Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon.

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

Autoregulated splicing of TRA2ß programs T cell fate in response to antigen-receptor stimulation.

T cell receptor (TCR) sensitivity to peptide-major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2ß in mouse and human. TRA2ß-PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2ß-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2ß-PE reinclusion allowed T cell survival. Finally, we found that TRA2ß-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2ß-PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.

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.

Genome of the endangered eastern quoll (Dasyurus viverrinus) reveals signatures of historical decline and pelage color evolution.

The eastern quoll (Dasyurus viverrinus) is an endangered marsupial native to Australia. Since the extirpation of its mainland populations in the 20th century, wild eastern quolls have been restricted to two islands at the southern end of their historical range. Eastern quolls are the subject of captive breeding programs and attempts have been made to re-establish a population in mainland Australia. However, few resources currently exist to guide the genetic management of this species. Here, we generated a reference genome for the eastern quoll with gene annotations supported by multi-tissue transcriptomes. Our assembly is among the most complete marsupial genomes currently available. Using this assembly, we infer the species' demographic history, identifying potential evidence of a long-term decline beginning in the late Pleistocene. Finally, we identify a deletion at the ASIP locus that likely underpins pelage color differences between the eastern quoll and the closely related Tasmanian devil (Sarcophilus harrisii).

Unveiling the Genetic Blueprint of a Desert Scorpion: A Chromosome-level Genome of Hadrurus arizonensis Provides the First Reference for Parvorder Iurida.

Over 400 million years old, scorpions represent an ancient group of arachnids and one of the first animals to adapt to life on land. Presently, the lack of available genomes within scorpions hinders research on their evolution. This study leverages ultralong nanopore sequencing and Pore-C to generate the first chromosome-level assembly and annotation for the desert hairy scorpion, Hadrurus arizonensis. The assembled genome is 2.23 Gb in size with an N50 of 280 Mb. Pore-C scaffolding reoriented 99.6% of bases into nine chromosomes and BUSCO identified 998 (98.6%) complete arthropod single copy orthologs. Repetitive elements represent 54.69% of the assembled bases, including 872,874 (29.39%) LINE elements. A total of 18,996 protein-coding genes and 75,256 transcripts were predicted, and extracted protein sequences yielded a BUSCO score of 97.2%. This is the first genome assembled and annotated within the family Hadruridae, representing a crucial resource for closing gaps in genomic knowledge of scorpions, resolving arachnid phylogeny, and advancing studies in comparative and functional genomics.

Profiling genome-wide methylation in two maples: Fine-scale approaches to detection with nanopore technology.

DNA methylation is critical to the regulation of transposable elements and gene expression and can play an important role in the adaptation of stress response mechanisms in plants. Traditional methods of methylation quantification rely on bisulfite conversion that can compromise accuracy. Recent advances in long-read sequencing technologies allow for methylation detection in real time. The associated algorithms that interpret these modifications have evolved from strictly statistical approaches to Hidden Markov Models and, recently, deep learning approaches. Much of the existing software focuses on methylation in the CG context, but methylation in other contexts is important to quantify, as it is extensively leveraged in plants. Here, we present methylation profiles for two maple species across the full range of 5mC sequence contexts using Oxford Nanopore Technologies (ONT) long-reads. Hybrid and reference-guided assemblies were generated for two new Acer accessions: Acer negundo (box elder; 65x ONT and 111X Illumina) and Acer saccharum (sugar maple; 93x ONT and 148X Illumina). The ONT reads generated for these assemblies were re-basecalled, and methylation detection was conducted in a custom pipeline with the published Acer references (PacBio assemblies) and hybrid assemblies reported herein to generate four epigenomes. Examination of the transposable element landscape revealed the dominance of LTR Copia elements and patterns of methylation associated with different classes of TEs. Methylation distributions were examined at high resolution across gene and repeat density and described within the broader angiosperm context, and more narrowly in the context of gene family dynamics and candidate nutrient stress genes.

Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

Salpa genome and developmental transcriptome analyses reveal molecular flexibility enabling reproductive success in a rapidly changing environment.

Ocean warming favors pelagic tunicates, such as salps, that exhibit increasingly frequent and rapid population blooms, impacting trophic dynamics and composition and human marine-dependent activities. Salp blooms are a result of their successful reproductive life history, alternating seasonally between asexual and sexual protogynous (i.e. sequential) hermaphroditic stages. While predicting future salp bloom frequency and intensity relies on an understanding of the transitions during the sexual stage from female through parturition and subsequent sex change to male, these transitions have not been explored at the molecular level. Here we report the development of the first complete genome of S. thompsoni and the North Atlantic sister species S. aspera. Genome and comparative analyses reveal an abundance of repeats and G-quadruplex (G4) motifs, a highly stable secondary structure, distributed throughout both salp genomes, a feature shared with other tunicates that perform alternating sexual-asexual reproductive strategies. Transcriptional analyses across sexual reproductive stages for S. thompsoni revealed genes associated with male sex differentiation and spermatogenesis are expressed as early as birth and before parturition, inconsistent with previous descriptions of sequential sexual differentiation in salps. Our findings suggest salp are poised for reproductive success at birth, increasing the potential for bloom formation as ocean temperatures rise.

