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Background: Genomic imprinting results in parent-of-origin-specific gene expression and, among vertebrates, is found only in therian mammals: marsupials and eutherians. A differentially methylated region (DMR), in which the methylation status of CpG dinucleotides differs between the two alleles, can mark the parental identity of imprinted genes. We developed a computational pipeline that detected CpG islands (CGIs) marked by both methylated and unmethylated signals in whole genome bisulfite sequencing data. This approach identified candidate marsupial DMRs in a publicly available koala methylome. One of these candidate DMRs was associated with PRKACB, a gene encoding the protein kinase A catalytic subunit beta. Nothing is known about the imprinting status of PRKACB in eutherian mammals although mutations of this gene are associated with endocrine neoplasia and other developmental disorders., Results: In the tammar wallaby and brushtail possum there was parent-of-origin-specific DNA methylation in the PRKACB DMR in which the maternal allele was methylated and the paternal allele was unmethylated. There were multiple RNAs transcribed from this locus. Allele-specific expression analysis identified paternal expression of a PRKACB lncRNA and an mRNA isoform. Comparison of the PRKACB gene start site between marsupials and eutherians demonstrated that the CGI is longer in marsupials. The PRKACB gene product functions in the same signalling pathway as the guanine nucleotide-binding protein alpha subunit encoded at the GNAS locus, a known eutherian imprinted gene. In a mouse methylome Gnas had three differentially methylated CGIs, while in the koala methylome the GNAS locus had two unmethylated CGIs., Conclusions: We conclude that PRKACB is a novel, DMR-associated marsupial imprinted gene. Imprinting of PRKACB in marsupials and GNAS in eutherians may indicate a conserved selection pressure for imprinting of the protein kinase A signalling pathway in therians with the two lineages adapting by imprinting different genes., (© 2024. Crown.)

X chromosome inactivation (XCI) is an epigenetic process that results in the transcriptional silencing of one X chromosome in the somatic cells of females. This phenomenon is common to both eutherian and marsupial mammals, but there are fundamental differences. In eutherians, the X chosen for silencing is random. DNA methylation on the eutherian inactive X is high at transcription start sites (TSSs) and their flanking regions, resulting in universally high DNA methylation. This contrasts XCI in marsupials where the paternally derived X is always silenced, and in which DNA methylation is low at TSSs and flanking regions. Here, we examined the DNA methylation status of the tammar wallaby X chromosome during spermatogenesis to determine the DNA methylation profile of the paternal X prior to and at fertilization. Whole genome enzymatic methylation sequencing was carried out on enriched flow-sorted populations of premeiotic, meiotic, and postmeiotic cells. We observed that the X displayed a pattern of DNA methylation from spermatogonia to mature sperm that reflected the inactive X in female somatic tissue. Therefore, the paternal X chromosome arrives at the egg with a DNA methylation profile that reflects the transcriptionally silent X in adult female somatic tissue. We present this epigenetic signature as a candidate for the long sought-after imprint for paternal XCI in marsupials., Competing Interests: Competing interests statement:The authors declare no competing interest.

The possession of fur or hair is a defining characteristic of mammals and can occur in a variety of colours and patterns. While genetic determinants of coat colour are well described in eutherian 'placental' mammals, the other major mammalian infraclass, marsupials, is grossly understudied. The fur of the common brushtail possum ( Trichosurus vulpecula ), an iconic native mammal found throughout Australia and introduced into Aotearoa New Zealand, possesses two main colour morphs: grey and black. To identify genetic variants associated with coat colour, we performed a genome-wide association study (GWAS) with genotype by sequencing (GBS) data. Single nucleotide variants (SNVs) on chromosome 3, close to the agouti signalling protein ( ASIP ) gene that controls the temporal and spatial distribution of pigments in eutherian mammals, were identified. Fine-mapping identified a C>T variant at chr3:100483705 that results in a ASIP:p.Arg115Cys missense substitution, and animals homozygous for this variant have black fur. In addition to uncovering the first genetic determinant of coat colour in a natural marsupial population, comparative analysis of ASIP in divergent marsupial species identified the dasyurids as having accelerated evolution, reflecting their well described diversity of coat colour and pattern., Competing Interests: All authors declare that there are no competing financial interests. T.A.H. and D.M.B. are directors and shareholders of TOTOGEN Ltd, an agricultural genetics consultancy., (© 2024 The Authors.)

