Imprinting of distal mouse chromosome 2 is associated with phenotypic anomalies in utero

Previous studies have shown that the distal region on mouse chromosome (Chr) 2 is subject to imprinting as mice with maternal duplication/paternal deficiency (MatDp.dist2) and the reciprocal (PatDp.dist2) for this region exhibit phenotypic anomalies at birth and die neonatally. We show here that imprinting effects are detectable in utero. Notably PatDp.dist2 embryos show an increase in wet weight compared with normal, which peaks at 16.5 d post coitum (dpc), and diminishes by birth, whereas the wet weight of placenta is slightly reduced in the latter half of gestation. Newborns have increased length of the long bones. By contrast, the wet weight of MatDp.dist2 embryos decreases during the second half of gestation. Measurements of dry weights of embryos at 16.5 dpc have indicated that there is no difference in either PatDp.dist2 or MatDp.dist2 compared with normal so that the wet weight differences are due to fluid retention in PatDp.dist2 but fluid loss in MatDp.dist2. In PatDp.dist2 embryos excess fluid is particularly prominent in the subcuticular skin layer, whereas by birth fluid is evident around the neck and tongue. At 16.5 dpc the PatDp.dist2 embryos are severely oedematous, as the average fluid content per unit dry weight per embryo was increased by 40%, whereas the MatDp.dist2 embryos are dehydrated as the average water content per unit dry weight per embryo was reduced by 6%. A preliminary conclusion is that there is neither growth enhancement in PatDp.dist2 nor growth retardation in MatDp.dist2 offspring.     

Peg1/Mest locates distal to the currently defined imprinting region on mouse proximal chromosome 6 and identifies a new imprinting region affecting growth

Mice with maternal duplication for proximal chromosome 6 (Chr 6) die in utero before 11.5 dpc, an effect that can be attributed to genomic imprinting. Previous studies have defined the region of Chr 6 responsible as lying proximal to the T6Ad translocation breakpoint in G-band 6B3. Evidence presented here with a new Chr 6 translocation T77H has substantially reduced the size of the imprinting region, locating it between G-band 6A3.2 and the centromere. The paternally expressed imprinted gene Mest had been mapped within the original imprinting region and was therefore a candidate for the early embryonic lethality. FISH has shown that Mest locates distal to T77H and therefore outside the redefined imprinting region. This evidence confirms that Mest is not a candidate for the early embryonic lethality found with two maternal copies of proximal Chr 6. Furthermore mice with maternal duplication for Ch 6 distal to T77H (MatDp.dist6) were found to be growth retarded at birth, the weight reduction remaining similar until adulthood. It can be concluded that the growth retardation is established in utero and is maintained at a similar level from birth to adulthood. Therefore Mest locates in a new imprinting region, distal to G-band 6A3.2 which affects growth. A targeted mutation of Mest has been reported that exhibits growth retardation, reduced postnatal survival and abnormal maternal behaviour. Here the phenotype of MatDp.dist6 mice is compared to that of Mest-deficient mutant mice. Unlike the latter, MatDp.dist6 mice have good survival rates and females have normal maternal behaviour. Possible reasons for these differences are discussed.     

Murine Stim1 maps to distal Chromosome 7 and is not imprinted

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects. Received: 23 January 1998 / Accepted: 10 April 1998     

Towards unravelling the Igf2/H19 imprinted domain

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence⁽¹⁾ suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.     

