The admixed brushtail possum genome reveals invasion history in New Zealand and novel imprinted genes.

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.

A Point Mutation in Phytochromobilin synthase Alters the Circadian Clock and Photoperiodic Flowering of Medicago truncatula.

Plants use seasonal cues to initiate flowering at an appropriate time of year to ensure optimal reproductive success. The circadian clock integrates these daily and seasonal cues with internal cues to initiate flowering. The molecular pathways that control the sensitivity of flowering to photoperiods (daylengths) are well described in the model plant Arabidopsis. However, much less is known for crop species, such as legumes. Here, we performed a flowering time screen of a TILLING population of Medicago truncatula and found a line with late-flowering and altered light-sensing phenotypes. Using RNA sequencing, we identified a nonsense mutation in the Phytochromobilin synthase (MtPΦBS) gene, which encodes an enzyme that carries out the final step in the biosynthesis of the chromophore required for phytochrome (phy) activity. The analysis of the circadian clock in the MtpΦbs mutant revealed a shorter circadian period, which was shared with the MtphyA mutant. The MtpΦbs and MtphyA mutants showed downregulation of the FT floral regulators MtFTa1 and MtFTb1/b2 and a change in phase for morning and night core clock genes. Our findings show that phyA is necessary to synchronize the circadian clock and integration of light signalling to precisely control the timing of flowering.

Castration delays epigenetic aging and feminizes DNA methylation at androgen-regulated loci.

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.

Estimating Global Methylation and Erasure Using Low-Coverage Whole-Genome Bisulfite Sequencing (WGBS ).

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.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. 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 amniotes2,4. 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.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

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.

Infiltration-RNAseq: transcriptome profiling of Agrobacterium-mediated infiltration of transcription factors to discover gene function and expression networks in plants.

BACKGROUND: Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed. RESULTS: Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis. CONCLUSIONS: Infiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method to identify TF-mediated transcriptional changes and TF target genes in M. truncatula and Nicotiana benthamiana. This included the identification of target genes of a TF not normally expressed in leaves, and targets of TFs from other plant species. Infiltration-RNAseq can be easily adapted to other plant species where agroinfiltration protocols have been optimised. The ability to identify downstream genes, including positive and negative transcriptional regulators, will result in a greater understanding of TF function.

Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana.

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active flowering Wageningen epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.

Small RNAs and heritable epigenetic variation in plants.

Recent studies suggest that inheritance of phenotypes in plants is more likely to involve epigenetics than in mammals. There are two reasons for this difference. First, there is a RNA-based system in plants involving small (s)RNAs that influences de novo establishment and maintenance of DNA methylation at many sites in plant genomes. These regions of methylated DNA are epigenetic marks with the potential to affect gene expression that are transmitted between dividing cells of the same generation. Second, unlike mammals, DNA methyltransferases in plants are active during gametogenesis and embryogenesis so that patterns of DNA methylation can persist from parent to progeny and do not need to be reset. We discuss how the effects of stress and genome interactions in hybrid plants are two systems that illustrate how RNA-based mechanisms can influence heritable phenotypes in plants.

A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.

Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.

The low temperature response pathways for cold acclimation and vernalization are independent.

Vernalization is the promotion of flowering in response to the prolonged cold of winter. To survive sub-zero winter temperatures, plants must first acclimate to low, non-freezing temperatures (cold acclimation). Induction of VERNALIZATION INSENSITIVE 3 (VIN3), the first gene in the vernalization pathway, is initiated within the same time frame as the induction of genes in the cold acclimation pathway raising the question of whether there are common elements in the signal transduction pathways that activate these two responses to cold. We show that none of the signalling components required for cold acclimation, including the 'master regulator'INDUCTION OF CBF EXPRESSION1 (ICE1) or HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1), which has been described as a link between cold acclimation and vernalization, play a role in VIN3 induction. We also show that the hormone abscisic acid (ABA) does not modulate VIN3 induction, consistent with earlier reports that ABA signalling plays no role in the vernalization response. The cold acclimation pathway is activated at 12 °C, at which temperature there is no induction of VIN3 expression. Taken together, our data demonstrate that the responses to low temperatures leading to cold acclimation and vernalization are controlled by distinct signalling pathways.

Polycomb proteins regulate the quantitative induction of VERNALIZATION INSENSITIVE 3 in response to low temperatures.