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits., (© 2023. Springer Nature Limited.)

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals., (© 2023. The Author(s).)

Using DNA methylation profiles ( n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Background: Pluripotent stem cells (PSCs) can be an ideal source of differentiation of cardiomyocytes in vitro and during transplantation to induce cardiac regeneration. However, differentiation of PSCs into a heterogeneous population is associated with an increased incidence of arrhythmia following transplantation. We aimed to design a protocol to drive PSCs to a ventricular lineage by regulating Wnt and retinoic acid (RA) signalling pathways., Methods: Mouse embryonic stem cells were cultured either in monolayers or three-dimensional hanging drop method to form embryonic bodies (EBs) and exposed to different treatments acting on Wnt and retinoic acid signalling. Samples were collected at different time points to analyse cardiomyocyte-specific markers by RT-PCR, flow cytometry and immunofluorescence., Results: Treatment of monolayer and EBs with Wnt and RA signalling pathways and ascorbic acid, as a cardiac programming enhancer, resulted in the formation of an immature non-contractile cardiac population that expressed many of the putative markers of cardiac differentiation. The population exhibited upregulation of ventricular specific markers while suppressing the expression of pro-atrial and pro-sinoatrial markers. Differentiation of EBs resulted in early foetal like non-contractile ventricular cardiomyocytes with an inherent propensity to contract when stimulated., Conclusion: Our results provide the first evidence of in vitro differentiation that mimics the embryonic morphogenesis towards ventricular specific cardiomyocytes through regulation of Wnt and RA signalling pathways., (© 2022. The Author(s).)

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge., Competing Interests: Authors JU and JG are employed by Southern Community Laboratories, Dunedin, New Zealand. Authors WR and LL are employed by South Pacific Sera, Washdyke, Timaru, New Zealand. Authors RO'B and PC are employed by MicroGEM NZ Ltd., 201 Princes Street, Dunedin, New Zealand. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stanton, O'Brien, Hall, Chernyavtseva, Ha, Jelley, Mace, Klenov, Treece, Fraser, Clow, Clarke, Su, Kurup, Filichev, Rolleston, Law, Rendle, Harris, Wood, Scully, Ussher, Grant, Hore, Moser, Harfoot, Lawley, Quiñones-Mateu, Collins and Blaikie.)

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

In mammals, females generally live longer than males. Nevertheless, the mechanisms underpinning sex-dependent longevity are currently unclear. Epigenetic clocks are powerful biological biomarkers capable of precisely estimating chronological age and identifying novel factors influencing the aging rate using only DNA methylation data. In this study, we developed the first epigenetic clock for domesticated sheep ( Ovis aries ), which can predict chronological age with a median absolute error of 5.1 months. We have discovered that castrated male sheep have a decelerated aging rate compared to intact males, mediated at least in part by the removal of androgens. Furthermore, we identified several androgen-sensitive CpG dinucleotides that become progressively hypomethylated with age in intact males, but remain stable in castrated males and females. Comparable sex-specific methylation differences in MKLN1 also exist in bat skin and a range of mouse tissues that have high androgen receptor expression, indicating that it may drive androgen-dependent hypomethylation in divergent mammalian species. In characterizing these sites, we identify biologically plausible mechanisms explaining how androgens drive male-accelerated aging., Competing Interests: VS, JZ, PN, AL, OO, MG, CB, SR, AH, MG, KS, NW, XY, RS No competing interests declared, DB Director and shareholder of Totovision, a small agricultural consultancy. RH Commercial sheep famer, TH is a shareholder and director of Totovision Ltd, a small agricultural and biotechnology consultancy. SH is a founder of the non-profit Epigenetic Clock Development Foundation which plans to license several patents from his employer UC Regents, including a patent for the mammalian assay utilised in this study (WO2020150705)., (© 2021, Sugrue et al.)

