Biology subjects

Liu, A.

Publications and source records attributed to Liu, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis

Organelle transporters define metabolic compartmentalization and how this metabolite transport process can be modulated is poorly explored. Here, we discovered that SLC25A39, a mitochondrial transporter critical for mitochondrial glutathione uptake, is a short-lived protein under dual regulation at the protein level. Co-immunoprecipitation mass spectrometry and CRISPR KO in cells identified that mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1. SLC25A39 senses mitochondrial iron-sulfur cluster using four matrix cysteine residues and inhibits its degradation. SLC25A39 protein regulation is robust in developing and mature neurons. This dual transporter regulation, by protein quality control and metabolic sensing, allows modulating mitochondrial glutathione level in response to iron homeostasis, opening new avenues to explore regulation of metabolic compartmentalization. Neuronal SLC25A39 regulation connects mitochondrial protein quality control, glutathione and iron homeostasis, which were previously unrelated biochemical features in neurodegeneration.

cell biology↗

bioRxiv2023

Targeted Protein Relocalization via Protein Transport Coupling

Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2-4. The localization of numerous proteins has been annotated5-7, and pharmacologically relevant approaches for precise rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules which harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could provide an avenue for targeted protein relocalization for interactome-rewiring therapeutics. To realize this concept, we deploy a quantitative approach to identify features which govern the ability to hijack protein trafficking, develop a collection of shuttle proteins and ligands, and demonstrate relocalization of proteins bearing endogenous localization signals. Using a custom imaging analysis pipeline, we show that endogenous localization signals can be overcome through molecular coupling of target proteins to shuttle proteins containing sufficiently strong native localization sequences expressed in the necessary abundance. We develop nuclear hormone receptors as viable shuttles which can be harnessed with Targeted Relocalization Activating Molecules (TRAMs) to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43 {Delta}NLS, and FUSR495X. Small molecule-mediated relocalization of FUSR495X to the nucleus from the cytoplasm reduced the number of cellular stress granules in a model of cellular stress. Using Cas9-mediated knock-in tagging, we demonstrate nuclear enrichment of both low abundance (FOXO3a) and high abundance (FKBP12) endogenous proteins via molecular coupling to nuclear hormone receptor trafficking. Finally, small molecule-mediated redistribution of NMNAT1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the WldS gain-of-function phenotype from mice resistant to certain types of neurodegeneration8. The concept of targeted protein relocalization could therefore nucleate approaches for treating disease through interactome rewiring.

biochemistry↗

bioRxiv2023

Disruption of Epithelium Integrity by Inflammation-Associated Fibroblasts through Prostaglandin Signaling

Inflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed an in vitro model whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFs in vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury. TeaserInflammation-associated fibroblasts compromise colon epithelial barrier integrity and genome stability via PGE2-EP4 signaling.

cell biology↗

bioRxiv2023

Integrative PTEN Enhancer Discovery Reveals a New Model of Enhancer Organization

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

genomics↗

bioRxiv2023

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

EROS (essential for reactive oxygen species) is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in NOX2 expression or function due to genetic mutations leads to chronic granulomatous disease (CGD) with recurrent bacterial and fungal infections. To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex. EROS binds to NOX2 in plasma membrane through its anti-parallel -helices H1 and H2, and in cytoplasm through multiple {beta}-strands that form hydrogen bonds with the C terminal fragment of NOX2. EROS binding alters the conformation of the TM2 and TM6 transmembrane helices, increases the distance between the two hemes, and causes dislocation of the binding site for flavin adenine dinucleotide (FAD). EROS colocalizes with NOX2 on cell surface of neutrophil-like HL-60 cells and forms a heterotrimer with mature NOX2-p22phox in transfected cells. Phorbol myristate acetate, an activator of NOX2, induces dissociation of EROS from NOX2 in a NanoLuc complementation assay with concurrent production of superoxide in reconstituted cells. Taken together, these findings provide a structural basis for EROS-NOX2 interaction and suggest a previously unidentified function of EROS in regulating NOX2 activation.

immunology↗

bioRxiv2023

Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development

The development of multi-cellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic-helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling - the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knock-down of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

plant biology↗

bioRxiv2023

Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin

Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane in a GTP hydrolysis-dependent manner. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Phosphorylation of S579 is widely regarded as activating, while S600 phosphorylation is commonly considered inhibiting. However, the direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. In this study, we compare the effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and in vitro phosphorylation. The oligomerization state of both phospho-mimetic mutants is shifted toward smaller oligomers. Both phospho-mimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wild-type Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant that lacks GTPase activity, the K38A mutant, stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions, similar to previous results for dynamin-1. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly, and likely requires additional factors for stimulation of mitochondrial fission in cells. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.

