Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1β when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1β could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1β.     

Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression

BackgroundSelective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence.ResultsHere, we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice, assigning allele specificity to approximately two-thirds of all binding sites. While half of these are biallelic and include endogenous retrovirus (ERV) targets, the rest show monoallelic binding based either on parental origin or on genetic background of the allele. Parental-origin allele-specific binding is methylation-dependent and maps only to imprinting control differentially methylated regions (DMRs) established in the germline. We identify a novel imprinted gene, Fkbp6, which has a critical function in mouse male germ cell development. Genetic background-specific sequence differences also influence ZFP57 binding, as genetic variation that disrupts the consensus binding motif and its methylation is often associated with monoallelic expression of neighboring genes.ConclusionsThe work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0672-7) contains supplementary material, which is available to authorized users.     

In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions

The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.     

A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints

The mechanisms responsible for maintaining genomic methylation imprints in mouse embryos are not understood. We generated a knockout mouse in the Zfp57 locus encoding a KRAB zinc finger protein. Loss of just the zygotic function of Zfp57 causes partial neonatal lethality, whereas eliminating both the maternal and zygotic functions of Zfp57 results in a highly penetrant embryonic lethality. In oocytes, absence of Zfp57 results in failure to establish maternal methylation imprints at the Snrpn imprinted region. Intriguingly, methylation imprints are reacquired specifically at the maternally derived Snrpn imprinted region when the zygotic Zfp57 is present in embryos. This suggests that there may be DNA methylation-independent memory for genomic imprints. Zfp57 is also required for the postfertilization maintenance of maternal and paternal methylation imprints at multiple imprinted domains. The effects on genomic imprinting are consistent with the maternal-zygotic lethality of Zfp57 mutants.     

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.     

Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.     

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICR_BS) and many other loci (non-ICR_BS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICR_BS and non-ICR_BS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICR_BS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.     

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.     

Sequence-specific methylation of the mouse H19 gene in embryonic cells deficient in the Dnmt-1 gene

We have used Dnmt^c/c ES cells that are homozygous for disruption of the DNA methyltransferase gene to address how de novo methylation is propagated and whether it is directed to specific sites in the early embryo. We examined the imprinted H19 gene and the specific-sequence region implicated as an “imprinting mark” to determine whether de novo methylation was occurring at a restricted set of sites. Since the “imprinting mark” was found to be methylated differentially at all stages of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmt^c/c cells, even though the loss of maintenance methylase activity renders the H19 promoter active. We used bisulfite genomic sequencing to determine the methylation state of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstream region of the imprinted H19 sequence in the Dnmt^c/c mutant ES cells. Moreover, these CpG sites appeared to be favoured targets for further de novo methylation of neighbouring CpG sites in rescued ES cells, which possess apparently normal maintenance activity. Our data provide further evidence for a separate methylating activity in ES cells and indicate that this activity displays sequence specificity. Dev. Genet. 22:111–121, 1998. © 1998 Wiley-Liss, Inc.     

Shared role for differentially methylated domains of imprinted genes

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.     

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.     

Differential genomic imprinting and expression of imprinted microRNAs in testes-derived male germ-line stem cells in mouse

Background

Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings.

Methods and Findings

Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA.

Conclusions

Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.     

Establishment and maintenance of DNA methylation patterns in mouse Ndn: implications for maintenance of imprinting in target genes of the imprinting center

Ndn is located on chromosome 7C, an imprinted region of the mouse genome. Imprinting of Ndn and adjacent paternally expressed genes is regulated by a regional imprinting control element known as the imprinting center (IC). An IC also controls imprint resetting of target genes in the region of conserved synteny on human chromosome 15q11-q13, which is deleted or rearranged in the neurodevelopmental disorder Prader-Willi syndrome. Epigenetic modifications such as DNA methylation, which occur in gametes and can be stably propagated, are presumed to establish and maintain the imprint in target genes of the IC. While most DNA becomes substantially demethylated by the blastocyst stage, some imprinted genes have regions that escape global demethylation and may maintain the imprint. We have now analyzed the methylation of 39 CpG dinucleotide sequences in the 5' end of Ndn by sodium bisulfite sequencing in gametes and in preimplantation and adult tissues. While sperm DNA is completely unmethylated across this region, oocyte DNA is partially methylated. A distinctive but unstable maternal methylation pattern persists until the morula stage and is lost in the blastocyst stage, where low levels of methylation are present on most DNA strands of either parental origin. The methylation pattern is then substantially remodeled, and fewer than half of maternally derived DNA strands in adult brain resemble the oocyte pattern. We postulate that for Ndn, DNA methylation may initially preserve a gametic imprint during preimplantation development, but other epigenetic events may maintain the imprint later in embryonic development.     

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.     

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.     

Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome

Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.     

A KRAB domain zinc finger protein in imprinting and disease

The monoallelic expression of imprinted genes is regulated by DNA methylation marks that originate from the oocyte or sperm. Li et al. (2008) show in this issue of Developmental Cell that the KRAB zinc finger protein Zfp57 contributes to the embryonic maintenance of these imprints. At one locus, Zfp57 is also involved in imprint establishment. These findings provide a mechanistic interpretation for Mackay et al.'s recently reported ZFP57 mutations in patients with transient neonatal diabetes.     

母源效应蛋白在基因组印记维持中的作用

基因组印记是指生殖细胞发生过程中双亲基因组发生差异表观修饰,使带有亲代印记的等位基因出现父源或母源单等位基因表达。在配子发生和早期胚胎发育过程中,基因组印记甲基化经历一个去除、重建和维持的复杂过程。这个过程中的任何环节被干扰都将导致印记紊乱,造成胚胎发生、胎盘形成及出生后发育异常。近来研究表明,早期胚胎发育过程中一些母源效应蛋白在印记基因表观调控中起重要作用。为了更好地理解这些母源因子对印记基因建立及维持的作用与机制,文章综述了DPPA3、ZFP57、TRIM28和DNMT1等母源效应因子近年来的相关研究进展,并探讨了这些因子对基因组印记的表观调控机制。