Development of primordial germ cells (PGCs: precursors to adult gametes) is a key process in vertebrate sexual differentiation. Marsupials are ideal to investigate this phenomenon because much of PGC migration and development unusually occurs postnatally in pouch young. However, investigation of the molecular dynamics underpinning PGC development is restricted to one marsupial model species: the tammar wallaby (Macropus eugenii). Given the reproductive diversity among clades, marsupial PGCs likely exhibit diversity in molecular patterns that could help uncover their developmental dynamics. Here we characterise PGC marker expression (SSEA1 and DDX4) in developing ovaries of the brushtail possum, Trichosurus vulpecula. Female germ cells expressed DDX4 from 6 days postpartum (dpp) and almost all germ cells expressed DDX4 by meiosis (40 dpp), consistent with M. eugenii and eutherian mammals. In contrast, PGCs and oogonia expressed SSEA1 from 12 dpp, throughout proliferation and until entry into meiosis (40-63 dpp). SSEA1 expression was temporally distinct from that of M. eugenii, in which SSEA1 expression persists only until 14 dpp, indicating differential expression between marsupial species at equivalent stages of germ cell development. Hence, the molecular characteristics of M. eugenii germ cells cannot be assumed for all marsupials, as at least one key molecule exhibits species-specific expression.

Whole-genome bisulfite sequencing (WGBS) is a popular method for characterizing cytosine methylation because it is fully quantitative and has base-pair resolution. While WGBS is prohibitively expensive for experiments involving many samples, low-coverage WGBS can accurately determine global methylation and erasure at similar cost to high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assays (ELISA). Moreover, low-coverage WGBS has the capacity to distinguish between methylation in different cytosine contexts (e.g., CG, CHH, and CHG), can tolerate low-input material (<100 cells), and can detect the presence of overrepresented DNA originating from mitochondria or amplified ribosomal DNA. In addition to describing a WGBS library construction and quantitation approach, here we detail computational methods to predict the accuracy of low-coverage WGBS using empirical bootstrap samplers and theoretical estimators similar to those used in election polling. Using examples, we further demonstrate how non-independent sampling of cytosines can alter the precision of error calculation and provide methods to improve this.

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10 -7 ) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10 -26 ). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10 -8 ) and in the transgenic sheep model (p = 2.4 × 10 -88 ). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10 -7 ) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10 -9 ), GRIK4 (p = 3.0 × 10 -8 ), and COX4I2 (p = 6.5 × 10 -8 ). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana 1,2 -is an iconic species that is endemic to New Zealand 2,3 . A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes 2,4 . Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Fish show extraordinary sexual plasticity, changing sex naturally as part of their life cycle or reversing sex because of environmental stressors. This plasticity shows that sexual fate is not an irreversible process but the result of an ongoing tug-of-war for supremacy between male and female signaling networks. The behavioral, gonadal, and morphological changes involved in this process are well described, yet the molecular events that underpin those changes remain poorly understood. Epigenetic modifications emerge as a critical link between environmental stimuli, the onset of sex change, and subsequent maintenance of sexual phenotype. Here we synthesize current knowledge of sex change, focusing on the genetic and epigenetic processes that are likely involved in the initiation and regulation of sex change. We anticipate that better understanding of sex change in fish will shed new light on sex determination and development in vertebrates and on how environmental perturbations affect sexual fate.

