear to be common and clinically relevant events, since they occur in other human cancer cell lines and models of malignant transformation, as well as clinical cancer specimens. Aberrant DNA methylation in general occurred more often within histone H3 lysine-27 trimethylation stem cell domains. We found a striking association between enrichment of histone H3 lysine-9 trimethylation stem cell domains and toxicant-induced agglomerative DNA methylation, suggesting these epigenetic modifications may become aberrantly linked during malignant transformation. In summary, we found an association between toxicant-induced malignant transformation and agglomerative DNA methylation, which lends further support to the hypothesis that epigenetic dysfunction plays an important role in toxicant-induced malignant transformation.     

Dynamic chromatin modifications characterise the first cell cycle in mouse embryos

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.     

Agglomerates of aberrant DNA methylation are associated with toxicant-induced malignant transformation

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals or cadmium. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. The agglomerative DNA methylation changes induced by arsenicals appear to be common and clinically relevant events, since they occur in other human cancer cell lines and models of malignant transformation, as well as clinical cancer specimens. Aberrant DNA methylation in general occurred more often within histone H3 lysine-27 trimethylation stem cell domains. We found a striking association between enrichment of histone H3 lysine-9 trimethylation stem cell domains and toxicant-induced agglomerative DNA methylation, suggesting these epigenetic modifications may become aberrantly linked during malignant transformation. In summary, we found an association between toxicant-induced malignant transformation and agglomerative DNA methylation, which lends further support to the hypothesis that epigenetic dysfunction plays an important role in toxicant-induced malignant transformation.     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

The specification of imprints in mammals

At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.     

Cyclophilin a protects Peg3 from hypermethylation and inactive histone modification

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. Disruption of normal imprinting leads to abnormal embryogenesis, certain inherited diseases, and is associated with various cancers. In the context of screening for the gene(s) responsible for the alteration of phenotype in cyclophilin A knockdown (CypA-KD) P19 cells, we observed a silent paternally expressed gene, Peg3. Treatment of CypA-KD P19 cells with the DNA demethylating agent 5-aza-dC reversed the silencing of Peg3 biallelically. Genomic bisulfite sequencing and methylation-specific PCR revealed DNA hypermethylation in CypA-KD P19 cells, as the normally unmethylated paternal allele acquired methylation that resulted in biallelic methylation of Peg3. Chromatin immunoprecipitation assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 and MBD2 binding on the cytosine-guanine dinucleotide (CpG) islands of Peg3. Our results indicate that DNA hypermethylation on the paternal allele and allele-specific acquisition of histone methylation leads to silencing of Peg3 in CypA-KD P19 cells. This study is the first demonstration of the epigenetic function of CypA in protecting the paternal allele of Peg3 from DNA methylation and inactive histone modifications.     

Timing of establishment of paternal methylation imprints in the mouse

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.     

G9a histone methyltransferase contributes to imprinting in the mouse placenta

Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Epigenotype switching of imprintable loci in embryonic germ cells

 Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar. Received: 1 September 1997 / Accepted: 15 October 1997     

Early loss of Xist RNA expression and inactive X chromosome associated chromatin modification in developing primordial germ cells

BACKGROUND: The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges. PRINCIPAL FINDINGS/METHODOLOGY: We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27. CONCLUSIONS/SIGNIFICANCE: We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome.