Comparative sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2-H19 region.
ABSTRACT: The Dlk1-Gtl2 domain on mouse chromosome 12 contains reciprocally imprinted genes with the potential to contribute to our understanding of common features involved in imprinting control. We have sequenced this conserved region in the mouse and sheep and included the human sequence in a three species comparison. This analysis resulted in a precise conservation map and identification of highly conserved sequence elements, some of which we have shown previously to be differentially methylated in the mouse. Additionally, this analysis facilitated identification of a CpG-rich tandem repeat array located approximately 13-15 kb upstream of Gtl2. Furthermore, we have identified a third imprinted transcript that overlaps with the last Dlk1 exon in the mouse. This transcript lacks a conserved open reading frame and is probably generated by cleavage of extended Dlk1 transcripts. Because Dlk1 and Gtl2 share many of the imprinting properties of the well-characterized Igf2-H19 domain, it has been proposed that the two regions may be regulated in the same way. Comparative genomic examination of the two domains indicates that although there are similarities, other features are very different, including the location of conserved CTCF-binding sites, and the level of conservation at regulatory regions.
Project description: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.A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'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 Gtl2Delta5'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, Gtl2Delta5' 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.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 Gtl2Delta5'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.
Project description:Epigenetic alterations in the 11p15.5 imprinted gene cluster are frequent in human cancers and are associated with disordered imprinting of insulin-like growth factor (IGF)2 and H19. Recently, an imprinted gene cluster at 14q32 has been defined and includes two closely linked but reciprocally imprinted genes, DLK1 and GTL2, that have similarities to IGF2 and H19, respectively. Both GTL2 and H19 are maternally expressed RNAs with no protein product and display paternal allele promoter region methylation, and DLK1 and IGF2 are both paternally expressed. To determine whether methylation alterations within the 14q32 imprinted domain occur in human tumorigenesis, we investigated the status of the GTL2 promoter differentially methylated region (DMR) in 20 neuroblastoma tumours, 20 phaeochromocytomas and, 40 Wilms' tumours. Hypermethylation of the GTL2 promoter DMR was detected in 25% of neuroblastomas, 10% of phaeochromocytoma and 2.5% of Wilms' tumours. Tumours with GTL2 promoter DMR hypermethylation also demonstrated hypermethylation at an upstream intergenic DMR thought to represent a germline imprinting control element. Analysis of neuroblastoma cell lines revealed that GTL2 DMR hypermethylation was associated with transcriptional repression of GTL2. These epigenetic findings are similar to those reported in Wilms' tumours in which H19 repression and DMR hypermethylation is associated with loss of imprinting (LOI, biallelic expression) of IGF2. However, a neuroblastoma cell line with hypermethylation of the GTL2 promoter and intergenic DMR did not show LOI of DLK1 and although treatment with a demethylating agent restored GTL2 expression and reduced DLK1 expression. As described for IGF2/H19, epigenetic changes at DLK1/GTL2 occur in human cancers. However, these changes are not associated with DLK1 LOI highlighting differences in the imprinting control mechanisms operating in the IGF2-H19 and DLK1-GTL2 domains. GTL2 promoter and intergenic DMR hypermethylation is associated with the loss of GTL2 expression and this may contribute to tumorigenesis in a subset of human cancers.
Project description:Genes subject to genomic imprinting exist in large chromosomal domains, probably reflecting coordinate regulation of the genes within a cluster. Such regulation has been demonstrated for the H19, Igf2, and Ins2 genes that share a bifunctional imprinting control region. We have identified the Dlk1 gene as a new imprinted gene that is paternally expressed. Furthermore, we show that Dlk1 is tightly linked to the maternally expressed Gtl2 gene. Dlk1 and Gtl2 are coexpressed and respond in a reciprocal manner to loss of DNA methylation. These genes are likely to represent a new example of coordinated imprinting of linked genes.
Project description:Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.