biochemistry↗

bioRxiv2023

The effects of reference panel perturbations on the accuracy of genotype imputation

Reference-based genotype imputation is a standard technique that has become increasingly popular in large-scale studies involving genomic data. The two key elements involved in the process of genotype imputation are (1) the haplotype reference panel to which a target individual is being imputed, and (2) the imputation algorithm used to infer missing genotypes in the target individual. The imputation literature has historically focused mainly on (2), with a typical comparative study investigating the relative performance of various imputation algorithms while holding the reference panel constant. However, the role of the reference panel itself (1) on overall imputation performance is equally, if not more, important than the choice among many high-performing algorithms. Even though it is intuitive that the quality of a reference panel should play a role in the accuracy of imputation, it is nonetheless unclear to what extent common errors during panel creation (e.g., genotyping and phase error) lead to suboptimal imputation performance. In this study, we investigate the effects of applying three distinct modes of perturbations to a widely used haplotype reference panel in human genetics on the resulting imputation accuracy. Specifically, we perturb the reference panel by (1) randomly introducing phase errors, (2) randomly introducing genotype errors, and (3) randomly pruning variants from the panel (all at varying magnitudes). We then impute a set of diverse individuals at various sequencing coverages (0.5x, 1.0x, and 2.0x) to these various perturbed panels and evaluate imputation accuracy using the r2 metric for the entire cohort as well as ancestry-stratified subsets. We observe that both phase- and genotype-perturbations can dramatically affect imputation accuracy, particularly at very low allele frequencies, while pruning variants has a far smaller effect. We then empirically verified that our simulations reliably predict the impact of potential filtering techniques in a real-world dataset. In the context of haplotype reference panels, these results suggest that phasing and genotyping accuracy are far more important than the density of a reference panel used for imputation.

genomics↗

bioRxiv2023

Scaling between cell cycle duration and wing growth is regulated by Fat-Dachsous signaling in Drosophila

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila. Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth.

developmental biology↗

bioRxiv2023

Endocytosis gated by emergent properties of membrane-clathrin interactions

Clathrin-mediated endocytosis (CME), the major cellular entry pathway, starts when clathrin assembles on the plasma membrane into clathrin-coated pits (CCPs). Two populations of CCPs are detected within the same cell: productive CCPs that invaginate and pinch off, forming clathrin-coated vesicles (CCVs) [1, 2], and abortive CCPs [3, 4, 5] that prematurely disassemble. The mechanisms of gating between these two populations and their relations to the functions of dozens of early-acting endocytic accessory proteins (EAPs) [5, 6, 7, 8, 9] have remained elusive. Here, we use experimentally-guided modeling to integrate the clathrin machinery and membrane mechanics in a single dynamical system. We show that the split between the two populations is an emergent property of this system, in which a switch between an Open state and a Closed state follows from the competition between the chemical energy of the clathrin basket and the mechanical energy of membrane bending. In silico experiments revealed an abrupt transition between the two states that acutely depends on the strength of the clathrin basket. This critical strength is lowered by membrane-bending EAPs [10, 11, 12]. Thus, CME is poised to be shifted between abortive and productive events by small changes in membrane curvature and/or coat stability. This model clarifies the workings of a putative endocytic checkpoint whose existence was previously proposed based on statistical analyses of the lifetime distributions of CCPs [4, 13]. Overall, a mechanistic framework is established to elucidate the diverse and redundant functions of EAPs in regulating CME progression.

biophysics↗

bioRxiv2023

Long-chain fatty acyl-coenzyme A activates the mitochondrial fission factors MiD49 and MiD51 by inducing their oligomerization

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, activating their ability to stimulate DRP1 GTPase activity. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knock-down effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with the fatty acid analogue 2-bromopalmitate, which causes increased acyl-CoA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty acid-induced mitochondrial fragmentation. Finally, MiD49 or MiD51 oligomers synergize with MFF, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

cell biology↗

bioRxiv2023

Mecp2 Fine-tunes Quiescence Exit by Targeting Nuclear Receptors

Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit, as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. Genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly, disruption of two nuclear receptors (NRs), Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration.

