Abstract Genomic imprinting is an epigenetic process causing parent-of-origin specific gene expression. The Dlk1-Dio3 domain is one of the largest imprinted clusters. While DNA methylation at an intergenic CpG-island (IG-CGI) within the imprinting control region (ICR) controls expression from the paternal chromosome, mechanisms regulating the unmethylated maternal chromosome remain unknown. Within the transcriptional regulatory element (IG-TRE) of the ICR, deletions identified a minimal region in vitro exhibiting both silencing and enhancing activity, with SOX2 and ZFP281 contributing to enhancer function on the maternal chromosome. In vivo, however, this deletion did not affect maternal expression in mouse embryos; instead it activated Dlk1 on both parental chromosomes. Combining deletion of this IG-TRE with the lethal IG-CGI deletion rescued lethality in mice by balancing Dlk1 expression, despite persistent maternal gene upregulation. These results demonstrate that loss of expression at this domain is more detrimental than gain, highlighting the importance of in vivo analysis. Identification of active regulatory factors on the unmethylated maternal chromosome challenges the prevailing view that imprinting is primarily a methylation-driven phenomenon, further revealing the sophisticated hierarchical mechanisms governing imprinting control.

The mammary gland, which primarily develops postnatally, undergoes significant changes during pregnancy and lactation to facilitate milk production. Through the generation and analysis of 480 transcriptomes, we provide the most detailed allelic expression map of the mammary gland, cataloguing cell-type-specific expression from ex-vivo purified cell populations over 10 developmental stages, enabling comparative analysis. The work identifies genes involved in the mammary gland cycle, parental-origin-specific and genetic background-specific expression at cellular and temporal resolution, genes associated with human lactation disorders and breast cancer. Genomic imprinting, a mechanism regulating gene expression based on parental origin, is crucial for controlling gene dosage and stem cell potential throughout development. The analysis identified 25 imprinted genes monoallelically expressed in the mammary gland, with several showing allele-specific expression in distinct cell types. No novel imprinted genes were identified and the absence of biallelically expressed imprinted genes suggests that, unlike in brain, selective absence of imprinting does not regulate gene dosage in the mammary gland. This research highlights transcriptional dynamics within mammary gland cells and identifies novel candidate genes potentially significant in the tissue during pregnancy and lactation. Overall, this comprehensive atlas represents a valuable resource for future studies on expression and transcriptional dynamics in mammary cells.

Genomic imprinting is an epigenetic process whereby genes are monoallelically expressed in a parent-of-origin-specific manner. Imprinted genes are frequently found clustered in the genome, likely illustrating their need for both shared regulatory control and functional inter-dependence. The Dlk1-Dio3 domain is one of the largest imprinted clusters. Genes in this region are involved in development, behavior, and postnatal metabolism: failure to correctly regulate the domain leads to Kagami–Ogata or Temple syndromes in humans. The region contains many of the hallmarks of other imprinted domains, such as long non-coding RNAs and parental origin-specific CTCF binding. Recent studies have shown that the Dlk1-Dio3 domain is exquisitely regulated via a bipartite imprinting control region (ICR) which functions differently on the two parental chromosomes to establish monoallelic expression. Furthermore, the Dlk1 gene displays a selective absence of imprinting in the neurogenic niche, illustrating the need for precise dosage modulation of this domain in different tissues. Here, we discuss the following: how differential epigenetic marks laid down in the gametes cause a cascade of events that leads to imprinting in the region, how this mechanism is selectively switched off in the neurogenic niche, and why studying this imprinted region has added a layer of sophistication to how we think about the hierarchical epigenetic control of genome function.

In mouse and human, genes subjected to genomic imprinting have been shown to function in development, behavior, and post-natal adaptations. Failure to correctly imprint genes in human is associated with developmental syndromes, adaptive, and metabolic disorders during life as well as numerous forms of cancer. In recent years researchers have turned to RNA-seq technologies applied to reciprocal hybrid strains of mice to identify novel imprinted genes, causing a threefold increase in genes reported as having a parental origin-specific expression bias. The functional relevance of parental origin-specific expression bias is not fully appreciated especially since many are reported with only minimal parental bias (e.g. 51:49). Here, we present an in-depth meta-analysis of previously generated RNA-seq data and show that the methods used to generate and analyze libraries greatly influence the calling of allele-specific expression. Validation experiments show that most novel genes called with parental-origin-specific allelic bias are artefactual, with the mouse strain contributing a larger effect on expression biases than parental origin. Of the weak novel genes that do validate, most are located at the periphery of known imprinted domains, suggesting they may be affected by local allele- and tissue-specific conformation. Together these findings highlight the need for robust tools, definitions, and validation of putative imprinted genes to provide meaningful information within imprinting databases and to understand the functional and mechanistic implications of the process.

