Invasion and Amplification of Endogenous Retroviruses in Dasyuridae Marsupial Genomes.

Retroviruses are an ancient viral family that have globally coevolved with vertebrates and impacted their evolution. In Australia, a continent that has been geographically isolated for millions of years, little is known about retroviruses in wildlife, despite the devastating impacts of a retrovirus on endangered koala populations. We therefore sought to identify and characterize Australian retroviruses through reconstruction of endogenous retroviruses from marsupial genomes, in particular the Tasmanian devil due to its high cancer incidence. We screened 19 marsupial genomes and identified over 80,000 endogenous retrovirus fragments which we classified into eight retrovirus clades. The retroviruses were similar to either Betaretrovirus (5/8) or Gammaretrovirus (3/8) retroviruses, but formed distinct phylogenetic clades compared to extant retroviruses. One of the clades (MEBrv 3) lost an envelope but retained retrotranspositional activity, subsequently amplifying throughout all Dasyuridae genomes. Overall, we provide insights into Australian retrovirus evolution and identify a highly active endogenous retrovirus within Dasyuridae genomes.

Imprinted X chromosome inactivation in marsupials: The paternal X arrives at the egg with a silent DNA methylation profile.

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.

Incomplete transcriptional dosage compensation of chicken and platypus sex chromosomes is balanced by post-transcriptional compensation.

Heteromorphic sex chromosomes (XY or ZW) present problems of gene dosage imbalance between sexes and with autosomes. A need for dosage compensation has long been thought to be critical in vertebrates. However, this was questioned by findings of unequal mRNA abundance measurements in monotreme mammals and birds. Here, we demonstrate unbalanced mRNA levels of X genes in platypus males and females and a correlation with differential loading of histone modifications. We also observed unbalanced transcripts of Z genes in chicken. Surprisingly, however, we found that protein abundance ratios were 1:1 between the sexes in both species, indicating a post-transcriptional layer of dosage compensation. We conclude that sex chromosome output is maintained in chicken and platypus (and perhaps many other non therian vertebrates) via a combination of transcriptional and post-transcriptional control, consistent with a critical importance of sex chromosome dosage compensation.

Origin and maintenance of large ribosomal RNA gene repeat size in mammals.

The genes encoding ribosomal RNA are highly conserved across life and in almost all eukaryotes are present in large tandem repeat arrays called the rDNA. rDNA repeat unit size is conserved across most eukaryotes but has expanded dramatically in mammals, principally through the expansion of the intergenic spacer region that separates adjacent rRNA coding regions. Here, we used long-read sequence data from representatives of the major amniote lineages to determine where in amniote evolution rDNA unit size increased. We find that amniote rDNA unit sizes fall into two narrow size classes: "normal" (∼11-20 kb) in all amniotes except monotreme, marsupial, and eutherian mammals, which have "large" (∼35-45 kb) sizes. We confirm that increases in intergenic spacer length explain much of this mammalian size increase. However, in stark contrast to the uniformity of mammalian rDNA unit size, mammalian intergenic spacers differ greatly in sequence. These results suggest a large increase in intergenic spacer size occurred in a mammalian ancestor and has been maintained despite substantial sequence changes over the course of mammalian evolution. This points to a previously unrecognized constraint on the length of the intergenic spacer, a region that was thought to be largely neutral. We finish by speculating on possible causes of this constraint.

Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation.

The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST, which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

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.

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials.

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet configuration and synapsis progressed exclusively from the telomeres towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

Three dimensions of thermolabile sex determination.

The molecular mechanism of temperature-dependent sex determination (TSD) is a long-standing mystery. How is the thermal signal sensed, captured and transduced to regulate key sex genes? Although there is compelling evidence for pathways via which cells capture the temperature signal, there is no known mechanism by which cells transduce those thermal signals to affect gene expression. Here we propose a novel hypothesis we call 3D-TSD (the three dimensions of thermolabile sex determination). We postulate that the genome has capacity to remodel in response to temperature by changing 3D chromatin conformation, perhaps via temperature-sensitive transcriptional condensates. This could rewire enhancer-promoter interactions to alter the expression of key sex-determining genes. This hypothesis can accommodate monogenic or multigenic thermolabile sex-determining systems, and could be combined with upstream thermal sensing and transduction to the epigenome to commit gonadal fate.

Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals.

Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling.

Meiotic chromosome dynamics and double strand break formation in reptiles.

During meiotic prophase I, tightly regulated processes take place, from pairing and synapsis of homologous chromosomes to recombination, which are essential for the generation of genetically variable haploid gametes. These processes have canonical meiotic features conserved across different phylogenetic groups. However, the dynamics of meiotic prophase I in non-mammalian vertebrates are poorly known. Here, we compare four species from Sauropsida to understand the regulation of meiotic prophase I in reptiles: the Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first performed a histological characterization of the spermatogenesis process in both the bearded dragon and the painted turtle. We then analyzed prophase I dynamics, including chromosome pairing, synapsis and the formation of double strand breaks (DSBs). We show that meiosis progression is highly conserved in reptiles with telomeres clustering forming the bouquet, which we propose promotes homologous pairing and synapsis, along with facilitating the early pairing of micro-chromosomes during prophase I (i.e., early zygotene). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our results provide new insights into reptile meiosis.

Arctic introgression and chromatin regulation facilitated rapid Qinghai-Tibet Plateau colonization by an avian predator.

The Qinghai-Tibet Plateau (QTP), possesses a climate as cold as that of the Arctic, and also presents uniquely low oxygen concentrations and intense ultraviolet (UV) radiation. QTP animals have adapted to these extreme conditions, but whether they obtained genetic variations from the Arctic during cold adaptation, and how genomic mutations in non-coding regions regulate gene expression under hypoxia and intense UV environment, remain largely unknown. Here, we assemble a high-quality saker falcon genome and resequence populations across Eurasia. We identify female-biased hybridization with Arctic gyrfalcons in the last glacial maximum, that endowed eastern sakers with alleles conveying larger body size and changes in fat metabolism, predisposing their QTP cold adaptation. We discover that QTP hypoxia and UV adaptations mainly involve independent changes in non-coding genomic variants. Our study highlights key roles of gene flow from Arctic relatives during QTP hypothermia adaptation, and cis-regulatory elements during hypoxic response and UV protection.

Fragile, unfaithful and persistent Ys-on how meiosis can shape sex chromosome evolution.

Sex-linked inheritance is a stark exception to Mendel's Laws of Heredity. Here we discuss how the evolution of heteromorphic sex chromosomes (mainly the Y) has been shaped by the intricacies of the meiotic programme. We propose that persistence of Y chromosomes in distantly related mammalian phylogroups can be explained in the context of pseudoautosomal region (PAR) size, meiotic pairing strategies, and the presence of Y-borne executioner genes that regulate meiotic sex chromosome inactivation. We hypothesise that variation in PAR size can be an important driver for the evolution of recombination frequencies genome wide, imposing constraints on Y fate. If small PAR size compromises XY segregation during male meiosis, the stress of producing aneuploid gametes could drive function away from the Y (i.e., a fragile Y). The Y chromosome can avoid fragility either by acquiring an achiasmatic meiotic XY pairing strategy to reduce aneuploid gamete production, or gain meiotic executioner protection (a persistent Y). Persistent Ys will then be under strong pressure to maintain high recombination rates in the PAR (and subsequently genome wide), as improper segregation has fatal consequences for germ cells. In the event that executioner protection is lost, the Y chromosome can be maintained in the population by either PAR rejuvenation (extension by addition of autosome material) or gaining achiasmatic meiotic pairing, the alternative is Y loss. Under this dynamic cyclic evolutionary scenario, understanding the meiotic programme in vertebrate and invertebrate species will be crucial to further understand the plasticity of the rise and fall of heteromorphic sex chromosomes.

Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

Between the Devil and the Deep Blue Sea: Non-Coding RNAs Associated with Transmissible Cancers in Tasmanian Devil, Domestic Dog and Bivalves.