Assembly of 43 human Y chromosomes reveals extensive complexity and variation.

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.

Kangaroo endogenous retrovirus (KERV) forms megasatellite DNA with a simple repetition pattern in which the provirus structure is retained.

The kangaroo endogenous retrovirus (KERV) was previously reported to have undergone a rapid copy number increase in the red-necked wallaby; however, the mode of amplification was left to be clarified. The present study revealed that the long terminal repeat (LTR) (0.6 kb) and internal region (2.0 kb) of a provirus are repeated alternately, forming megasatellite DNA which we named kervRep. This repetition pattern was the same as that observed for walbRep, megasatellite DNA originating from another endogenous retrovirus. Their formation process can be explained using a simple model: pairing slippage followed by homologous recombination. This model features that the initial step is triggered by the presence of two identical sequences within a short distance; the possession of LTRs by endogenous retroviruses fulfills this condition. The discovery of two cases suggests that formation of this type of satellite DNA is one of non-negligible effects of endogenous retroviruses on their host genomes.

A long non-coding RNA Leat1 mediates the hormone responsiveness of EfnB2 during male urogenital development.

The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the testosterone surge from the developing testis and directly interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.

Deep statistical modelling of nanopore sequencing translocation times reveals latent non-B DNA structures.

MOTIVATION: Non-canonical (or non-B) DNA are genomic regions whose three-dimensional conformation deviates from the canonical double helix. Non-B DNA play an important role in basic cellular processes and are associated with genomic instability, gene regulation, and oncogenesis. Experimental methods are low-throughput and can detect only a limited set of non-B DNA structures, while computational methods rely on non-B DNA base motifs, which are necessary but not sufficient indicators of non-B structures. Oxford Nanopore sequencing is an efficient and low-cost platform, but it is currently unknown whether nanopore reads can be used for identifying non-B structures. RESULTS: We build the first computational pipeline to predict non-B DNA structures from nanopore sequencing. We formalize non-B detection as a novelty detection problem and develop the GoFAE-DND, an autoencoder that uses goodness-of-fit (GoF) tests as a regularizer. A discriminative loss encourages non-B DNA to be poorly reconstructed and optimizing Gaussian GoF tests allows for the computation of P-values that indicate non-B structures. Based on whole genome nanopore sequencing of NA12878, we show that there exist significant differences between the timing of DNA translocation for non-B DNA bases compared with B-DNA. We demonstrate the efficacy of our approach through comparisons with novelty detection methods using experimental data and data synthesized from a new translocation time simulator. Experimental validations suggest that reliable detection of non-B DNA from nanopore sequencing is achievable. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/bayesomicslab/ONT-nonb-GoFAE-DND.

Introducing the READY Study: DHH Young people's Well-Being and Self-Determination.

READY is a self-report prospective longitudinal study of deaf and hard of hearing (DHH) young people aged 16 to 19 years on entry. Its overarching aim is to explore the risk and protective factors for successful transition to adulthood. This article introduces the cohort of 163 DHH young people, background characteristics and study design. Focusing on self-determination and subjective well-being only, those who completed the assessments in written English (n = 133) score significantly lower than general population comparators. Sociodemographic variables explain very little of the variance in well-being scores; higher levels of self-determination are a predictor of higher levels of well-being, outweighing the influence of any background characteristics. Although women and those who are LGBTQ+ have statistically significantly lower well-being scores, these aspects of their identity are not predictive risk factors. These results add to the case for self-determination interventions to support better well-being amongst DHH young people.

Whole-genome sequence and assembly of the Javan gibbon (Hylobates moloch).

The Javan gibbon, Hylobates moloch, is an endangered gibbon species restricted to the forest remnants of western and central Java, Indonesia, and one of the rarest of the Hylobatidae family. Hylobatids consist of 4 genera (Holoock, Hylobates, Symphalangus, and Nomascus) that are characterized by different numbers of chromosomes, ranging from 38 to 52. The underlying cause of this karyotype plasticity is not entirely understood, at least in part, due to the limited availability of genomic data. Here we present the first scaffold-level assembly for H. moloch using a combination of whole-genome Illumina short reads, 10X Chromium linked reads, PacBio, and Oxford Nanopore long reads and proximity-ligation data. This Hylobates genome represents a valuable new resource for comparative genomics studies in primates.

