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.     

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     

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.     

Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Background

Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.

Methodology/Principal Findings

We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.

Conclusions/Significance

These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.     

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.     

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.     

Increased plasticity of genomic imprinting of Dlk1 in brain is due to genetic and epigenetic factors

The expression of six imprinted genes (Dlk1, Gtl2, Igf2r, Kcnq1, Nnat, and Peg1) was examined in brains of 21 mice derived from N₂ × N₂ intercrosses between C57BL/6 and MOLF/Ei strains. Imprinting of Igf2r, Kcnq1, Gtl2, and Dlk1 varied among individuals. As three of these genes are implicated in cell–cell signaling or cell–environment interactions, variation in their imprinting may influence a wide range of biological processes from cell differentiation to behavior. To elucidate the mechanisms underlying the interindividual imprinting variation in the brain, we focused our effort on the paternally expressed gene Dlk1. We investigated expression of Dlk1 in the brains of animals from N₉ and N₁₀ backcrosses and found that reactivation of the normally silent maternal Dlk1 allele in the N₉ and N₁₀ mice occurred less often than in N₂ × N₂ animals. Our data suggest that trans-acting genetic factors of MOLF/Ei origin facilitate the reactivation of the normally silent maternal allele of Dlk1. We mapped one of these factors to the proximal part of Chr 7. The results of bisulfite sequencing methylation analysis show that reactivation of the maternal allele was also associated with hypermethylation of the intragenic differentially methylated region (IG DMR), which is the imprinting control region for the Dlk1–Gtl2 domain. Thus, the imprinting status of Dlk1 in the brain depends upon trans-acting genetic influences and correlates with the methylation status of a specific subregion of the IG DMR.The GenBank accession numbers for sequences described in this article are AY644388–644394.     

Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1-Dio3 cluster.     

The IG-DMR and the MEG3-DMR at Human Chromosome 14q32.2: Hierarchical Interaction and Distinct Functional Properties as Imprinting Control Centers

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR.     

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.     

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.     

Alternative splicing and imprinting control of the Meg3/Gtl2-Dlk1 locus in mouse embryos

The distal part of the mouse Chr 12 contains a cluster of reciprocally imprinted genes. Recently we found a grandparental origin-dependent, transmission-ratio distortion (TRD) in this region. The TRD resulted from postimplantation loss of embryos that inherited the distal Chr 12 alleles from the maternal grandfather. These data suggested that imprinting of one or more genes in this region was not uniformly well established or maintained in all the embryos. To elucidate the mechanism underlying such a variation, we examined the expression of two genes from the distal Chr 12 imprinted region, the maternally expressed gene 3/gene-trap locus 2 ( Meg3/ Gtl2), and the delta-like homolog 1 ( Dlk1) gene. We demonstrated that the Meg3/ Gtl2 gene had two major mRNA forms. One form, Meg3-proximal ( Meg3p), contained exons 1-3. The second form, Meg3-distal ( Meg3d) did not contain exons 1-3 and was present in oocytes and in 1- and 2-cell embryos. We observed cross-dependent and splice form-specific relaxation of imprinting of the Dlk1 and Meg3d, but not Meg3p. Expression patterns of Dlk1 and Meg3/ Gtl2 in embryos from crosses between different mouse strains suggest that 1). imprinting of the Dlk1 and Meg3/ Gtl2 genes is not strictly coordi- nated; 2). parental origin-dependent expression of these genes is under control of a strain-specific, cis-acting modifier located in a 1.5-Mb region that includes the Meg3/ Gtl2-Dlk1 locus. Biallelic expression of Dlk1 and Meg3d did not affect embryo viability and, therefore, cannot be responsible for the lethal phenotypes in UPD12 embryos or for the transmission-ratio distortion.