cell biology↗

bioRxiv2023

The role of embryonic origins and suture proximity in repair of cranial bone

The cranial sutures are proposed to be a stem cell niche, harbouring skeletal stem cells (SSCs) that are directly involved in development, homeostasis, and healing. Like the craniofacial bones, the sutures are formed from both mesoderm and neural crest. During cranial repair, neural crest cells have been proposed to be key players; however, neural crest contributions to the sutures are not well defined, and the relative importance of suture proximity is unclear. Here, we combine calvarial wounding experiments with genetically labelled mouse models to demonstrate that suture proximity predicts the efficiency of cranial repair. Furthermore, we define the neural crest-mesoderm boundary within the sutures and demonstrate that Gli1+ and Axin2+ SSCs are present in all calvarial sutures examined. We propose that the position of the defect determines the availability of neural crest-derived progenitors, which appear to be a key element in the repair of calvarial defects. Summary statementThis study provides a new examination of the embryonic origins of cranial sutures in mouse. Using genetic lineage labelling, we identify specific contributions of neural crest cells and associated skeletal stem cells during repair of calvarial wounds. This is extremely relevant considering recent findings regarding the role of suture-residing stem cells.

developmental biology↗

bioRxiv2023

A statistical method for image-mediated association studies discovers genes and pathways associated with four brain disorders

Brain imaging and genomics are critical tools enabling characterization of the genetic basis of brain disorders. However, imaging large cohorts is expensive, and may be unavailable for legacy datasets used for genome-wide association studies (GWAS). Using an integrated feature selection/aggregation model, we developed Image-Mediated Association Study (IMAS), which utilizes borrowed imaging/genomics data to conduct association mapping in legacy GWAS cohorts. By leveraging the UK Biobank image-derived phenotypes (IDPs), IMAS discovered genetic bases underlying four neuropsychiatric disorders and verified them by analyzing annotations, pathways, and expression Quantitative Trait Loci (eQTLs). A cerebellar-mediated mechanism was identified to be common to the four disorders. Simulations show that, if the goal is identifying genetic risk, IMAS is more powerful than a hypothetical protocol in which the imaging results were available in the GWAS dataset. This implies the feasibility of reanalyzing legacy GWAS datasets without conducting additional imaging, yielding cost-savings for integrated analysis of genetics and imaging.

bioinformatics↗

bioRxiv2023

Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) - Gi protein complex bound to the chemerin C-terminal nonapeptide

Chemerin is an adipokine with chemotactic activity to a subset of leukocytes. Chemerin mainly acts through its C-terminal nonapeptide (YFPGQFAFS, C9) that bind to three G protein-coupled receptors including chemokine-like receptor 1 (CMKLR1), G-protein coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2). We examined C9 signaling through GPR1 and found this receptor capable of Gi signaling but very weak {beta}-arrestin signaling. Here we report high-resolution cryo-EM structures of GPR1-Gi complexes bound to full-length chemerin and to C9, respectively. Unlike C9 that inserts directly into a transmembrane binding pocket, full-length chemerin uses its N-terminal globular core for extensive interaction with the N terminus of GPR1. Within the binding pocket, the C terminal loop containing the nonapeptide takes the same "S-shape" pose as synthetic C9 nonapeptide. These findings explain why the nonapeptide is a full agonist of GPR1, and demonstrate that chemerin uses a "two-site" model for interaction with GPR1. An analysis of the GPR1-Gi protein interface found high similarities to the CMKLR1-Gi complex, as confirmed by site-directed mutagenesis with functional verifications. Our structural analysis demonstrates shared features with chemokines in that chemerin acts as a "reverse chemokine" with switched functions of its N and C termini in the interaction with GPR1.

pharmacology and toxicology↗

bioRxiv2023

Nematostella vectensis exemplifies the exceptional expansion and diversity of opsins in the eyeless Hexacorallia

