Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.

Temporal dynamics of woolly mammoth genome erosion prior to extinction.

A number of species have recently recovered from near-extinction. Although these species have avoided the immediate extinction threat, their long-term viability remains precarious due to the potential genetic consequences of population declines, which are poorly understood on a timescale beyond a few generations. Woolly mammoths (Mammuthus primigenius) became isolated on Wrangel Island around 10,000 years ago and persisted for over 200 generations before becoming extinct around 4,000 years ago. To study the evolutionary processes leading up to the mammoths' extinction, we analyzed 21 Siberian woolly mammoth genomes. Our results show that the population recovered quickly from a severe bottleneck and remained demographically stable during the ensuing six millennia. We find that mildly deleterious mutations gradually accumulated, whereas highly deleterious mutations were purged, suggesting ongoing inbreeding depression that lasted for hundreds of generations. The time-lag between demographic and genetic recovery has wide-ranging implications for conservation management of recently bottlenecked populations.

Large-scale snake genome analyses provide insights into vertebrate development.

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

A widely distributed gene cluster compensates for uricase loss in hominids.

Approximately 15% of US adults have circulating levels of uric acid above its solubility limit, which is causally linked to the disease gout. In most mammals, uric acid elimination is facilitated by the enzyme uricase. However, human uricase is a pseudogene, having been inactivated early in hominid evolution. Though it has long been known that uric acid is eliminated in the gut, the role of the gut microbiota in hyperuricemia has not been studied. Here, we identify a widely distributed bacterial gene cluster that encodes a pathway for uric acid degradation. Stable isotope tracing demonstrates that gut bacteria metabolize uric acid to xanthine or short chain fatty acids. Ablation of the microbiota in uricase-deficient mice causes severe hyperuricemia, and anaerobe-targeted antibiotics increase the risk of gout in humans. These data reveal a role for the gut microbiota in uric acid excretion and highlight the potential for microbiome-targeted therapeutics in hyperuricemia.

Mesoscale DNA feature in antibody-coding sequence facilitates somatic hypermutation.

Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.

Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.

HGT is widespread in insects and contributes to male courtship in lepidopterans.

Horizontal gene transfer (HGT) is an important evolutionary force shaping prokaryotic and eukaryotic genomes. HGT-acquired genes have been sporadically reported in insects, a lineage containing >50% of animals. We systematically examined HGT in 218 high-quality genomes of diverse insects and found that they acquired 1,410 genes exhibiting diverse functions, including many not previously reported, via 741 distinct transfers from non-metazoan donors. Lepidopterans had the highest average number of HGT-acquired genes. HGT-acquired genes containing introns exhibited substantially higher expression levels than genes lacking introns, suggesting that intron gains were likely involved in HGT adaptation. Lastly, we used the CRISPR-Cas9 system to edit the prevalent unreported gene LOC105383139, which was transferred into the last common ancestor of moths and butterflies. In diamondback moths, males lacking LOC105383139 courted females significantly less. We conclude that HGT has been a major contributor to insect adaptation.

Repeat-based holocentromeres influence genome architecture and karyotype evolution.

The centromere represents a single region in most eukaryotic chromosomes. However, several plant and animal lineages assemble holocentromeres along the entire chromosome length. Here, we compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula, and R. tenuis) and their closest monocentric relative, Juncus effusus. We demonstrate that transition to holocentricity affected 3D genome architecture by redefining genomic compartments, while distributing centromere function to thousands of repeat-based centromere units genome-wide. We uncover a complex genome organization in R. pubera that hides its unexpected octoploidy and describe a marked reduction in chromosome number for R. tenuis, which has only two chromosomes. We show that chromosome fusions, facilitated by repeat-based holocentromeres, promoted karyotype evolution and diploidization. Our study thus sheds light on several important aspects of genome architecture and evolution influenced by centromere organization.

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

The Roots of Genetic Coding in Aminoacyl-tRNA Synthetase Duality.

Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.

Tunnels for Protein Export from the Endoplasmic Reticulum.

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

The expanding amyloid family: Structure, stability, function, and pathogenesis.

The hidden world of amyloid biology has suddenly snapped into atomic-level focus, revealing over 80 amyloid protein fibrils, both pathogenic and functional. Unlike globular proteins, amyloid proteins flatten and stack into unbranched fibrils. Stranger still, a single protein sequence can adopt wildly different two-dimensional conformations, yielding distinct fibril polymorphs. Thus, an amyloid protein may define distinct diseases depending on its conformation. At the heart of this conformational variability lies structural frustrations. In functional amyloids, evolution tunes frustration levels to achieve either stability or sensitivity according to the fibril's biological function, accounting for the vast versatility of the amyloid fibril scaffold.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Ancient and modern genomes unravel the evolutionary history of the rhinoceros family.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.

A mouse-specific retrotransposon drives a conserved Cdk2ap1 isoform essential for development.

Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.

Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.