Currently there are nine known examples of transmissible cancers in nature. They have been observed in domestic dog, Tasmanian devil, and six bivalve species. These tumours can overcome host immune defences and spread to other members of the same species. Non-coding RNAs (ncRNAs) are known to play roles in tumorigenesis and immune system evasion. Despite their potential importance in transmissible cancers, there have been no studies on ncRNA function in this context to date. Here, we present possible applications of the CRISPR/Cas system to study the RNA biology of transmissible cancers. Specifically, we explore how ncRNAs may play a role in the immortality and immune evasion ability of these tumours.

Microchromosomes are building blocks of bird, reptile, and mammal chromosomes.

Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical.

Ancient viral integrations in marsupials: a potential antiviral defence.

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed 'endogenous viral elements' (EVEs), could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing data sets (n = 35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae, or Parvoviridae EVEs. The other twelve transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n = 188) were mapped to marsupial genomes (where available, n = 5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum, and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n = 71). Every marsupial has Bornaviridae, Filoviridae, and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence data sets, we detected Bornaviridae replicase and Filoviridae nucleoprotein produced small RNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Pseudogenes: A Novel Source of Trans-Acting Antisense RNAs.

Several recent studies support a functional role for pseudogenes, a copy of a parent gene that has lost protein-coding potential, which was for a long time thought to represent only "junk" DNA. Several hundreds of pseudogenes have now been reported as transcribed RNAs in a large variety of tissues and tumor types. Most studies have focused on pseudogenes expressed in sense direction, relative to their protein-coding gene counterpart, but some reports suggest that pseudogenes can be also transcribed as antisense RNAs (asRNAs). Key regulatory genes, such as PTEN and OCT4, have in fact been reported to be under the regulation of pseudogene-expressed asRNAs. Here, we review what is known about pseudogene-expressed asRNAs, we discuss the functional role that these transcripts may have in gene regulation and we summarize the techniques that are available to study them.

MicroRNA dynamics during hibernation of the Australian central bearded dragon (Pogona vitticeps).

Hibernation is a physiological state employed by many animals that are exposed to limited food and adverse winter conditions. Controlling tissue-specific and organism wide changes in metabolism and cellular function requires precise regulation of gene expression, including by microRNAs (miRNAs). Here we profile miRNA expression in the central bearded dragon (Pogona vitticeps) using small RNA sequencing of brain, heart, and skeletal muscle from individuals in late hibernation and four days post-arousal. A total of 1295 miRNAs were identified in the central bearded dragon genome; 664 of which were novel to central bearded dragon. We identified differentially expressed miRNAs (DEmiRs) in all tissues and correlated mRNA expression with known and predicted target mRNAs. Functional analysis of DEmiR targets revealed an enrichment of differentially expressed mRNA targets involved in metabolic processes. However, we failed to reveal biologically relevant tissue-specific processes subjected to miRNA-mediated regulation in heart and skeletal muscle. In brain, neuroprotective pathways were identified as potential targets regulated by miRNAs. Our data suggests that miRNAs are necessary for modulating the shift in cellular metabolism during hibernation and regulating neuroprotection in the brain. This study is the first of its kind in a hibernating reptile and provides key insight into this ephemeral phenotype.

Meiotic Executioner Genes Protect the Y from Extinction.

The Y has been described as a wimpy degraded relic of the X, with imminent demise should it lose sex-determining function. Why then has it persisted in almost all mammals? Here we present a novel mechanistic explanation for its evolutionary perseverance: the persistent Y hypothesis. The Y chromosome bears genes that act as their own judge, jury, and executioner in the tightly regulated meiotic surveillance pathways. These executioners are crucial for successful meiosis, yet need to be silenced during the meiotic sex chromosome inactivation window, otherwise germ cells die. Only rare transposition events to the X, where they remain subject to obligate meiotic silencing, are heritable, posing strong evolutionary constraint for the Y chromosome to persist.