Vernalization, the promotion of flowering in response to low temperatures, is one of the best characterized examples of epigenetic regulation in plants. The promotion of flowering is proportional to the duration of the cold period, but the mechanism by which plants measure time at low temperatures has been a long-standing mystery. We show that the quantitative induction of the first gene in the Arabidopsis vernalization pathway, VERNALIZATION INSENSITIVE 3 (VIN3), is regulated by the components of Polycomb Response Complex 2, which trimethylates histone H3 lysine 27 (H3K27me3). In differentiated animal cells, H3K27me3 is mostly associated with long-term gene repression, whereas, in pluripotent embyonic stem cells, many cell lineage-specific genes are inactive but exist in bivalent chromatin that carries both active (H3K4me3) and repressive (H3K27me3) marks on the same molecule. During differentiation, bivalent domains are generally resolved to an active or silent state. We found that H3K27me3 maintains VIN3 in a repressed state prior to cold exposure; this mark is not removed during VIN3 induction. Instead, active VIN3 is associated with bivalently marked chromatin. The continued presence of H3K27me3 ensures that induction of VIN3 is proportional to the duration of the cold, and that plants require prolonged cold to promote the transition to flowering. The observation that Polycomb proteins control VIN3 activity defines a new role for Polycomb proteins in regulating the rate of gene induction.

Hypoxia: a novel function for VIN3.

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana. Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC). During vernalization VIN3 binds to the chromatin of the FLC locus, and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2). This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3), a repressive chromatin mark that increases at the FLC locus as a result of vernalization. In our recent paper we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.

Histone acetylation, VERNALIZATION INSENSITIVE 3, FLOWERING LOCUS C, and the vernalization response.

The quantitative induction of VIN3 by low temperatures is required for PRC2 repression of FLC and promotion of flowering (vernalization) in Arabidopsis. Histone acetylation, a chromatin modification commonly associated with gene transcription, increased on VIN3 chromatin in two spatially and temporally distinct phases in response to low temperatures. During short-term cold exposure, histone H3 acetylation at the transcription start site rapidly increased, implying that it is required for VIN3 induction. Subsequent changes in histone H3 and H4 acetylation occurred following continued VIN3 transcription during prolonged cold exposure. Members of the SAGA-like transcriptional adaptor complex, including the histone acetyltransferase GCN5, which induces expression of the cold acclimation pathway genes, do not regulate VIN3 induction during cold exposure, indicating that the cold acclimation pathway and the cold-induction of VIN3 are regulated by different transcriptional mechanisms. Mutations in the other 11 histone acetyltransferase genes did not affect VIN3 induction. However, nicotinamide, a histone deacetylase inhibitor, induced VIN3 and altered histone acetylation at the VIN3 locus. VIN3 induction was proportional to the length of nicotinamide treatment, which was associated with an early-flowering phenotype and repression of FLC. However, unlike vernalization, the repression of FLC was independent of VIN3 activity. Nicotinamide treatment did not cause a change in the expression of any genes in the autonomous pathway or members of the PRC2 complex, the well characterized repressors of FLC. Our data suggest that FLC is repressed via a novel pathway involving the SIR2 class of histone deacetylases.

VERNALIZATION INSENSITIVE 3 (VIN3) is required for the response of Arabidopsis thaliana seedlings exposed to low oxygen conditions.

VERNALIZATION INSENSITIVE 3 (VIN3), which is required for the vernalization-mediated epigenetic repression of FLOWERING LOCUS C (FLC) in Arabidopsis thaliana, is quantitatively induced in response to low temperatures. We found that hypoxic conditions also induce VIN3 in a quantitative manner but high salt, high temperatures and osmotic stress do not. Inhibition of mitochondrial respiration did not induce VIN3 expression, consistent with the lack of VIN3 induction in response to other stresses that affect the rate of mitochondrial respiration. De novo protein synthesis is required for VIN3 induction during hypoxic conditions; this situation is not the case for VIN3 induction by low temperatures, indicating that different mechanisms act to induce VIN3 expression in response to cold and hypoxic conditions. Without VIN3 activity, fewer seedlings survived following a 72-h period of hypoxic treatment, indicating that VIN3 is required for the survival of Arabidopsis thaliana in response to hypoxic stress. Complementation of the vin3 mutant with a VIN3 transgene restored the wild-type response to low oxygen and confirmed the role of VIN3 in protecting both shoots and roots during low oxygen conditions. Loss of VIN3 protein did not affect the transcriptional regulation of genes known to be important in the response to low oxygen stress, which suggests that there is a novel mechanism to combat hypoxia that involves VIN3. This mechanism is likely to involve chromatin remodelling and may be similar to the role of VIN3 in the epigenetic repression of FLC during the vernalization response.

Passing the message on: inheritance of epigenetic traits.

Epigenetic modifiers play an important role in genome organization, stability and the control of gene expression. Three research groups that are exploring the transfer of epigenetic information between generations have recently published papers. Mary Alleman et al. have shown that RNA-directed chromatin changes mediate paramutation in maize, and Minoo Rassoulzadegan et al. have demonstrated that RNA also plays a role in paramutation in mice. A new aspect of epigenetic regulation has been revealed by Jean Molinier et al. - they have demonstrated that the memory of exposure to stress is transferred through several generations.