Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

Background

The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.

Results

A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.

Conclusion

These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

DNA methylation dynamics of a maternally methylated DMR in the mouse Dlk1–Dio3 domain

The mouse delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1–Dio3) imprinted domain contains three known paternally methylated differentially methylated regions (DMRs): intergenic DMR (IG-DMR), maternally expressed 3-DMR (Gtl2-DMR), and Dlk1-DMR. Here, we report the first maternally methylated DMR, CpG island 2 (CGI-2), is located approximately 800 bp downstream of miR-1188. CGI-2 is highly methylated in sperm and oocytes, de-methylated in pre-implantation embryos, and differentially re-methylated during post-implantation development. CGI-2, similarly to Gtl2-DMR and Dlk1-DMR, acquires differential methylation prior to embryonic day 7.5 (E7.5). Both H3K4me3 and H3K9me3 histone modifications are enriched at CGI-2. Furthermore, CCCTC-binding factor (CTCF) binds to both alleles of CGI-2 in vivo. These results contribute to the investigation of imprinting regulation in this domain.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.Key words: genomic imprinting, DNA methylation, Gtl2, secondary DMR, epigenetics     

MIRA-SNuPE,a quantitative,multiplex method for measuring allele-specific DNA methylation

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well-known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.Key words: epigenetics, imprinting, DMR, MIRA, MBD, DNA methylation, SNuPE     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.     

Aberrant regulation of imprinted gene expression in Gtl2lacZ mice

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.     

High-frequency generation of viable mice from engineered bi-maternal embryos

Mammalian development to adulthood typically requires both maternal and paternal genomes, because genomic imprinting places stringent limitations on mammalian development, strictly precluding parthenogenesis. Here we report the generation of bi-maternal embryos that develop at a high success rate equivalent to the rate obtained with in vitro fertilization of normal embryos. These bi-maternal mice developed into viable and fertile female adults. The bi-maternal embryos, distinct from parthenogenetic or gynogenetic conceptuses, were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. The results provide conclusive evidence that imprinted genes regulated by these two paternally methylated imprinting-control regions are the only paternal barrier that prevents the normal development of bi-maternal mouse fetuses to term.     

Identification of imprinting regulators at the Meg3 differentially methylated region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos.     

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.     

A 178-kb BAC transgene imprints the mouse Gtl2 gene and localizes tissue-specific regulatory elements

The regulation of genomic imprinting, the allele-specific expression of an autosomal gene, is complex and poorly understood. Imprinted genes are organized in clusters, where cis-acting regulatory elements are believed to interact to control multiple genes. We have used BAC transgenesis in the mouse to begin to delineate the region of DNA required for proper expression and imprinting of the mouse Delta-like1 (Dlk1) and Gene-trap locus2 (Gtl2) imprinted genes. We demonstrate that the Gtl2 gene is expressed from a BAC transgene in mouse embryo and placenta only upon maternal inheritance, as is the endogenous Gtl2 gene. Gtl2 is therefore properly imprinted on the BAC in an ectopic chromosomal location and must carry with it all necessary imprinting regulatory elements. Furthermore, we show that the BAC Gtl2 gene is expressed at levels approaching those of the endogenous gene only in the brain of adult animals, not in other sites of endogenous expression such as the pituitary, adrenal, and skeletal muscle. These data localize the enhancer(s) for brain Gtl2 expression, but not those for other tissues, to the DNA contained within the BAC clone. As the Dlk1 gene is not expressed from the BAC in any tissues, it must require additional elements that are different from those necessary for Gtl2 expression. Our data refine the interval for future investigation of Gtl2 imprinting and provide evidence for distinct regulation of the linked Dlk1 and Gtl2 genes.