At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1

John P Hagan,Colin L Stewart,Carlo M Croce,Brittany L O'Neill,Serguei V Kozlov,Rodolfo Aramayo

doi:10.1371/journal.pone.0004352

Abstract

BackgroundGenomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.Methodology/Principal FindingsTo identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.Conclusions/SignificanceTen imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/“Rian”, AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.

Highlights

Unlike other vertebrates, mammalian embryos have an absolute dependency on both parental genomes for successful development
We report the isolation and characterization of a maternally expressed RNA called Imprinted RNA near Meg3/Gtl2 (Irm) (Imprinted RNA near Meg3) that is located on the distal region of mouse chromosome 12 near the known imprinted genes, Delta-like homolog 1 (Dlk1) and Maternally expressed gene 3/Gene trap locus 2 (Meg3/Gtl2)
A+ RNA purified from both AG and PG parthenogenetic mouse embryonic fibroblast (PMEF) were subjected to RNA expression profiling using an Incyte mouse GEM1 array, containing 8,638 cDNA probes corresponding to identified genes [47]

Summary

Introduction

Mammalian embryos have an absolute dependency on both parental genomes for successful development. In which the entire genome is of maternal (parthenogenetic) or paternal (androgenetic) origin, do not develop past embryonic day 10 [1,2]. The basis for this developmental failure is that some genes are subject to genomic imprinting and are expressed from only one parental allele. Female mice deficient in Paternally expressed gene 1/Mesoderm specific transcript (Peg1/MEST) [10] or Paternally expressed gene 3 (Peg3) [11] have aberrant maternal nurturing behavior with low incidences of pup retrieval, nest building, and milk ejection These findings clearly illustrate the necessity to gain a better understanding of imprinting in mammalian biology. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/ Angelman syndromes and cancer

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