Identification and characterisation of imprinted genes in the mouse.

Imprinted genes are expressed specifically from one or other parental allele. Over 70 are now known, and about one-half of these are expressed from the paternal allele and one-half from the maternal allele. Most imprinted genes are clustered within imprinting regions of the mouse genome, regions which are associated with abnormal phenotypes when inherited uniparentally. Imprinted genes have been identified from surveys based on differential expression or differential methylation according to parental origin, as well as analyses of candidate genes, mutants and imprinted gene clusters. Many imprinted genes affect growth and development, and more than 25 per cent determine non-coding RNAs that may have a function in controlling imprinted gene expression.     

Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.     

Regulatory links between imprinted genes: evolutionary predictions and consequences

Genomic imprinting is essential for development and growth and plays diverse roles in physiology and behaviour. Imprinted genes have traditionally been studied in isolation or in clusters with respect to cis-acting modes of gene regulation, both from a mechanistic and evolutionary point of view. Recent studies in mammals, however, reveal that imprinted genes are often co-regulated and are part of a gene network involved in the control of cellular proliferation and differentiation. Moreover, a subset of imprinted genes acts in trans on the expression of other imprinted genes. Numerous studies have modulated levels of imprinted gene expression to explore phenotypic and gene regulatory consequences. Increasingly, the applied genome-wide approaches highlight how perturbation of one imprinted gene may affect other maternally or paternally expressed genes. Here, we discuss these novel findings and consider evolutionary theories that offer a rationale for such intricate interactions among imprinted genes. An evolutionary view of these trans-regulatory effects provides a novel interpretation of the logic of gene networks within species and has implications for the origin of reproductive isolation between species.     

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.     

MiR-296-5p ameliorates deep venous thrombosis by inactivating S100A4

Deep venous thrombosis is one of the most common venous thromboembolic diseases and has a low cure rate and a high postoperative recurrence rate. Furthermore, emerging evidence indicates that microRNAs are involved in deep venous thrombosis. miR-296-5p is an important microRNA that plays a critical role in various cellular functions, and S100A4 is closely related to vascular function. miR-296-5p is downregulated in deep venous thrombosis patients, and its predicted target S100A4 is upregulated in deep venous thrombosis patients. Therefore, it was hypothesized that miR-296-5p may play a vital role in the development of deep venous thrombosis by targeting S100A4. An Ox-LDL-stimulated HUVEC and deep venous thrombosis mouse model was employed to detect the biological functions of miR-296-5p and S100A4. Dual luciferase reporter assays and pull-down assays were used to authenticate the interaction between miR-296-5p and S100A4. ELISA and Western blotting were employed to detect the protein levels of thrombosis-related factors and the endothelial-to-mesenchymal transition (EndMT)-related factors. The miR-296-5p levels were reduced, while the S100A4 levels were enhanced in deep venous thrombosis patients, and the miR-296-5p levels were negatively correlated with the S100A4 levels in deep venous thrombosis patients. miR-296-5p suppressed S100A4 expression by targeting the 3ʹ UTR of S100A4. MiR-296-5p knockdown accelerated ox-LDL-induced HUVEC apoptosis, oxidative stress, thrombosis-related factor expression, and EndMT, while S100A4 knockdown antagonized these effects in ox-LDL-induced HUVECs. S100A4 knockdown reversed the effect induced by miR-296-5p knockdown. Moreover, the in vivo studies revealed that miR-296-5p knockdown in deep venous thrombosis mice exacerbated deep venous thrombosis formation, whereas S100A4 knockdown had the opposite effect. These results indicate that elevated miR-296-5p inhibits deep venous thrombosis formation by inhibiting S100A4 expression. Both miR-296-5p and S100A4 may be potential diagnostic markers and therapeutic targets for deep venous thrombosis.     

Loss of Gnas imprinting differentially affects REM/NREM sleep and cognition in mice

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM-linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM-dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes.     

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos

The mouse chromosome 7C, orthologous to the human 15q11–q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3–4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1^ps alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.     

Mechanism of imprinting on mouse distal chromosome 7

Genomic imprinting is an epigenetic mode of gene regulation that results in expression of the autosomal 'imprinted' genes from only a single allele, determined exclusively by parental origin. To date over 20 imprinted genes have been identified in mouse and man and these appear to lie in clusters in restricted regions on a subset of chromosomes. This may be a critical feature of imprinting suggesting a domain-type mode of regulation. Imprinted domains are replicated asynchronously, show sex-specific meiotic recombination frequencies and have CpG-rich regions that are differentially methylated, often associated with the imprinted genes themselves. Mouse distal chromosome 7 is one such domain, containing at least nine imprinted genes spanning over 1 Mb of DNA. For the maternally expressed p57Kip2 gene, passage through the female germline is essential to generate the active state, whereas passage through the male germline is needed to force the maternally expressed H19 gene into an inactive state. It is therefore possible that the mouse distal chromosome 7 imprinted domain is actually composed of two or more independently regulated subdomains.     

Regulation of growth and metabolism by imprinted genes

A small sub-set of mammalian genes are subject to regulation by genomic imprinting such that only one parental allele is active in at least some sites of expression. Imprinted genes have diverse functions, notably including the regulation of growth. Much attention has been devoted to the insulin-like growth factor signalling pathway that has a major influence on fetal size and contains two components encoded by the oppositely imprinted genes, Igf2 (a growth promoting factor expressed from the paternal allele) and Igf2r (a growth inhibitory factor expressed from the maternal allele). These genes fit the parent-offspring conflict hypothesis for the evolution of genomic imprinting. Accumulated evidence indicates that at least one other fetal growth pathway exists that has also fallen under the influence of imprinting. It is clear that not all components of growth regulatory pathways are encoded by imprinted genes and instead it may be that within a pathway the influence of a single gene by each of the parental genomes may be sufficient for parent-offspring conflict to be enacted. A number of imprinted genes have been found to influence energy homeostasis and some, including Igf2 and Grb10, may coordinate growth with glucose-regulated metabolism. Since perturbation of fetal growth can be correlated with metabolic disorders in adulthood these imprinted genes are considered as candidates for involvement in this phenomenon of fetal programming.