Imprinted genes in mice and humans mainly occur in clusters that are associated with differential DNA methylation of an imprint control element (ICE) and at least one nonprotein-coding RNA (ncRNA). Imprinted gene silencing is achieved by parental-specific insulator activity of the unmethylated ICE mediated by CTCF (CCCTC-binding factor) binding, or by ncRNA expression from a promoter in the unmethylated ICE. In many imprinted clusters, some genes, particularly those located furthest away from the ICE, show imprinted expression only in extraembryonic tissues. Recent research indicates that genes showing imprinted expression only in extraembryonic tissues may be regulated by different epigenetic mechanisms compared with genes showing imprinted expression in extraembryonic tissues and in embryonic/adult tissues. The study of extraembryonic imprinted expression, thus, has the potential to illuminate novel epigenetic strategies, but is complicated by the need to collect tissue from early stages of mouse development, when extraembryonic tissues may be contaminated by maternal cells or be present in limited amounts. Research in this area would be advanced by the development of an in vitro model system in which genetic experiments could be conducted in less time and at a lower cost than with mouse models. Here, we summarize what is known about the mechanisms regulating imprinted expression in mouse extraembryonic tissues and explore the possibilities for developing an in vitro model.
genomic imprinting; placenta; yolk sac; noncoding RNA; insulator
The mouse achaete-scute homolog-2 gene (Ascl2 or Mash2) encodes a transcription factor playing a role in the development of the trophoblast. The Ascl2 is an imprinted gene with maternal expression and assigned to an imprinting gene cluster region (ICR) at a distal region of mouse chromosome 7. We previously isolated a phage clone carrying the human homolog, ASCL2, and mapped it to human chromosome 11p15.5, a human ICR. In the present study, we demonstrate the expression patterns of the human ASCL2 in the fetus at a stage between first and second trimesters and in the placental tissues. In addition, it has been shown that the human ASCL2 gene escapes genomic imprinting.
ASCL2; imprinting; expression; placenta
Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles.
Embryonic stem (ES) cells; Epigenetics; Genomic imprinting; ES in vitro imprinting model; ncRNA; Histone modifications; DNA methylation; Allele-specific expression; Airn (Air)
Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11–q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.
Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC.
Genomic Imprinting; Sfmbt2; PcG gene; Extraembryonic tissues; placenta; MMU2; YY1; microarrays; uniparental embryos
Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice.
Considering the important functions of several IC2-regulated genes in placentation, we set out to determine whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae.
Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully rescuing trans placental abnormalities that are caused by imprinting defects.
Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse.
Evolutionarily conserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA)/(2·CpG) ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements.
We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in epigenetic features and a special response to selective pressure. In addition, our data support the potential role of intronic CpG islands as epigenetic key regulatory elements and suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions.
A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.
genomic imprinting; Rasgrf1; DNA methylation; epigenetics
Nuclear architecture and chromatin geography are important factors in the regulation of gene expression, as these components may play a vital epigenetic role both in normal physiology as well as in the initiation and progression of malignancies. Using a modification of the chromosome conformation capture (3C) technique, we examined long-range chromatin interactions of the imprinted human IGF2 gene. We demonstrate that numerous intrachromosomal interactions occur along both parental alleles in normal tissues, where the IGF2 is paternally expressed, as well as in normal liver where gene expression is biallelic. Long-range and allele-specific interactions occur between the IGF2/H19 imprinting control region-1 (ICR1) and ICR2, a region which regulates an imprinted gene cluster nearly a megabase distant from IGF2. Loss of genomic imprinting is a common epigenetic event in cancer, and long-range interactions have not been examined in malignant cells. In cancer cell lines in which IGF2 imprinting is maintained (MOI), essentially all of the 3C interactions seen in normal cells were preserved. However, in cells in which IGF2 imprinting was lost (LOI), nearly all of the long-range chromatin interactions involving IGF2 were abrogated. A three-dimensional computer model depicts the physical interactions between the IGF2 promoter and ICR1 in MOI cells, while the model of LOI lung cancer cells is flattened with few long-range interactions. This dramatic change in the three-dimension configuration of the chromatin at the IGF2 locus in LOI cancer cells suggests that the loss of imprinting may lead to a variety of changes in gene expression in addition to changes in IGF2 transcription.
Tssc3 is a maternally expressed imprinted gene.