Cytosine methylation is a DNA modification that is critical for vertebrate development and provides a plastic yet stable information module in addition to the DNA code. DNA methylation memory establishment, maintenance and erasure is carefully balanced by molecular machinery highly conserved among vertebrates. In mammals, extensive erasure of epigenetic marks, including 5-methylcytosine (5mC), is a hallmark of early embryo and germline development. Conversely, global cytosine methylation patterns are preserved in at least some non-mammalian vertebrates over comparable developmental windows. The evolutionary mechanisms which drove this divergence are unknown, nevertheless a direct consequence of retaining epigenetic memory in the form of 5mC is the enhanced potential for transgenerational epigenetic inheritance (TEI). Given that DNA methylation dynamics remains underexplored in most vertebrate lineages, the extent of information transferred to offspring by epigenetic modification might be underestimated., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology ( Oberacker et al. , 2019 ). Updated protocols can be found on the associated webpage (https://bomb.bio)., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

The germline is the only cellular lineage capable of transferring genetic information from one generation to the next. Intergenerational transmission of epigenetic memory through the germline, in the form of DNA methylation, has been proposed; however, in mammals this is largely prevented by extensive epigenetic erasure during germline definition. Here we report that, unlike mammals, the continuously-defined 'preformed' germline of zebrafish does not undergo genome-wide erasure of DNA methylation during development. Our analysis also uncovers oocyte-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA (fem-rDNA). The peak of fem-rDNA amplification coincides with the initial expansion of stage IB oocytes, the poly-nucleolar cell type responsible for zebrafish feminisation. Given that fem-rDNA overlaps with the only zebrafish locus identified thus far as sex-linked, we hypothesise fem-rDNA expansion could be intrinsic to sex determination in this species.

Bluehead wrasses undergo dramatic, socially cued female-to-male sex change. We apply transcriptomic and methylome approaches in this wild coral reef fish to identify the primary trigger and subsequent molecular cascade of gonadal metamorphosis. Our data suggest that the environmental stimulus is exerted via the stress axis and that repression of the aromatase gene (encoding the enzyme converting androgens to estrogens) triggers a cascaded collapse of feminizing gene expression and identifies notable sex-specific gene neofunctionalization. Furthermore, sex change involves distinct epigenetic reprogramming and an intermediate state with altered epigenetic machinery expression akin to the early developmental cells of mammals. These findings reveal at a molecular level how a normally committed developmental process remains plastic and is reversed to completely alter organ structures.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement., Competing Interests: TH and DB are directors of a small agricultural and biotech consultancy (TOTOGEN Ltd), at which some BOMB protocols were developed and have been donated to this project. SRH is director of an agricultural engineering firm (CENENG Ltd).

Sexual fate can no longer be considered an irreversible deterministic process that once established during early embryonic development, plays out unchanged across an organism's life. Rather, it appears to be a dynamic process, with sexual phenotype determined through an ongoing battle for supremacy between antagonistic male and female developmental pathways. That sexual fate is not final and is actively regulated via the suppression or activation of opposing genetic networks creates the potential for flexibility in sexual phenotype in adulthood. Such flexibility is seen in many fish, where sex change is a usual and adaptive part of the life cycle. Many fish are sequential hermaphrodites, beginning life as one sex and changing sometime later to the other. Sequential hermaphrodites include species capable of female-to-male (protogynous), male-to-female (protandrous), or bidirectional (serial) sex change. These natural forms of sex change involve coordinated transformations across multiple biological systems, including behavioral, anatomical, neuroendocrine and molecular axes. Here we review the biological processes underlying this amazing transformation, focusing particularly on the molecular aspects, where new genomic technologies are beginning to help us understand how sex change is initiated and regulated at the molecular level., (© 2019 Elsevier Inc. All rights reserved.)

Vitamins A and C represent unrelated sets of small molecules that are essential to the human diet and have recently been shown to intensify erasure of epigenetic memory in naive embryonic stem cells. These effects are driven by complementary enhancement of the ten-eleven translocation (TET) demethylases - vitamin A stimulates TET expression, whereas vitamin C potentiates TET catalytic activity. Vitamin A and C cosupplementation synergistically enhances reprogramming of differentiated cells to the naive state, but overuse may exaggerate instability of imprinted genes. As such, optimizing their use in culture media will be important for regenerative medicine and mammalian transgenics. In addition, mechanistic perception of how these vitamins interact with the epigenome may be relevant for understanding cancer and improving patient treatment.