Mechanisms of Rapid Karyotype Evolution in Mammals.

Chromosome reshuffling events are often a foundational mechanism by which speciation can occur, giving rise to highly derivative karyotypes even amongst closely related species. Yet, the features that distinguish lineages prone to such rapid chromosome evolution from those that maintain stable karyotypes across evolutionary time are still to be defined. In this review, we summarize lineages prone to rapid karyotypic evolution in the context of Simpson's rates of evolution-tachytelic, horotelic, and bradytelic-and outline the mechanisms proposed to contribute to chromosome rearrangements, their fixation, and their potential impact on speciation events. Furthermore, we discuss relevant genomic features that underpin chromosome variation, including patterns of fusions/fissions, centromere positioning, and epigenetic marks such as DNA methylation. Finally, in the era of telomere-to-telomere genomics, we discuss the value of gapless genome resources to the future of research focused on the plasticity of highly rearranged karyotypes.

Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution.

Alternative splicing of RNA occurs frequently in eukaryotic cells and can result in multiple protein isoforms that are nearly identical in amino acid sequence, but have unique biological roles. Moreover, the relative abundance of these unique isoforms can be correlative with diseased states and potentially used as biomarkers or therapeutic targets. However, due to high sequence similarities among isoforms, current proteomic methods are incapable of differentiating native protein isoforms derived from most alternative splicing events. Herein, a strategy employing a nonsynonymous, non-native amino acid (nnAA) pseudo-hapten (i.e. an amino acid or amino acid derivative that is different from the native amino acid at a particular position) as a targeting epitope in splice junction-spanning peptides was successful in directed antibody derivation. After isolating nnAA-specific antibodies, directed evolution reduced the antibody's binding dependence on the nnAA pseudo-hapten and improved binding to the native splice junction epitope. The resulting antibodies demonstrated codependent binding affinity to each exon of the splice junction and thus are splice junction- and isoform-specific. Furthermore, epitope scanning demonstrated that positioning of the nnAA pseudo-hapten within a peptide antigen can be exploited to predetermine the isolated antibody's specificity at, or near, amino acid resolution. Thus, this nnAA targeting strategy has the potential to robustly derive splice junction- and site-specific antibodies that can be used in a wide variety of research endeavors to unambiguously differentiate native protein isoforms.

Epigenetic patterns in a complete human genome.

The completion of a telomere-to-telomere human reference genome, T2T-CHM13, has resolved complex regions of the genome, including repetitive and homologous regions. Here, we present a high-resolution epigenetic study of previously unresolved sequences, representing entire acrocentric chromosome short arms, gene family expansions, and a diverse collection of repeat classes. This resource precisely maps CpG methylation (32.28 million CpGs), DNA accessibility, and short-read datasets (166,058 previously unresolved chromatin immunoprecipitation sequencing peaks) to provide evidence of activity across previously unidentified or corrected genes and reveals clinically relevant paralog-specific regulation. Probing CpG methylation across human centromeres from six diverse individuals generated an estimate of variability in kinetochore localization. This analysis provides a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation.

From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.

Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.

Comparative Analyses of Gibbon Centromeres Reveal Dynamic Genus-Specific Shifts in Repeat Composition.

Centromeres are functionally conserved chromosomal loci essential for proper chromosome segregation during cell division, yet they show high sequence diversity across species. Despite their variation, a near universal feature of centromeres is the presence of repetitive sequences, such as DNA satellites and transposable elements (TEs). Because of their rapidly evolving karyotypes, gibbons represent a compelling model to investigate divergence of functional centromere sequences across short evolutionary timescales. In this study, we use ChIP-seq, RNA-seq, and fluorescence in situ hybridization to comprehensively investigate the centromeric repeat content of the four extant gibbon genera (Hoolock, Hylobates, Nomascus, and Siamang). In all gibbon genera, we find that CENP-A nucleosomes and the DNA-proteins that interface with the inner kinetochore preferentially bind retroelements of broad classes rather than satellite DNA. A previously identified gibbon-specific composite retrotransposon, LAVA, known to be expanded within the centromere regions of one gibbon genus (Hoolock), displays centromere- and species-specific sequence differences, potentially as a result of its co-option to a centromeric function. When dissecting centromere satellite composition, we discovered the presence of the retroelement-derived macrosatellite SST1 in multiple centromeres of Hoolock, whereas alpha-satellites represent the predominate satellite in the other genera, further suggesting an independent evolutionary trajectory for Hoolock centromeres. Finally, using de novo assembly of centromere sequences, we determined that transcripts originating from gibbon centromeres recapitulate the species-specific TE composition. Combined, our data reveal dynamic shifts in the repeat content that define gibbon centromeres and coincide with the extensive karyotypic diversity within this lineage.