BackgroundOpsins are the primary proteins responsible for light detection in animals. Cnidarians (jellyfish, sea anemones, corals) have diverse visual systems that have evolved in parallel with bilaterians (squid, flies, fish) for hundreds of millions of years. Medusozoans (e.g. jellyfish, hydroids) have evolved eyes multiple times, each time independently incorporating distinct opsin orthologs. Anthozoans (e.g. corals, sea anemones,) have diverse light-mediated behaviors and, despite being eyeless, exhibit more extensive opsin duplications than medusozoans. To better understand the evolution of photosensitivity in animals without eyes we increased anthozoan representation in the phylogeny of animal opsins and investigated the large but poorly characterized opsin family in the sea anemone Nematostella vectensis. ResultsWe analyzed genomic and transcriptomic data from 16 species of cnidarians to generate a large opsin phylogeny (708 sequences) with the largest sampling of anthozoan sequences to date. We identified 29 opsins from N. vectensis (NvOpsins) with high confidence, using transcriptomic and genomic datasets. We found that lineage-specific opsin duplications are common across Cnidaria, with anthozoan lineages exhibiting among the highest numbers of opsins in animals. To establish putative photosensory function of NvOpsins, we identified canonically conserved protein domains and amino acid sequences essential for opsin function in other animal species. We show high sequence diversity among NvOpsins at sites important for photoreception and transduction, suggesting potentially diverse functions. We further examined the spatiotemporal expression of NvOpsins and found both dynamic expression of opsins during embryonic development and sexually dimorphic opsin expression in adults. ConclusionsThese data show that lineage-specific duplication and divergence has led to expansive diversity of opsins in eyeless cnidarians, suggesting opsins from these animals may exhibit novel biochemical functions. The variable expression patterns of opsins in N. vectensis suggest opsin gene duplications allowed for a radiation of unique sensory cell types with tissue-and stage-specific functions. This diffuse network of distinct sensory cell types could be an adaptive solution for varied sensory tasks experienced in distinct life history stages in Anthozoans.

evolutionary biology↗

bioRxiv2023

Tractable and Expressive Generative Models of Genetic Variation Data

Generative models play an important role in population genetics, being used to generate artificial genomes (AGs) that help benchmark methods, test evolutionary hypotheses, and construct reference panels for imputation while working around data-sharing restrictions. Existing generative models of genetic variation, however, struggle to faithfully express dependencies in the data while retaining tractability and preserving privacy. We introduce Genetic Probabilistic Circuits (GPC), a deep generative model for genetic variation data based on hidden Chow-Liu trees represented as probabilistic circuits. GPC generalizes traditional hidden Markov models by allowing arbitrary tree structures over latent variables, enabling it to capture long-range dependencies among SNPs. GPC is tractable, supporting exact computation of marginal and conditional probabilities that enables both AG generation and direct genotype imputation (avoiding the need for simulating AGs). We show that GPC generates AGs that accurately reproduce population structure and linkage disequilibrium patterns across a range of length scales. Compared to other deep generative approaches, GPC consistently improves imputation accuracy, with particularly strong gains for low-frequency variants and in populations that are not well-represented in public reference panels. Finally, we show that GPC better preserves privacy of the training data, thereby providing a practical framework for AG generation in settings with limited data access.

genetics↗

bioRxiv2023

Extreme genome scrambling in cryptic Oikopleura dioica species

Genes are not randomly distributed throughout chromosomes. How gene order evolves and how selective constraints act to preserve or vary gene order, both at the macrosyntenic level of whole chromosomes or microsyntenic level of gene blocks, are central questions of evolutionary biology and genomics that remain largely unsolved. Here, after sequencing several genomes of the appendicularian tunicate Oikopleura dioica from different locations around the globe, we show an unprecedented amount of genome scrambling in animals with no obvious morphological differences, consistent with cryptic speciation. Our assemblies suggest that all members of this clade possess a common 3-chromosome karyotype, and that different species largely preserve gene content, despite the presence of thousands of rearrangements in gene order. The movements of genes are largely restricted to chromosome arms and sex-specific regions, which appear to be the primary unit of macrosynteny conservation, and examples of these within-arm movements can be seen in the Hox and Fgf gene families. Our approach employing whole-genome alignments demonstrates that segments containing protein-coding elements tend to be preserved at the microsyntenic scale, consistent with strong purifying selection, with appreciably less preservation of non-coding elements. Unexpectedly, scrambling did not preserve operon structure across species, suggesting an absence of selective pressure to maintain operon structure. As well, genome scrambling does not occur uniformly across all chromosomes, as short chromosome arms possess shorter genes, smaller operons, more breakpoints, and elevated dN/dS values compared to long chromosome arms. Estimation of divergence times among the cryptic O. dioica lineages yielded an estimated breakpoint accumulation rate of 6 to 25 breakpoints per megabase per million years, which is an order of magnitude higher than the rates for other ascidian tunicates or Drosophila species. Therefore, O. dioica appears to be an attractive animal system to unravel the mechanisms that underlie gene order and synteny conservation, as well as exploring the limits of genome scrambling without an apparent impact on phenotypic evolution.

genomics↗