Results: TSSC3 regulates Mash2 transcription in TS cells through phosphorylation of AKT and Sp1 translocation from cytoplasm to nucleus.
Conclusion: TSSC3 determines the fate of TS cells in terms of development into trophoblast progenitors and/or labyrinth trophoblasts.
Significance: TSSC3 regulates TS cell differentiation through the AKT/Sp1/MASH2 signaling pathway.
Tssc3 is a maternally expressed/paternally silenced imprinted gene. Recent evidence suggests that the loss of TSSC3 results in placental overgrowth in mice. These findings showed that the TSSC3 gene functions as a negative regulator of placental growth. In this study, we describe the function of TSSC3 and its signaling pathway in mouse trophoblast stem (TS) cell differentiation. First of all, we tested Tssc3 expression levels in TS cells. TS cells expressed Tssc3, and its expression level was the highest from day 1 to 4 but was down-regulated at day 5 after the induction of differentiation. Overexpression of TSSC3 in TS cells up-regulated Gcm1 and Mash2, which are marker genes of mouse trophoblast differentiation. Down-regulation of TSSC3 by siRNA enhanced Pl1 and Tpbpa expression in TS cells cultured under stem cell conditions, suggesting the contribution of TSSC3 to the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts. TSSC3 activated the PI3K/AKT pathway through binding with phosphatidylinositol phosphate lipids and enhanced the activity of a promoter containing an E-box structure, which is the binding sequence of the Mash2 downstream target gene promoter. PI3K inhibitor suppressed the promoter activity induced by TSSC3. TSSC3 induced Sp1 translocation from cytoplasm to nucleus through the PI3K/AKT pathway. Nuclear Sp1 activated the Mash2 transcription by Sp1 binding with a consensus Sp1-binding motif. This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway.
Molecular Cell Biology; Placenta; Stem Cells; Transcription Factors; Trophoblast
Approximately 100 mouse genes undergo genomic imprinting, whereby one of the two parental alleles is epigenetically silenced. Imprinted genes influence processes including development, X chromosome inactivation, obesity, schizophrenia, and diabetes, motivating the identification of all imprinted loci. Local sequence features have been used to predict candidate imprinted genes, but rigorous testing using reciprocal crosses validated only three, one of which resided in previously identified imprinting clusters. Here we show that specific epigenetic features in mouse cells correlate with imprinting status in mice, and we identify hundreds of additional genes predicted to be imprinted in the mouse. We used a multitiered approach to validate imprinted expression, including use of a custom single nucleotide polymorphism array and traditional molecular methods. Of 65 candidates subjected to molecular assays for allele-specific expression, we found 10 novel imprinted genes that were maternally expressed in the placenta.
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.
Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.
In this study, we characterised the phenotype of mice carrying different copy number integrations of a bacterial artificial chromosome spanning Cdkn1c. Excess Cdkn1c resulted in embryonic growth retardation that was dosage-dependent and also responsive to the genetic background. Two-fold expression of Cdkn1c in a subset of tissues caused a 10–30% reduction in embryonic weight, embryonic lethality and was associated with a reduction in the expression of the potent, non-imprinted embryonic growth factor, Igf1. Conversely, loss of expression of Cdkn1c resulted in embryos that were 11% heavier with a two-fold increase in Igf1.
We have shown that embryonic growth in mice is exquisitely sensitive to the precise dosage of Cdkn1c. Cdkn1c is a maternally expressed gene and our findings support the prediction of the parental conflict hypothesis that that the paternal genome silences genes that have an inhibitory role in embryonic growth. Within the IC2 imprinted domain, Cdkn1c encodes the major regulator of embryonic growth and we propose that Cdkn1c was the focal point of the selective pressure for imprinting of this domain.
Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
Genomic imprinting is a specialized system of gene regulation whereby only one copy of a gene is used, either the maternal or the paternal copy. Misregulation of imprinting in humans results in developmental disorders such as Beckwith-Wiedemann Syndrome, and is implicated in many cancers. Study of imprinted genes in mice can lead to a greater understanding of these diseases as well as insight into gene regulation. Many imprinted genes are associated with methylation on the silenced allele. The imprinted gene H19 is maternally expressed and paternally methylated in a region upstream of the promoter known as the differentially methylated domain. This region is required for proper imprinted expression of H19 and its upstream imprinted neighbor Igf2. Our studies have explored the requirement for methyl-CpG binding protein 3 (MBD3) in silencing of the paternal allele. MBD3 is known to be part of a repressive complex that resides at silenced genes. In our experiments, we have shown that MBD3 is required for imprinting of H19, and is also required for the maintenance of methylation on the paternal allele. Finally, the MBD3 protein can be found at the differentially methylated domain. The identification of a protein required for silencing of the paternal allele of H19 is an important step in understanding regulation of this gene.