At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cell lines to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery however we did not observe an enrichment of inactive alleles in the immediate vicinity of the nuclear envelope. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe that expressed alleles are significantly further away from the nuclear periphery. However, within individual nuclei, alleles closer to the periphery are equally likely to be expressed as those further away. In other words, absolute position does not predict expression. Taken together, this suggests that whilst stochastic activation can cause subtle shifts in localisation for this locus, there is no dramatic relocation of alleles upon gene activation. Our results suggest that transcriptional activity, rather than the parent-of-origin, defines subnuclear localisation at an endogenous imprinted domain.

Mammalian parental imprinting represents an exquisite form of epigenetic control regulating the parent-specific monoallelic expression of genes in clusters. While imprinting perturbations are widely associated with developmental abnormalities, the intricate regional interplay between imprinted genes makes interpreting the contribution of gene dosage effects to phenotypes a challenging task. Using mouse models with distinct deletions in an intergenic region controlling imprinting across the Dlk1-Dio3 domain, we link changes in genetic and epigenetic states to allelic-expression and phenotypic outcome in vivo. This determined how hierarchical interactions between regulatory elements orchestrate robust parent-specific expression, with implications for non-imprinted gene regulation. Strikingly, flipping imprinting on the parental chromosomes by crossing genotypes of complete and partial intergenic element deletions rescues the lethality of each deletion on its own. Our work indicates that parental origin of an epigenetic state is irrelevant as long as appropriate balanced gene expression is established and maintained at imprinted loci.

In mouse and human, genes subjected to genomic imprinting have been shown to function in development, behaviour, and post-natal adaptations. Failure to correctly imprint genes in human is associated with developmental syndromes, adaptive and metabolic disorders during life as well as numerous forms of cancer. In recent years researchers have turned to RNA-seq technologies applied to reciprocal hybrid strains of mice to identify novel imprinted genes, causing a 3-fold increase in genes reported as having a parental origin specific expression bias. The functional relevance of parental origin-specific expression bias is not fully appreciated especially since many are reported with only minimal parental bias (e.g. 51:49). Here we present an in-depth meta-analysis of previously generated RNA-seq data and show that the methods used to generate and analyse libraries greatly influence the calling of allele-specific expression. Validation experiments show that most novel genes called with parental-origin specific allelic bias are artefactual, with the mouse strain contributing a larger effect on expression biases than parental origin. Of the weak novel genes that do validate, most are located at the periphery of known imprinted domains, suggesting they may be affected by local allele- and tissue-specific conformation. Together these findings highlight the need for robust tools, definitions, and validation of putative imprinted genes to provide meaningful information within imprinting databases and to understand the functional and mechanistic implications of the process.

Following on from the success of last year’s event, Babraham Bioinformatics organised the second Cambridge Bioinformatics Hackathon, held on the 10-12 th December 2018. As before, the hackathon was open to students, postdocs and PIs from academic institutions or companies. We spent the three days working together on our respective projects which, this year, included an increased number of AI-related studies, highlighting the burgeoning interest in this field. The event proved an excellent forum for people to exchange ideas, start coding new software, build collaborations and of course have some fun. Furthermore, we held the hackathon at the Centre Computing History in Cambridge, giving the event a delightful retro feel. Participants were encouraged to make their code freely available on version control websites, such as GitHub. We expect many of these projects will be maintained for the foreseeable future and, hopefully, may lead to peer-reviewed publications.

