Imprinting of an evolutionarily conserved antisense transcript gene APeg3.
ABSTRACT: APeg3 is an antisense transcript gene of Peg3, which has been recently identified from rat brain. Careful analyses of EST databases indicated that a homologous transcript also exists in other mammalian species, including mouse, cow and human. 5'-and 3'-RACE experiments have subsequently identified a 900-bp cDNA sequence of APeg3 from mouse brain. Mouse APeg3 is localized in the 3'UTR of Peg3 with an intronless genomic structure. The expression of mouse APeg3 is derived mainly from the paternal allele, indicating the imprinting of this antisense transcript gene in brain. Strand-specific RNA analyses also revealed the expression of both human and cow APEG3 in adult brains. In sum, our study confirms that the mammalian PEG3 locus harbors an antisense transcript gene displaying paternal allele-specific expression, and the evolutionary conservation further suggests potential roles of this transcript gene for the function of this imprinted domain.
Project description:Peg3 (paternally expressed gene 3) is an imprinted gene localized within an evolutionarily conserved 500-kb domain in human chromosome 19q13.4 and mouse proximal chromosome 7. In the current study, we have identified three alternative promoters for mouse Peg3 and one alternative promoter for human PEG3. These alternative promoters are localized within the 200-kb upstream region of human and mouse PEG3, which is well conserved and thus predicted to harbor several cis-regulatory elements for the PEG3 domain. In the mouse, two of these alternative promoters drive maternal-specific expression of Peg3 specifically in the hypothalamus of the adult brain, while the remaining third promoter drives bi-allelic expression of Peg3 with a paternal bias only in the neonatal-stage brain. In human, an alternative transcript is also detected at relatively very low levels in adult brain and placenta. Overall, the identification of alternative promoters in both mouse and human models suggests that these alternative promoters may be functionally selected features for the PEG3 imprinted domain during mammalian evolution.
Project description:The imprinting and transcription of the 500 kb genomic region surrounding the mouse Peg3 is predicted to be regulated by the Peg3-differentially methylated region (DMR). In the current study, this prediction was tested using a mutant mouse line lacking this potential imprinting control region (ICR). At the organismal level, paternal and maternal transmission of this knockout (KO) allele caused either reduced or increased growth rates in the mouse, respectively. In terms of the imprinting control, the paternal transmission of the KO allele resulted in bi-allelic expression of the normally maternally expressed Zim2, whereas the maternal transmission switched the transcriptionally dominant allele for Zfp264 (paternal to maternal). However, the allele-specific DNA methylation patterns of the DMRs of Peg3, Zim2 and Zim3 were not affected in the mice that inherited the KO allele either paternally or maternally. In terms of the transcriptional control, the paternal transmission caused a dramatic down-regulation in Peg3 expression, but overall up-regulation in the other nearby imprinted genes. Taken together, deletion of the Peg3-DMR caused global changes in the imprinting and transcription of the Peg3 domain, confirming that the Peg3-DMR is an ICR for this imprinted domain.
Project description:The parental allele specificity of mammalian imprinted genes has been evolutionarily well conserved, although its functional constraints and associated mechanisms are not fully understood. In the current study, we generated a mouse mutant with switched active alleles driving the switch from paternal-to-maternal expression for Peg3 and the maternal-to-paternal expression for Zim1. The expression levels of Peg3 and Zim1, but not the spatial expression patterns, within the brain showed clear differences between wild type and mutant animals. We identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and that also participate in allele-biased chromosomal conformations with regional promoters. Most importantly, these data suggest for the first time that long-distance enhancers may contribute to allelic expression within imprinted domains through allele-biased interactions with regional promoters.
Project description:The expression of mouse Peg3 (Paternally expressed gene 3) is driven by 4 promoters, including its main and three alternative promoters. The sexual, temporal and spatial specificity of these promoters was characterized in the current study. According to the results, the main promoter displays ubiquitous expression patterns throughout different stages and tissues. In contrast, the expression of Peg3 driven by the alternative promoter U2 was detected mainly in muscle and skin, but not in brain, starting from the late embryonic stage, revealing its tissue and stage specificity. The expression levels of both the main and U2 promoters are also sexually biased: the levels in females start higher but become lower than those in males during early postnatal stages. As an imprinted locus, the paternal alleles of these promoters are active whereas the maternal alleles are silent. Interestingly, deletion of the repressed maternal allele of the main promoter has an unusual effect on the opposite paternal allele, causing the up-regulation of both the main and U2 promoters. Overall, the promoters of Peg3 derive sexually biased and tissue-specific expression patterns.
Project description:Genomic imprinting, the preferential expression of maternal or paternal alleles of imprinted genes, is often maintained through expression of imprinted long non-coding (lnc) "antisense" RNAs. These may overlap imprinted transcripts, and are expressed from the opposite allele. Previously we have described brain region-specific imprinted expression of the Dio3 gene in rat, which is preferentially modified by fetal ethanol exposure. The Dio3os (opposite strand) transcript is transcribed in opposite orientation to Dio3 in mouse and human, partially overlaps the Dio3 promoter, and mirrors total Dio3 developmental expression levels. Here, we present that the rat Dio3os transcript(s) exhibits brain region-specific imprinted expression patterns similar to those of Dio3. Rat Dio3os transcript expression is also similarly modified by fetal ethanol exposure. Uniquely, both Dio3 and Dio3os expression occur on the same, rather than opposite, alleles, as determined by strand-specific RT-PCR. Future studies will require direct manipulation of the Dio3os transcript to determine whether the novel paralleling of total and allele-specific expression patterns of this sense/antisense imprinted gene pair reflects an as-yet undefined regulatory mechanism for lncRNA mediated tissue-specific imprinted expression, or rather is a consequence of a more straightforward, but previously undescribed transcriptional coregulation process.