Analysis of mouse conceptuses with uniparental duplication/deficiency for distal chromosome 12: comparison with chromosome 12 uniparental disomy and implications for genomic imprinting

Distal mouse chromosome 12 is imprinted. Phenotypic analysis of mouse embryos with maternal or paternal uniparental disomy for the whole of chromosome 12 has characterized the developmental defects associated with the altered dosage of imprinted genes on this chromosome. Here we conduct a characterization of maternal and paternal Dp(dist12) mice using the reciprocal translocation T(4;12)47H. This limits the region analysed to the chromosomal domain distal to the T47H breakpoint in B3 on mouse chromosome 12. Both MatDp(dist12)T47H and PatDp(dist12)T47H conceptuses are non-viable and the frequency of recovery of Dp(dist12) conceptuses by 10.5 days post coitum (dpc) was lower than expected after normal adjacent-1 disjunction. A subset of MatDp(dist12) embryos can survive up to one day post partum. In contrast to paternal uniparental disomy 12 embryos, no live PatDp (dist12) embryos were recovered after 16.5 days of gestation. Other phenotypes observed in maternal and paternal chromosome 12 uniparental disomy mice are recapitulated in the Dp(dist12) mice and include placental, muscle and skeletal defects. Additional defects were also noted in the skin of both MatDp(dist12) and maternal uniparental disomy 12 embryos. This study shows that the developmental abnormalities associated with the altered parent of origin for mouse chromosome 12 can be attributed to the genomic region distal to the T47H breakpoint.     

Analysis of imprinting in mice with uniparental duplication of proximal chromosomes 7 and 15 by use of a custom oligonucleotide microarray

We have developed an imprinting assay combining the use of mice carrying maternal or paternal duplication of chromosomal regions of interest with custom oligonucleotide microarrays. As a model system, we analyzed RNA from CNS tissue of neonatal mice carrying the reciprocal translocation T(7;15)9H and uniparental duplication of proximal Chr 7 and 15. The duplicated region includes the locus on proximal Chr 7 corresponding to the human Prader-Willi/Angelman Syndrome. The microarray contained 322 oligonucleotides, including probes to detect major genes involved in neural excitability and synaptic transmission, as well as known imprinted genes mapping to proximal Chr 7: Ndn, Snrpn, Mkrn3, Magel2, Peg3, and Ube3a. Imprinting of these genes in neonatal cortex and cerebellum was first confirmed by quantitative RT-PCR. Their inclusion on the microarray thus provided positive controls for evaluating the effect of background on the sensitivity of the assay, and for establishing the minimum level of expression required to detect imprinting. Our analysis extended previous work by revealing bi-allelic expression in CNS tissue of those queried genes mapping to proximal Chr 7 or 15, including the Gabrb3 gene, for which there have been conflicting reports. Microarray analysis also revealed no effect of the maternal or paternal disomy on expression levels of the unlinked genes detected, including those potentially implicated in the Prader-Willi or Angelman Syndrome. In addition, quantitative RT-PCR revealed a gene dosage effect in both cerebellum and cortex for all of the known imprinted genes assayed, except for Ube3a in cerebellum.     

Epigenetic and phenotypic consequences of a truncation disrupting the imprinted domain on distal mouse chromosome 7

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

Imprinted methylation profiles for proximal mouse Chromosomes 11 and 7 as revealed by methylation-sensitive representational difference analysis

Proximal mouse Chromosome (Chr) 11 shares regions of orthology with the candidate gene region for the imprinting growth disorder Silver-Russell syndrome (SRS) on human Chr 7p. It has previously been shown that mice with two maternal or two paternal copies (duplications, Dp) of proximal Chr 11 exhibit reciprocal growth phenotypes. Those with two paternal copies show fetal and placental overgrowth, while those with two maternal copies are growth retarded. The growth retardation observed in the latter is reminiscent of the intrauterine growth restriction (IUGR) observed in SRS patients with maternal uniparental disomy for Chr 7 (mUPD7). We have carried out a methylation-sensitive representational difference analysis (Me-RDA) screen to look for regions of differential methylation (DMRs) associated with imprinted genes. For these experiments, we have used mouse embryos with uniparental duplications of Chrs 11 and 7 proximal to the breakpoint of the reciprocal translocation T(7;11)40Ad. Two previously known imprinted loci associated with paternal allele hypomethylation were recovered on proximal mouse Chr 11, U2af1-rs1 and Meg1/Grb10. These two genes map 15 cM apart, so it seems likely that they are within separate imprinted domains that do not contain additional DMRs. The known imprinted gene Peg3, located on mouse proximal Chr 7, was also detected in our screen. The finding that Peg3 was differentially methylated in embryos with uniparental inheritance of proximal Chr 7 confirms that Peg3 is located proximal to the breakpoint of T40Ad in G-band 7A2. Because GRB10 has previously been reported to be a candidate gene for SRS, we analysed 22 patients for epimutations of the GRB10 differentially methylated region that could lead to the altered expression of this gene. No such mutations were found.     

Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation

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.     

Ubiquitous expression and imprinting of Snrpn in the mouse

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.     

A reassessment of imprinting regions and phenotypes on mouse chromosome 6: Nap1l5 locates within the currently defined sub-proximal imprinting region

Previous studies (Beechey, 2000) have shown that mouse proximal chromosome (Chr) 6 has two imprinting regions. An early embryonic lethality is associated with two maternal copies of the more proximal imprinting region, while mice with two maternal copies of the sub-proximal imprinting region are growth retarded at birth, the weight reduction remaining similar to adulthood. No detectable postnatal imprinting phenotype was seen in these earlier studies with two paternal copies of either region. The sub-proximal imprinting region locates distal to the T77H reciprocal translocation breakpoint in G-band 6A3.2 and results reported here show that it does not extend beyond the breakpoint of the more distal T6Ad translocation in 6C2. It has been confirmed that the postnatal growth retardation observed with two maternal copies of the sub-proximal region is established in utero, although placental size was normal. A new finding is that 16.5-18.5-dpc embryos, with two paternal copies of the sub-proximal imprinting region, were larger than their normal sibs, although placental size was normal. As no postnatal growth differences have been observed in these mice, the fetal overgrowth must normalize by birth. The imprinted genes Peg1/Mest, Copg2, Copg2as and Mit1/Lb9 map to the sub-proximal imprinting region and are thus candidates for the observed imprinting phenotypes. Another candidate is the recently reported imprinted gene Nap1l5. Expression studies of Nap1l5 in mice with two maternal or two paternal copies of different regions of Chr 6 have demonstrated that the gene locates within the sub-proximal imprinting region. FISH has mapped Nap1l5 to G-band 6C1, within the sub-proximal imprinting region but several G-bands distal to the Peg1/Mest cluster. This location, and the 30-Mb separation of these loci on the sequence map, makes it probable that Nap1l5 defines a new imprinting domain within the currently defined sub-proximal imprinting region.     

Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome

Background  

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.     

Gene homologs on human chromosome 15q21-q26 and a chicken microchromosome identify a new conserved segment

The genes for insulin-like growth factor 1 receptor (IGF1R), aggrecan (AGC1), β₂-microglobulin (B2M), and an H6-related gene have been mapped to a single chicken microchromosome by genetic linkage analysis. In addition, a second H6-related gene was mapped to chicken macrochromosome 3. The Igf1r and Agc1 loci are syntenic on mouse Chr 7, together with Hmx3, an H6-like locus. This suggests that the H6-related locus, which maps to the chicken microchromosome in this study, is the homolog of mouse Hmx3. The IGF1R, AGC1, and B2M loci are located on human Chr 15, probably in the same order as found for this chicken microchromosome. This conserved segment, however, is not entirely conserved in the mouse and is split between Chr 7 (Igf1r-Agc) and 2 (B2m). This comparison also predicts that the HMX3 locus may map to the short arm of human Chr 15. The conserved segment defined by the IGF1R–AGC1–HMX3—B2M loci is approximately 21–35 Mb in length and probably covers the entire chicken microchromosome. These results suggest that a segment of human Chr 15 has been conserved as a chicken microchromosome. The significance of this result is discussed with reference to the evolution of the avian and mammalian genomes. Received: 7 December 1996 / Accepted: 7 February 1997