Anne C. Ferguson-Smith

The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted—monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

Parental origin of sequence variants associated with complex diseases.

Effects of susceptibility variants may depend on from which parent they are inherited. Although many associations between sequence variants and human traits have been discovered through genome-wide associations, the impact of parental origin has largely been ignored. Here we show that for 38,167 Icelanders genotyped using single nucleotide polymorphism (SNP) chips, the parental origin of most alleles can be determined. For this we used a combination of genealogy and long-range phasing. We then focused on SNPs that associate with diseases and are within 500 kilobases of known imprinted genes. Seven independent SNP associations were examined. Five—one with breast cancer, one with basal-cell carcinoma and three with type 2 diabetes—have parental-origin-specific associations. These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. Furthermore, we observed a novel association between the SNP rs2334499 at 11p15 and type 2 diabetes. Here the allele that confers risk when paternally inherited is protective when maternally transmitted. We identified a differentially methylated CTCF-binding site at 11p15 and demonstrated correlation of rs2334499 with decreased methylation of that site.

Genomic imprinting: the emergence of an epigenetic paradigm

The emerging awareness of the contribution of epigenetic processes to genome function in health and disease is underpinned by decades of research in model systems. In particular, many principles of the epigenetic control of genome function have been uncovered by studies of genomic imprinting. The phenomenon of genomic imprinting, which results in some genes being expressed in a parental--origin-specific manner, is essential for normal mammalian growth and development and exemplifies the regulatory influences of DNA methylation, chromatin structure and non-coding RNA. Setting seminal discoveries in this field alongside recent progress and remaining questions shows how the study of imprinting continues to enhance our understanding of the epigenetic control of genome function in other contexts.

A Maternal-Zygotic Effect Gene, Zfp57, Maintains Both Maternal and Paternal Imprints

The mechanisms responsible for maintaining genomic methylation imprints in mouse embryos are not understood. We generated a knockout mouse in the Zfp57 locus encoding a KRAB zinc finger protein. Loss of just the zygotic function of Zfp57 causes partial neonatal lethality, whereas eliminating both the maternal and zygotic functions of Zfp57 results in a highly penetrant embryonic lethality. In oocytes, absence of Zfp57 results in failure to establish maternal methylation imprints at the Snrpn imprinted region. Intriguingly, methylation imprints are reacquired specifically at the maternally derived Snrpn imprinted region when the zygotic Zfp57 is present in embryos. This suggests that there may be DNA methylation-independent memory for genomic imprints. Zfp57 is also required for the postfertilization maintenance of maternal and paternal methylation imprints at multiple imprinted domains. The effects on genomic imprinting are consistent with the maternal-zygotic lethality of Zfp57 mutants.

Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene

MicroRNAs (miRNAs) are an abundant class of RNAs that are ∼21–25 nucleotides (nt) long, interact with mRNAs and trigger either translation repression or RNA cleavage (RNA interference, RNAi) depending on the degree of complementarity with their targets. Here we show that the imprinted mouse distal chromosome 12 locus encodes two miRNA genes expressed from the maternally inherited chromosome and antisense to a retrotransposon-like gene (Rtl1) expressed only from the paternal allele.

Distinct fibroblast lineages determine dermal architecture in skin development and repair

Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibres of the extracellular matrix (ECM). Even within a single tissue, fibroblasts exhibit considerable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing in mice, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle, which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesize the bulk of the fibrillar ECM, and the preadipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialization. Epidermal β-catenin activation stimulates the expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

Mammalian Genomic Imprinting

Normal mammalian development requires a maternal and paternal contribution, which is attributed to imprinted genes, or genes that are expressed from a single parental allele. Approximately 100 imprinted genes have been reported in mammals thus far. Imprinted genes are controlled by cis-acting regulatory elements, termed imprinting control regions (ICRs), which have parental-specific epigenetic modifications, including DNA methylation. ICRs are methylated by de novo DNA methyltransferases during germline development; these parental-specific modifications must be maintained following fertilization when the genome is extensively reprogrammed. Many imprinted genes reside in ∼1-megabase clusters, with two major mechanisms of imprinting regulation currently recognized, CTCF-dependent insulators and long noncoding RNAs. Unclustered imprinted genes are generally regulated by germline-derived differential promoter methylation. Here, we describe the identification and functions of imprinted genes, cis-acting control sequences, trans-acting factors, and imprinting mechanisms in clusters. Finally, we define questions that require more extensive research.