Genomic imprinting is a process that causes genes to be expressed according to their parental origin. Imprinting appears to have evolved gradually in two of the three mammalian subclasses, with no imprinted genes yet identified in prototheria and only six found to be imprinted in marsupials to date. By interrogating the genomes of eutherian suborders, we determine that imprinting evolved at the majority of eutherian specific genes before the eutherian radiation. Theories considering the evolution of imprinting often relate to resource allocation and recently consider maternal-offspring interactions more generally, which, in marsupials, places a greater emphasis on lactation. In eutherians, the imprint memory is retained at least in part by zinc finger protein 57 (ZFP57), a Kruppel associated box (KRAB) zinc finger protein that binds specifically to methylated imprinting control regions. Some imprints are less dependent on ZFP57invivo and it may be no coincidence that these are the imprints that are found in marsupials. Because marsupials lack ZFP57, this suggests another more ancestral protein evolved to regulate imprints in non-eutherian subclasses, and contributes to imprinting control in eutherians. Hence, understanding the mechanisms acting at imprinting control regions across mammals has the potential to provide valuable insights into our understanding of the origins and evolution of genomic imprinting.

Approximately half the mammalian genome is composed of repetitive sequences, and accumulating evidence suggests that some may have an impact on genome function. Here, we characterized a large array class of repeats of long-interspersed elements (LINE-1). Although widely distributed in mammals, locations of such arrays are species specific. Using targeted deletion, we asked whether a 170-kb LINE-1 array located at a mouse imprinted domain might function as a modulator of local transcriptional control. The LINE-1 array is lamina associated in differentiated ES cells consistent with its AT-richness, and although imprinting occurs both proximally and distally to the array, active LINE-1 transcripts within the tract are biallelically expressed. Upon deletion of the array, no perturbation of imprinting was observed, and abnormal phenotypes were not detected in maternal or paternal heterozygous or homozygous mutant mice. The array does not shield nonimprinted genes in the vicinity from local imprinting control. Reduced neural expression of protein-coding genes observed upon paternal transmission of the deletion is likely due to the removal of a brain-specific enhancer embedded within the LINE array. Our findings suggest that presence of a 170-kb LINE-1 array reflects the tolerance of the site for repeat insertion rather than an important genomic function in normal development.

The paternally expressed imprinted Retrotransposon-like 1 (Rtl1/Peg11) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127 and Rtl1 deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1 mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.

Genomic imprinting is an epigenetic process causing expression of a subset of genes in a parent-of-origin-specific manner. Among vertebrates, only therian mammals have been demonstrated to imprint, indicating that placentation and imprinting arose at similar time points in evolution and that imprinting may be involved in key mammal-specific processes. However, although several theories have been posited to explain the evolution of imprinting, each has shortcomings and none fully explains the wide variety of genes regulated by imprinting. In this review, we catalog the phenotypes associated with genetic mutation and overexpression at particular imprinted loci in order to consider the wide impact of imprinted genes on development. In addition to the well-described roles of imprinted genes in prenatal growth and placentation, more recent data emphasize that imprinted genes are critical for specific aspects of postnatal mammalian development involving adaptive processes, metabolism, and behavior.

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of "simple" cancer-associated chromosome deletions.

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.

A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.

BACKGROUND:The evolution of genomic imprinting, the parental-origin specific expression of genes, is the subject of much debate. There are several theories to account for how the mechanism evolved including the hypothesis that it was driven by the evolution of X-inactivation, or that it arose from an ancestrally imprinted chromosome.RESULTS:Here we demonstrate that mammalian orthologues of imprinted genes are dispersed amongst autosomes in both monotreme and marsupial karyotypes.CONCLUSION:These data, along with the similar distribution seen in birds, suggest that imprinted genes were not located on an ancestrally imprinted chromosome or associated with a sex chromosome. Our results suggest imprinting evolution was a stepwise, adaptive process, with each gene/cluster independently becoming imprinted as the need arose.

Clustered imprinted genes are regulated by differentially methylated imprinting control regions (ICRs) that affect gene activity and repression in cis over a large region. Although a primary imprint signal for each of these clusters is DNA methylation, different mechanisms are used to establish and maintain these marks. The majority of ICRs are methylated in the maternal germline and are usually promoters for antisense transcripts whose elongation is associated with imprinting control in the domain. In contrast, ICRs methylated in the paternal germline do not appear to act as promoters and are located between genes. At least one, at the Igf2/H19 locus, is known to function as an insulator. Analysis of ICRs suggests that maternal and paternal methylation imprints function in distinct ways.