Genomic imprinting is an epigenetic phenomenon affecting a small number of genes which leads to differential expression from the two parental alleles. Imprinted genes are known to regulate fetal growth and a ‘kinship’ or ‘parental conflict’ model predicts that paternally- and maternally-expressed imprinted genes promote and inhibit fetal growth, respectively. In this review we examine the role of imprinted genes in postnatal growth and metabolism, with an emphasis on the GNAS/Gnas locus. GNAS is a complex imprinted locus with multiple oppositely imprinted gene products, including the G protein α-subunit Gsα which is expressed primarily from the maternal allele in some tissues and the Gsα isoform XLαs which is expressed only from the paternal allele. Maternal, but not paternal, Gsα mutations lead to obesity in Albright hereditary osteodystrophy. Mouse studies show that this phenomenon is due to Gsα imprinting in the central nervous system leading to a specific defect in the ability of central melanocortins to stimulate sympathetic nervous system activity and energy expenditure. In contrast mutation of paternally-expressed XLαs leads to opposite metabolic effects in mice. While these findings conform to the ‘kinship’ model, the effects of other imprinted genes on body weight regulation do not conform to this model.
G protein; genomic imprinting; obesity; pseudohypoparathyroidism; Prader-Willi syndrome; Angelmans syndrome
Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X-chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.
The closely linked H19 and Igf2 genes show highly similar patterns of gene expression but are reciprocally imprinted. H19 is expressed almost exclusively from the maternally inherited chromosome, while Igf2 expression is mostly from the paternal chromosome. In humans, loss of imprinting at this locus is associated with tumors and with developmental disorders. Monoallelic expression at the imprinted Igf2/H19 locus occurs by at least two distinct mechanisms: a developmentally regulated silencing of the paternal H19 promoter, and transcriptional insulation of the maternal Igf2 promoters. Both mechanisms of allele-specific silencing are ultimately dependent on a common cis-acting element located just upstream of the H19 promoter. The coordinated expression patterns and some experimental data support the idea that positive regulatory elements are also shared by the two genes. To clarify the organization and function of positive and negative regulatory elements at the H19/Igf2 locus, we analyzed two mouse mutations. First, we generated a deletion allele to localize enhancers used in vivo for expression of both H19 and Igf2 in mesodermal tissues to sequences downstream of the H19 gene. Coincidentally, we demonstrated that some expression of Igf2 is independent of the shared enhancer element. Second, we used this new information to further characterize an ectopic H19 differentially regulated region and the associated insulator. We demonstrated that its activity is parent-of-origin dependent. In contrast to recent results from Drosophila model systems; we showed that this duplication of a mammalian insulator does not interfere with its normal function. Implications of these findings for current models for monoallelic gene expression at this locus are discussed.
By combining a tissue-specific microarray screen with mouse uniparental duplications, we have identified a novel imprinted gene, Dopa decarboxylase (Ddc), on chromosome 11. Ddc_exon1a is a 2-kb transcript variant that initiates from an alternative first exon in intron 1 of the canonical Ddc transcript and is paternally expressed in trabecular cardiomyocytes of the embryonic and neonatal heart. Ddc displays tight conserved linkage with the maternally expressed and methylated Grb10 gene, suggesting that these reciprocally imprinted genes may be coordinately regulated. In Dnmt3L mutant embryos that lack maternal germ line methylation imprints, we show that Ddc is overexpressed and Grb10 is silenced. Their imprinting is therefore dependent on maternal germ line methylation, but the mechanism at Ddc does not appear to involve differential methylation of the Ddc_exon1a promoter region and may instead be provided by the oocyte mark at Grb10. Our analysis of Ddc redefines the imprinted Grb10 domain on mouse proximal chromosome 11 and identifies Ddc_exon1a as the first example of a heart-specific imprinted gene.