RNAi-Mediated Allelic trans-Interaction at the Imprinted Rtl1/Peg11 Locus

The Dlk1-Gtl2 imprinted domain, encompassing the callipyge (CLPG) locus in sheep, has recently been shown to harbor a large number of maternally expressed miRNA genes [1, 2]. Two of these (mir127 and mir136) are processed from a transcript (antiPeg11) that is antisense to Rtl1/Peg11, a paternally expressed intronless gene with homology to the gag and pol polyproteins of Sushi-like retroelements [3]. We herein demonstrate that several additional miRNAs are processed from antiPeg11 and that these regulate Rtl1/Peg11 in trans by guiding RISC-mediated cleavage of its mRNA. This is the first demonstration of miRNA-mediated RNAi involving imprinted genes in mammals.

Genomic imprinting at the mammalian Dlk1-Dio3 domain

Genomic imprinting causes genes to be expressed or repressed depending on their parental origin. The majority of imprinted genes identified to date map in clusters and much of our knowledge of the mechanisms, function and evolution of imprinting have emerged from their analysis. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (Dlk1-Dio3) is located on distal mouse chromosome 12 and human chromosome 14. Its developmental importance is exemplified by severe phenotypes associated with altered dosage of these genes in mice and humans. The domain contains three imprinted protein-coding genes, Dlk1, Rtl1 and Dio3, expressed from the paternally inherited chromosome and several imprinted large and small noncoding RNA genes expressed from the maternally inherited homolog. Here, we discuss the function and regulation of imprinting at this domain.

In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism

Introduction The rapid global rise in metabolic disease suggests that nongenetic environmental factors contribute to disease risk. Early life represents a window of phenotypic plasticity important for adult metabolic health and that of future generations. Epigenetic inheritance has been implicated in the paternal transmission of environmentally induced phenotypes, but the mechanisms responsible remain unknown. In utero undernourishment alters the adult germ cell methylome. Undernourishment during PGC reprogramming results in hypomethylation of discrete loci in adult sperm. These regions are enriched in nucleosomes and are low-methylated regions. Although partially resistant to blastocyst reprogramming, differential methylation does not persist in the next generation. However, dysregulated expression of genes neighboring DMRs is observed in F2 offspring. Rationale We investigated the role of DNA methylation in epigenetic inheritance in an established murine model of intergenerational developmental programming. F1 offspring of undernourished dams (UN) have low birth weight and multiple metabolic defects. Metabolic phenotypic inheritance to the F2 generation is observed through the paternal line, even though the F1 mice did not experience postnatal environmental perturbation. The timing of nutritional restriction coincides with methylation reacquisition in F1 male primordial germ cells (PGCs). Therefore, we assessed F1 sperm whole-genome methylation using immunoprecipitation of methylated DNA, combined with high-throughput sequencing, followed by independent validation. We characterized the regions susceptible to methylation change and investigated the legacy of such methylation change in the phenotypic development of the next generation. Results In UN mice, 111 regions are hypomethylated relative to control sperm, and these changes are validated by bisulfite pyrosequencing. Methylation differences span multiple CpGs, with robust absolute changes of 10 to 30% (relative reduction ~50%). The absolute methylation level is consistent with differentially methylated regions (DMRs) being “low-methylated regions,” known to be enriched in regulatory elements. Indeed, luciferase assays suggest a role for these DMRs in transcriptional regulation. Hypomethylated DMRs are significantly depleted from coding and repetitive regions and enriched in intergenic regions and CpG islands. They are also enriched in nucleosome-retaining regions, which suggests that, at some loci, paternal germline hypomethylation induced by in utero undernutrition is transmitted in a chromatin context. DMRs are late to regain methylation in normal male PGCs. This may render them particularly susceptible to environmental perturbations that delay or impair remethylation in late gestation. Except for imprinted loci, gene-associated male germline methylation has generally been thought to be largely erased in the zygote,although recent studies suggest that resistance to reprogramming is more widespread. Indeed, 43% of hypomethylated DMRs persist and thus have the potential to affect development of the next generation. We show that differential methylation is lost in late-gestation F2 tissues, but considerable tissue-specific differences in expression of metabolic genes neighboring DMRs are present. Thus, it is unlikely that these expression changes are directly mediated by altered methylation; rather, the cumulative effects of dysregulated epigenetic patterns earlier in development may yield sustained alterations in chromatin architecture, transcriptional regulatory networks, differentiation, or tissue structure. Conclusion Prenatal undernutrition can compromise male germline epigenetic reprogramming and thus permanently alter DNA methylation in the sperm of adult offspring at regions resistant to zygotic reprogramming. However, persistence of altered DNA methylation into late-gestation somatic tissues of the subsequent generation is not observed. Nonetheless, alterations in gamete methylation may serve as a legacy of earlier developmental exposures and may contribute to the intergenerational transmission of environmentally induced disease. The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science, this issue p. 10.1126/science.1255903 Prenatal assaults change DNA methylation and chromatin structure in sperm and affect offspring. [Also see Perspective by Susiarjo and Bartolomei] Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.