The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome. We perform deep RNA-Seq and de novo transcriptome assembly at different stages of mouse oogenesis. This reveals thousands of novel non-annotated genes, as well as alternative promoters, for approximately 10 % of reference genes expressed in oocytes. In addition, a large fraction of novel promoters coincide with MaLR and ERVK transposable elements. Integration with our transcriptome assembly reveals that transcription correlates accurately with DNA methylation and accounts for approximately 85–90 % of the methylome. We generate a mouse model in which transcription across the Zac1/Plagl1 locus is abrogated in oocytes, resulting in failure of DNA methylation establishment at all CpGs of this locus. ChIP analysis in oocytes reveals H3K4me2 enrichment at the Zac1 imprinted control region when transcription is ablated, establishing a connection between transcription and chromatin remodeling at CpG islands by histone demethylases. By precisely defining the mouse oocyte transcriptome, this work not only highlights transcription as a cornerstone of DNA methylation establishment in female germ cells, but also provides an important resource for developmental biology research.

Grb10/GRB10 encodes a cytoplasmic adapter protein which modulates coupling of a number of cell surface receptor tyrosine kinases with specific signalling pathways. Mouse Grb10 is an imprinted gene with maternal-specific expression. In contrast, human GRB10 is expressed biallelically in most tissues, except for maternal-specific expression of one isoform in muscle and paternal expression in fetal brain. Owing to its location in 7p11.2–p12, GRB10 has been considered a candidate gene for the imprinted growth disorder, the Silver–Russell syndrome (SRS), but its predominantly biallelic expression argues against involvement in the syndrome. To investigate the discrepant imprinting between mouse and human, we compared the sequence organization of their upstream regions, and examined their allelic methylation patterns and the splice variant organization of the mouse locus. Contrary to expectation, we detected both maternal and paternal expression of mouse Grb10. Expression of the paternal allele arises from a different promoter region than the maternal and, as in human, is restricted to the brain. The upstream regions are well conserved, especially the presence of two CpG islands. Surprisingly, both genes have a similar imprinted methylation pattern, the second CpG island is a differentially methylated region (DMR) with maternal methylation in both species. Analysis of 24 SRS patients did not reveal methylation anomalies in the DMR. In the mouse this DMR is a gametic methylation mark. Our results suggest that the difference in imprinted expression in mouse and human is not due to acquisition of an imprint mark but in differences in the reading of this mark.

Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith–Wiedemann and Silver–Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

Mono-allelic expression of imprinted genes from either the paternal or the maternal allele is mediated by imprinting control regions (ICRs), which are epigenetically marked in an allele-specific fashion. Although, in somatic cells, these epigenetic marks comprise both DNA methylation and histone methylation, the relationship between these two modifications in imprint acquisition and maintenance remains unclear. To address this important question, we analyzed histone modifications at ICRs in mid-gestation embryos that were obtained from Dnmt3L^−/− females, in which DNA methylation imprints at ICRs are not established during oogenesis. The absence of maternal DNA methylation imprints in these conceptuses led to a marked decrease and loss of allele-specificity of the repressive H3K9me3, H4K20me3 and H2A/H4R3me2 histone modifications, providing the first evidence of a mechanistic link between DNA and histone methylation at ICRs. The existence of this relationship was strengthened by the observation that when DNA methylation was still present at the Snrpn and Peg3 ICRs in some of the progeny of Dnmt3L^−/− females, these ICRs were associated with the usual patterns of histone methylation. Combined, our data establish that DNA methylation is involved in the acquisition and/or maintenance of histone methylation at ICRs.

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.