The H19/IGFf2 locus belongs to a large imprinted domain located on human chromosome 11p15.5 (homologue to mouse distal chromosome 7). The H19 gene is expressed from the maternal allele, while IGF2 is paternally expressed. Natural antisense transcripts and intergenic transcription have been involved in many aspects of eukaryotic gene expression, including genomic imprinting and RNA interference. However, apart from the identification of some IGF2 antisense transcripts, few data are available on that topic at the H19/IGF2 locus. We identify here a novel transcriptional activity at both the human and the mouse H19/IGF2 imprinted loci. This activity occurs antisense to the H19 gene and has the potential to produce a single 120-kb transcript that we called the 91H RNA. This nuclear and short-lived RNA is not imprinted in mouse but is expressed predominantly from the maternal allele in both mice and humans within the H19 gene region. Moreover, the transcript is stabilized in breast cancer cells and overexpressed in human breast tumors. Finally, knockdown experiments showed that, in humans, 91H, rather than affecting H19 expression, regulates IGF2 expression in trans.
To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.
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.
Loss of genomic imprinting is involved in a number of developmental abnormalities and cancers. ZAC is an imprinted gene expressed from the paternal allele of chromosome 6q24 within a region known to harbor a tumor suppressor gene for several types of neoplasia. p57KIP2 (CDKN1C) is a maternally expressed gene located on chromosome 11p15.5 which encodes a cyclin-dependent kinase inhibitor that may also act as a tumor suppressor gene. Mutations in ZAC and p57KIP2 have been implicated in transient neonatal diabetes mellitus (TNDB) and Beckwith–Wiedemann syndrome, respectively. Patients with these diseases share many characteristics. Here we show that mouse Zac1 and p57Kip2 have a strikingly similar expression pattern. ZAC, a sequence-specific DNA-binding protein, binds within the CpG island of LIT1 (KCNQ1OT1), a paternally expressed, anti-sense RNA thought to negatively regulate p57KIP2 in cis. ZAC induces LIT1 transcription in a methylation-dependent manner. Our data suggest that ZAC may regulate p57KIP2 through LIT1, forming part of a novel signaling pathway regulating cell growth. Mutations in ZAC may, therefore, contribute to Beckwith–Wiedemann syndrome. Furthermore, we find changes in DNA methylation at the LIT1 putative imprinting control region in two patients with TNDB.
Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.
Genomic imprinting results in preferential expression of the paternal, or maternal allele of certain genes. We have performed a genome-wide characterization of imprinting in the mouse embryonic and adult brain. This approach uncovered parent-of-origin allelic effects in over 1300 loci. We identified parental bias in the expression of individual genes and of specific transcript isoforms, with differences between brain regions. Many imprinted genes are expressed in neural systems associated with feeding and motivated behaviors, and parental biases preferentially target genetic pathways governing metabolism and cell adhesion. We observed a preferential maternal contribution to gene expression in the developing brain and a major paternal contribution in the adult brain. Thus, parental expression bias emerges as a major mode of epigenetic regulation in the brain.
H19 and Igf2 are reciprocally imprinted genes that lie 90 kb apart on mouse chromosome 7. The two genes are coexpressed during development, with the H19 gene expressed exclusively from the maternal chromosome and Igf2 from the paternal chromosome. It has been proposed that their reciprocal imprinting is governed by a competition between the genes for a common set of enhancers. The competition on the paternal chromosome is influenced by extensive allele-specific methylation of the H19 gene and its 5′ flank, which acts to inhibit H19 transcription and thus indirectly leads to the activation of the Igf2 gene. In contrast, no allele-specific methylation has been detected on the maternal chromosome, and the basis for the preference for H19 transcription on that chromosome is unresolved. In this investigation, the mechanism controlling the silencing of the Igf2 gene on the maternal chromosome was explored by studying the transcriptional activity of a yeast artificial chromosome (YAC) containing Igf2 and H19 following transfer into differentiated tissue culture cells. Contrary to expectations, both H19 and Igf2 were expressed from a single integrated copy of the YAC. Furthermore, Igf2 expression appeared to be independent of the H19 locus, based on deletions of the H19 gene promoter and its enhancers. These results suggest that an active process is responsible for the transcriptional bias toward H19 on the maternal chromosome and that the hypomethylated state of this chromosome cannot be viewed as a “default” state. Moreover, the active process is not reproduced in a differentiated cell and may require passage through the female germ line.