Background: Methylation of CG dinucleotides constitutes a critical system of epigenetic memory in bony vertebrates, where it modulates gene expression and suppresses transposon activity. The genomes of studied vertebrates are pervasively hypermethylated, with the exception of regulatory elements such as transcription start sites (TSSs), where the presence of methylation is associated with gene silencing. This system is not found in the sparsely methylated genomes of invertebrates, and establishing how it arose during early vertebrate evolution is impeded by a paucity of epigenetic data from basal vertebrates., Methods:  We perform whole-genome bisulfite sequencing to generate the first genome-wide methylation profiles of a cartilaginous fish, the elephant shark Callorhinchus milii . Employing these to determine the elephant shark methylome structure and its relationship with expression, we compare this with higher vertebrates and an invertebrate chordate using published methylation and transcriptome data.  Results: Like higher vertebrates, the majority of elephant shark CG sites are highly methylated, and methylation is abundant across the genome rather than patterned in the mosaic configuration of invertebrates. This global hypermethylation includes transposable elements and the bodies of genes at all expression levels. Significantly, we document an inverse relationship between TSS methylation and expression in the elephant shark, supporting the presence of the repressive regulatory architecture shared by higher vertebrates., Conclusions:  Our demonstration that methylation patterns in a cartilaginous fish are characteristic of higher vertebrates imply the conservation of this epigenetic modification system across jawed vertebrates separated by 465 million years of evolution. In addition, these findings position the elephant shark as a valuable model to explore the evolutionary history and function of vertebrate methylation., Competing Interests: Competing interests: No competing interests were disclosed.

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe 2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome., Competing Interests: W.R. and S.B. serve as consultants for Cambridge Epigenetix Ltd.

Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aim: Extra-pancreatic infectious complications in acute pancreatitis increase morbidity, but their incidence and association with infected pancreatic necrosis is unknown. Half of bacterial cultures of pancreatic necrosis are of non-enteric origin, raising the possibility of other sources of infection. The aim of this systematic review was to assess the incidence of extra-pancreatic infectious complications in acute pancreatitis, their timing, and relation to severity of pancreatitis and mortality., Methods: A systematic review was performed using Ovid MEDLINE, Embase and Cochrane Libraries, following PRISMA guidelines. Search terms were "Pancreatitis" AND "Infection" AND ("Complication" OR "Outcome")., Results: 19 studies with 1741 patients were included. Extra-pancreatic infectious complication incidence was 32% (95% CI 23-41%), with the commonest being respiratory infection (9.2%) and bacteraemia (8.4%). Extra-pancreatic infectious complications were not associated with the predicted severity or the mortality of acute pancreatitis. Only 3 studies reported a relation of timing between extra-pancreatic and pancreatic infectious complications., Conclusions: This is the first systematic review to evaluate the incidence of extra-pancreatic infectious complications in acute pancreatitis, which a third of patients with acute pancreatitis will develop. Implications are vigilance and prompt treatment of extra-pancreatic infection, to reduce possibility of progression to infected pancreatic necrosis., (Copyright © 2014 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2014, American Association for the Advancement of Science.)

The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian development. Epigenetic reprogramming results in global hypomethylation of the genome together with a profound loss of memory, which underlies naive pluripotency. Such global reprogramming occurs in primordial germ cells, early embryos, and embryonic stem cells where reciprocal molecular links connect the methylation machinery to pluripotency. Priming for differentiation is initiated upon exit from pluripotency, and we propose that epigenetic mechanisms create diversity of transcriptional states, which help with symmetry breaking during cell fate decisions and lineage commitment., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development., Results: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements., Conclusions: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

