Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas , which encodes a G protein α subunit, and there is clinical and biochemical evidence that the human homologue GNAS1 , mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein α subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl , Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas . This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs. Genomic imprinting is a process resulting in expression of genes according to parental origin. Some imprinted genes are expressed when paternally derived and others when maternally derived. Thus imprinted genes are monoallelically expressed and one copy has to be silenced. There is evidence that some long non-coding RNAs, acting in cis, have a role in silencing. We investigated the role of Nespas, a gene for a non-coding RNA that is only expressed from the paternally derived chromosome in the Gnas cluster and runs antisense to its sense counterpart, Nesp. Expression of Nespas is associated with silencing of Nesp and a repressive methylation mark on the Nesp DNA. We generated a Nespas mutant with reduced levels of activity and showed that it down-regulated its sense counterpart Nesp, in the absence of a DNA methylation mark, but in the presence of an altered chromatin mark. We conclude that Nespas can repress Nesp by a mechanism independent of DNA methylation, by modulating a chromatin mark.

Genomic imprinting is the phenomenon whereby a subset of genes is differentially expressed according to parental origin. Imprinted genes tend to occur in clusters, and microRNAs are associated with the majority of well-defined clusters of imprinted genes. We show here that two microRNAs, miR-296 and miR-298, are part of the imprinted Gnas/GNAS clusters in both mice and humans. Both microRNAs show imprinted expression and are expressed from the paternally derived allele, but not the maternal allele. They arise from a long, noncoding antisense transcript, Nespas, with a promoter more than 27 kb away. Nespas had been shown previously to act in cis to regulate imprinted gene expression within the Gnas cluster. Using microarrays and luciferase assays, IKBKE, involved in many signaling pathways, and Tmed9, a protein transporter, were verified as new targets of miR-296. Thus, Nespas has two clear functions: as a cis-acting regulator within an imprinted gene cluster and as a precursor of microRNAs that modulate gene expression in trans. Furthermore, imprinted microRNAs, including miR-296 and miR-298, impose a parental specific modulation of gene expression of their target genes.

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.