Project description:In the current study, the imprinting control region of the mouse Peg3 domain was deleted to test its functional impact on animal growth and survival. The paternal transmission of the deletion resulted in complete abolition of the transcription of two paternally expressed genes, Peg3 and Usp29, causing the reduced body weight of the pups. In contrast, the maternal transmission resulted in the unexpected transcriptional up-regulation of the remaining paternal allele of both Peg3 and Usp29, causing the increased body weight and survival rates. Thus, the imprinted maternal allele of the ICR may be a suppressor antagonistic to the active paternal allele of the ICR, suggesting a potential intralocus allelic conflict. The opposite outcomes between the two transmissions also justify the functional compromise that the maternal allele has become epigenetically repressed rather than genetically deleted during mammalian evolution. The mice homozygous for the deletion develop normally but with a skewed sex ratio, one male per litter, revealing its sex-biased effect. Overall, the Peg3 locus may have evolved to an imprinted domain to cope with both parental and sexual conflicts driven by its growth-stimulating paternal versus growth-suppressing maternal alleles.
Project description:The imprinting of the mouse Peg3 domain is controlled through a 4-kb genomic region encompassing the bidirectional promoter and 1st exons of Peg3 and Usp29. In the current study, this ICR was inverted to test its orientation dependency for the transcriptional and imprinting control of the Peg3 domain. The inversion resulted in the exchange of promoters and 1st exons between Peg3 and Usp29. Paternal transmission of this inversion caused 10-fold down-regulation of Peg3 and 2-fold up-regulation of Usp29 in neonatal heads, consistent with its original promoter strength in each direction. The paternal transmission also resulted in reduced body size among the animals, which was likely contributed by the dramatic down-regulation of Peg3. Transmission through either allele caused no changes in the DNA methylation and imprinting status of the Peg3 domain except that Zfp264 became bi-allelic through the maternal transmission. Overall, the current study suggests that the orientation of the Peg3-ICR may play no role in its allele-specific DNA methylation, but very critical for the transcriptional regulation of the entire imprinted domain.
Project description:Using mouse BAC clones spanning an imprinted interval of proximal mouse chromosome 7 and the genomic sequence of the related interval of human chromosome 19q13.4, we have identified a novel mouse gene, Usp29 (ubiquitin-specific processing protease 29), near two known imprinted genes, Peg3 and Zim1. Gene Usp29 is located directly adjacent to Peg3 in a "head-to-head" orientation, and comprises exons distributed over a genomic distance of at least 400 kb. A similar human gene is also found in the homologous location in human chromosome 19q13.4. The mouse Usp29 gene is also imprinted and is transcribed mainly from the paternal allele with highest expression levels in adult brain, especially in the cerebral cortex and hippocampus, and in the forebrain, face, and limb buds of midgestation mouse embryos. Analysis of a full-length 7.6-kb cDNA clone revealed that Usp29 encodes an 869-amino-acid protein that displays significant homology with yeast and nematode ubiquitin carboxyl-terminal hydrolases. These data suggest that, like the candidate Angelman syndrome gene Ube3a (ubiquitin ligase), Usp29 may represent another imprinted gene involved in the ubiquitination pathway. This identification of a third imprinted gene, Usp29, from the Peg3/Zim1-region confirms the presence of a conserved imprinted domain spanning at least 500 kb in the proximal portion of mouse chromosome 7 (Mmu7).
Project description:In this study, we identified an antisense transcript to ZIM2 (zinc finger imprinted gene 2) in the human, called ZIM2as. Sequence analysis of the 110 kb region spanned by this transcript revealed a cluster of tandemly repeated sequence in the human, orangutan, and chimpanzee as well as a loss of approximately 70 kb from the corresponding region in the rhesus. The homologous region in most mammals contains a cluster of olfactory receptor (OLFR) genes, but this gene cluster has been lost from the primate lineage. Expression analyses confirmed that ZIM2as is expressed in the human brain and testis. Two CpG islands near the promoter region of ZIM2as showed different methylation patterns in these three species. The CpG island distal to ZIM2as showed an allele-specific DNA methylation pattern in the human testis, while the CpG island proximal to the ZIM2as promoter showed a mosaic methylation pattern in the chimpanzee. The methylation status of several nearby zinc finger genes was unchanged among the primates tested. Overall, this study reports the presence of a previously unreported primate-specific antisense transcript in the PEG3 imprinted domain, suggesting that the formation of this transcript may coincide with the loss of the OLFR cluster.
Project description:The ICR (Imprinting Control Region) of the Peg3 (Paternally Expressed Gene 3) domain contains an unusual cluster of YY1 binding sites. In the current study, these YY1 binding sites were mutated to characterize the unknown roles in the mouse Peg3 domain. According to the results, paternal and maternal transmission of the mutant allele did not cause any major effect on the survival of the pups. In the mutants, the maternal-specific DNA methylation on the ICR was properly established and maintained, causing no major effect on the imprinting of the domain. In contrast, the paternal transmission resulted in changes in the expression levels of several genes: down-regulation of Peg3 and Usp29 and up-regulation of Zim1. These changes were more pronounced during the neonatal stage than during the adult stage. In the case of Peg3 and Zim1, the levels of the observed changes were also different between males and females, suggesting the different degrees of YY1 involvement between two sexes. Overall, the results indicated that YY1 is mainly involved in controlling the transcriptional levels, but not the DNA methylation, of the Peg3 domain.