Determining the evolutionary origin of X inactivation mechanisms in mammals requires knowledge of features of X inactivation across all three major mammal lineages; monotremes, marsupials and eutherians. In the past, research into X inactivation in marsupials and monotremes lagged far behind the major advances made in understanding the mechanisms of X inactivation in human and mouse. Fragmentary knowledge of the genic content and sequence of marsupial and monotreme X chromosomes has been alleviated by the recent release of genome sequences for two marsupials and one monotreme. This has lead to a number of important findings, among which is the absence of XIST in marsupials and monotremes, and the surprising finding that X-borne genes in platypus are subject to stochastic transcriptional inhibition rather than whole chromosome inactivation. Availability of sequence data, and new techniques for studying expression and chromatin modification, now make rapid advance possible.

Parent-of-origin gene expression (genomic imprinting) is widespread among eutherian mammals and also occurs in marsupials. Most imprinted genes are expressed in the placenta, but the brain is also a favored site. Although imprinting evolved in therian mammals before the marsupial-eutherian split, the mechanisms have continued to evolve in each lineage to produce differences between the two groups in terms of the number and regulation of imprinted genes. As yet there is no evidence for genomic imprinting in the egg-laying monotreme mammals, although these mammals also form a placenta (albeit short-lived) and transfer nutrients from mother to embryo. Therefore, imprinting was not essential for the evolution of the placenta and its importance in nutrient transfer but the elaboration of imprinted genes in marsupials and eutherians is associated with viviparity. Here we review the recent analyses of imprinted gene clusters in marsupials and monotremes, which have served to shed light on the origin and evolution of imprinting mechanisms in mammals.

CTCF is an essential, ubiquitously expressed DNA-binding protein responsible for insulator function, nuclear architecture, and transcriptional control within vertebrates. The gene CTCF was proposed to have duplicated in early mammals, giving rise to a paralogue called "brother of regulator of imprinted sites" (BORIS or CTCFL) with DNA binding capabilities similar to CTCF, but testis-specific expression in humans and mice. CTCF and BORIS have opposite regulatory effects on human cancer-testis genes, the anti-apoptotic BAG1 gene, the insulin-like growth factor 2/H19 imprint control region (IGF2/H19 ICR), and show mutually exclusive expression in humans and mice, suggesting that they are antagonistic epigenetic regulators. We discovered orthologues of BORIS in at least two reptilian species and found traces of its sequence in the chicken genome, implying that the duplication giving rise to BORIS occurred much earlier than previously thought. We analysed the expression of CTCF and BORIS in a range of amniotes by conventional and quantitative PCR. BORIS, as well as CTCF, was found widely expressed in monotremes (platypus) and reptiles (bearded dragon), suggesting redundancy or cooperation between these genes in a common amniote ancestor. However, we discovered that BORIS expression was gonad-specific in marsupials (tammar wallaby) and eutherians (cattle), implying that a functional change occurred in BORIS during the early evolution of therian mammals. Since therians show imprinting of IGF2 but other vertebrate taxa do not, we speculate that CTCF and BORIS evolved specialised functions along with the evolution of imprinting at this and other loci, coinciding with the restriction of BORIS expression to the germline and potential antagonism with CTCF., Competing Interests: The authors have declared that no competing interests exist.

Dosage compensation has been thought to be a ubiquitous property of sex chromosomes that are represented differently in males and females. The expression of most X-borne genes is equalized between XX females and XY males in therian mammals (marsupials and "placentals") by inactivating one X chromosome in female somatic cells. However, compensation seems not to be strictly required to equalize the expression of most Z-borne genes between ZZ male and ZW female birds. Whether dosage compensation operates in the third mammal lineage, the egg-laying monotremes, is of considerable interest, since the platypus has a complex sex chromosome system in which five X and five Y chromosomes share considerable genetic homology with the chicken ZW sex chromosome pair, but not with therian XY chromosomes. The assignment of genes to four platypus X chromosomes allowed us to examine X dosage compensation in this unique species. Quantitative PCR showed a range of compensation, but SNP analysis of several X-borne genes showed that both alleles are transcribed in a heterozygous female. Transcription of 14 BACs representing 19 X-borne genes was examined by RNA-FISH in female and male fibroblasts. An autosomal control gene was expressed from both alleles in nearly all nuclei, and four pseudoautosomal BACs were usually expressed from both alleles in male as well as female nuclei, showing that their Y loci are active. However, nine X-specific BACs were usually transcribed from only one allele. This suggests that while some genes on the platypus X are not dosage compensated, other genes do show some form of compensation via stochastic transcriptional inhibition, perhaps representing an ancestral system that evolved to be more tightly controlled in placental mammals such as human and mouse., Competing Interests: The authors have declared that no competing interests exist.

