Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.     

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.     

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.     

Genomic imprinting at the mammalian Dlk1-Dio3 domain

Genomic imprinting causes genes to be expressed or repressed depending on their parental origin. The majority of imprinted genes identified to date map in clusters and much of our knowledge of the mechanisms, function and evolution of imprinting have emerged from their analysis. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (Dlk1-Dio3) is located on distal mouse chromosome 12 and human chromosome 14. Its developmental importance is exemplified by severe phenotypes associated with altered dosage of these genes in mice and humans. The domain contains three imprinted protein-coding genes, Dlk1, Rtl1 and Dio3, expressed from the paternally inherited chromosome and several imprinted large and small noncoding RNA genes expressed from the maternally inherited homolog. Here, we discuss the function and regulation of imprinting at this domain.     

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline

Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.     

Localizing transcriptional regulatory elements at the mouse Dlk1 locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression.     

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.     

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     

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.     

Selective autophagy regulates insertional mutagenesis by the Ty1 retrotransposon in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic components and is ubiquitously found in eukaryotic cells. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia superfamily, is the most abundant retrotransposon in the yeast Saccharomyces cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like particles (VLPs) localized in the cytoplasm. Here we show that selective autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to autophagosomes by an interaction with Atg19. We propose that selective autophagy safeguards genome integrity against excessive insertional mutagenesis caused during nutrient starvation by transposable elements in eukaryotic cells.