Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Partial Loss of Genomic Imprinting Reveals Important Roles for Kcnq1 and Peg10 Imprinted Domains in Placental Development

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte–derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.     

Targeting of 5-aza-2′-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme

5-Aza-2′-deoxycytidine (5-aza-dC) is a nucleoside analogue with cytotoxic and DNA demethylating effects. Here we show that 5-aza-dC induces the proteasomal degradation of free (non-chromatin bound) DNMT1 through a mechanism which is dependent on DNA synthesis and the targeting of incorporated 5-aza-dC residues by DNMT1 itself. Thus, 5-aza-dC induces Dnmt1 degradation in wild-type mouse ES cells, but not in Dnmt [3a^–/–, 3b^–/–] mouse ES cells which express Dnmt1 but lack DNA methylation (<0.7% of CpG methylated) and contain few hemi-methylated CpG sites, these being the preferred substrates for Dnmt1. We suggest that adducts formed between DNMT1 and 5-aza-dC molecules in DNA induce a ubiquitin-E3 ligase activity which preferentially targets free DNMT1 molecules for degradation by the proteasome. The proteasome inhibitor MG132 prevents DNMT1 degradation and reduces hypomethylation induced by 5-aza-dC.     

Distinct roles of DMAP1 in mouse development

DMAP1 (DNMT1-associated protein 1) is a member of the TIP60-p400 complex that maintains embryonic stem (ES) cell pluripotency and a complex containing the somatic form of DNA methyltransferase 1 (DNMT1s). DMAP1 interacts with DNMT1s through a domain that is absent in Dnmt1(V)(/)(V) mice expressing just the oocyte form (DNMT1o). A Dmap1-null allele was generated to study the role of DMAP1 in development. Consistent with the phenotypes of loss of other members of the TIP60-p400 complex, Dmap1(-/-) mice died during preimplantation in both Dnmt1(+/+) and Dnmt1(V)(/)(V) backgrounds. Unexpectedly, in the Dnmt1(V)(/)(V) background, Dmap1(+/-) parents produced mainly Dmap1(+/-) mice. Most Dmap1(+/+) progeny died during midgestation, with loss of DNA methylation on imprinted genes, suggesting that DMAP1 influences maintenance methylation mediated by DNMT1o. In this regard, a DMAP1-DNMT1o complex was detected in ES cells when DNMT1o was stably expressed but not when transiently expressed, indicating a novel interaction between DMAP1 and DNMT1o. These results suggest that DMAP1-DNMT1s and DMAP1-DNMT1o interactions are essential for normal development and that DMAP1-DNMT1o complexes are not readily formed in the embryo. Therefore, DMAP1 mediates distinct preimplantation epigenetic reprogramming processes: TIP60-p400 nucleosome remodeling and DNMT1 maintenance methylation.     

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1^−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1^−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.     

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1^−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information.     

Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1^kd,3a^−/−,3b^−/−]). Sequencing of 960 RRBS clones from Dnmt[1^kd,3a^−/−,3b^−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1^kd,3a^−/−,3b^−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.     

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos

The mouse chromosome 7C, orthologous to the human 15q11–q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3–4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1^ps alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.     

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α^−/−;Gsk-3β^−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α^−/−;Gsk-3β^−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.     

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.     

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.     

Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.