Genomic imprinting first evolved in mammals around the time that humans last shared a common ancestor with marsupials and monotremes (180-210 million years ago). Recent comparisons of large imprinted domains in these divergent mammalian groups have shown that imprinting evolved haphazardly at various times in different lineages, perhaps driven by different selective forces. Surprisingly, some imprinted domains were formed relatively recently, using non-imprinted components acquired from unexpected genomic regions. Rearrangement and the insertion of retrogenes, small nucleolar RNAs, microRNAs, differential CpG methylation and control by non-coding RNA often accompanied the acquisition of imprinting. Here, we use comparisons between different mammalian groups to chart the course of evolution of two related epigenetic regulatory systems in mammals: genomic imprinting and X-chromosome inactivation.

Rhizobium leguminosarum bv. trifolii forms nitrogen-fixing root nodules on the pasture legume Trifolium repens, and T. repens seed is often coated with a compatible R. leguminosarum bv. trifolii strain prior to sowing. However, significant losses in bacterial viability occur during the seed-coating process and during storage of the coated seeds, most likely due to desiccation stress. The disaccharide trehalose is known to function as an osmoprotectant, and trehalose accumulation due to de novo biosynthesis is a common response to desiccation stress in bacteria. In this study we investigated the role of endogenous trehalose synthesis in desiccation tolerance in R. leguminosarum bv. trifolii strain NZP561. Strain NZP561 accumulated trehalose as it entered the stationary phase due to the combined actions of the TreYZ and OtsAB pathways. Mutants deficient in either pathway showed near-wild-type levels of trehalose accumulation, but double otsA treY mutants failed to accumulate any trehalose. The double mutants were more sensitive to the effects of drying, and their survival was impaired compared to that of the wild type when glass beads were coated with the organisms and stored at relative humidities of 5 and 32%. The otsA treY mutants were also less competitive for nodule occupancy. Gene expression studies showed that the otsA and treY genes were expressed constitutively and that expression was not influenced by the growth phase, suggesting that trehalose accumulation is controlled at the posttranscriptional level or by control of trehalose breakdown rates. Our results indicate that accumulated trehalose plays an important role in protecting R. leguminosarum bv. trifolii cells against desiccation stress and against stress encountered during nodulation.

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

Marsupial, as well as eutherian, mammals are subject to X chromosome inactivation in the somatic cells of females, although the phenotype and the molecular mechanism differ in important respects. Monotreme mammals appear to subscribe at least to a form of dosage compensation of X-borne genes. An important question is whether inactivation in these non-eutherian mammals involves co-ordination by a control locus homologous to the XIST gene and neighbouring genes, which play a key regulatory role in human and mouse X inactivation. We mapped BACs containing several orthologues of protein-coding genes that flank human and mouse XIST and genes that lie in the homologous region in chicken and frog. We found that these genes map to two distant locations on the opossum X, and also to different locations on a platypus autosome. We failed to find any trace of an XIST orthologue in any marsupial or monotreme or on any flanking BAC, confirming the conclusion from recent work that non-eutherian mammals lack XIST. We propose the region homologous to the human and mouse X-inactivation centre expanded in early mammals, and this unstable region was disrupted independently in marsupial and monotreme lineages. In the eutherian lineage, inserted and existing sequences provided the starting material for the non-translated RNAs of the X-inactivation centre, including XIST.