Project description:The evolution of genomic imprinting in mammals occurred more than 100 million years ago, and resulted in the formation of genes that are functionally haploid because of parent-of-origin-dependent expression. Despite ample evidence from studies in a number of species suggesting the presence of imprinted genes on human chromosome 14, their identity has remained elusive. Here we report the identification of two reciprocally imprinted genes, GTL2 and DLK1, which together define a novel imprinting cluster on human chromosome 14q32. The maternally expressed GTL2 (gene trap locus 2) gene encodes for a nontranslated RNA. DLK1 (delta, Drosophila, homolog-like 1) is a paternally expressed gene that encodes for a transmembrane protein containing six epidermal growth factor (EGF) repeat motifs closely related to those present in the delta/notch/serrate family of signaling molecules. The paternal expression, chromosomal localization, and biological function of DLK1 also make it a likely candidate gene for the callipyge phenotype in sheep. Many of the predicted structural and regulatory features of the DLK1/GTL2 domain are highly analogous to those implicated in IGF2/H19 imprint regulation, including two hemimethylated consensus binding sites for the vertebrate enhancer blocking protein, CTCF. These results provide evidence that a common mechanism and domain organization may be used for juxtapositioned, reciprocally imprinted genes.
Project description:Background:Embryo vitrification is a key instrument in assisted reproductive technologies (ARTs). However, there is increasing concern that vitrification adversely affects embryo development. This study intends to assess the effect of vitrification on developmental competence, in addition to expressions of long non-coding RNA (lncRNA) gene trap locus 2 (Gtl2) and its reciprocal imprinted gene delta-like homolog 1 (Dlk1), in mouse blastocysts. Materials and Methods:In this experimental study, we have designed three experimental groups: control (fresh blastocysts collected from superovulated mice), in vitro fertilization (IVF; blastocysts derived from IVF) and vitrification (IVF derived blastocysts subjected to vitrification/warming at the 2-cell stage). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to assess the expression levels of Gtl2 and Dlk1 in the blastocysts. Results:The results showed that vitrification group had significantly lower blastocyst and hatching rates compared to the IVF group (P<0.037) and (P<0.041), respectively. Gtl2 was down-regulated and Dlk1 was up-regulated following the IVF and vitrification (P<0.05). Conclusion:These results suggested that IVF and vitrification disturbed genomic imprinting and lncRNA gene expressions, which might affect the health of IVF children.
Project description:The Dlk1-Gtl2 imprinting locus is located on mouse distal chromosome 12 and consists of multiple maternally expressed non-coding RNAs and several paternally expressed protein-coding genes. The imprinting of this locus plays a crucial role in embryonic development and postnatal growth. At least one cis-element, the intergenic differentially methylated region (IG-DMR) is required for expression of maternally expressed genes and repression of silenced paternally expressed genes. The mechanism by which the IG-DMR functions is largely unknown. However, it has been suggested that the unmethylated IG-DMR acts as a positive regulator activating expression of non-coding RNAs. Gtl2 is the first non-coding RNA gene downstream of the IG-DMR. Although its in vivo function in the mouse is largely unknown, its human ortholog MEG3 has been linked to tumor suppression in human tumor-derived cell lines. We generated a knockout mouse model, in which the first five exons and adjacent promoter region of the Gtl2 gene were deleted. Maternal deletion of Gtl2 resulted in perinatal death and skeletal muscle defects, indicating that Gtl2 plays an important role in embryonic development. The maternal deletion also completely abolished expression of downstream maternally expressed genes, activated expression of silenced paternally expressed genes and resulted in methylation of the IG-DMR. By contrast, the paternal inherited deletion did not have this effect. These data strongly indicate that activation of Gtl2 and its downstream maternal genes play an essential role in regulating Dlk1-Gtl2 imprinting, possibly by maintaining active status of the IG-DMR.
Project description:The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.
Project description:Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/"Rian", AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.
Project description:Expression regulation of the Dlk1-Dio3 imprinted domain by the intergenic differentially methylated region (IG-DMR) is essential for normal embryonic development in mammals. In this study, we investigated conserved IG-DMR genomic sequences in eutherians to elucidate their role in genomic imprinting of the Dlk1-Dio3 domain. Using a comparative genomics approach, we identified three highly conserved sequences in IG-DMR. To elucidate the functions of these sequences in vivo, we generated mutant mice lacking each of the identified highly conserved sequences using the CRISPR/Cas9 system. Although mutant mice did not exhibit the gross phenotype, deletions of the conserved sequences altered the expression levels of paternally expressed imprinted genes in the mutant embryos without skewing imprinting status. These results suggest that the conserved sequences in IG-DMR are involved in the expression regulation of some of the imprinted genes in the Dlk1-Dio3 domain.