Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal (cis) folding and less characterized inter-chromosomal (trans) interfaces. Current predictive models of 3D genome folding overlook trans-genome organization. We present TwinC, an interpretable convolutional neural network model that reliably predicts trans contacts measurable through proximity ligation-dependent (in situ and intact Hi-C) and independent (DNA SPRITE) genome-wide chromatin conformation assays. TwinC achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from in situ and intact Hi-C experiments in heart tissue. Furthermore, we train TwinC using in situ Hi-C data from the widely used GM12878 cell line and validate its performance with orthogonal DNA SPRITE assay in the same cell type. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding, and G-quadruplexes in forming trans contacts. In summary, TwinC models and interprets trans genome architecture, illuminating this poorly understood aspect of gene regulation.

Advances in the sequencing and assembly of chromosome-level genome assemblies has enabled the study of non-model animals, providing further insights into the evolution of genomes and chromosomes. Here, we present the waterbuck ( Kobus ellipsiprymnus ) as an emerging model antelope for studying population dynamics and chromosome evolution. Antelope evolutionary history has been shaped by Robertsonian (Rb) fusions, with waterbuck also showing variation in karyotype due to two polymorphic Rb fusions. These polymorphisms are variable between and within the two recognised subspecies, the common and defassa waterbuck. To provide new insights into waterbuck evolution, we firstly assembled a chromosome-level genome assembly for the defassa subspecies using PacBio HiFi and Hi-C sequencing. We then utilised museum collections to carry out whole genome sequencing (WGS) of 24 historical waterbuck skins from both subspecies. Combined with a previous WGS dataset (n = 119), this represents the largest study of waterbuck populations to date. We found novel population structure and gene flow between waterbuck populations and regions across the genome with high genomic differentiation between the two subspecies. Several of these regions were found around the centromeres of fixed and polymorphic Rb fusions, exhibiting signatures of low recombination and local population structure. Interestingly, these regions contain genes involved in development, fertility, and recombination. Our results highlight the importance of assembling genomes to the chromosome-level, the utility and value of historical collections in sampling a wide-ranging species to uncover fine-scale population structure, and the potential impacts of Rb fusions on genomic differentiation and the recombination landscape.

The stone marten (Martes foina) is an important species for cytogenetic studies in the order Carnivora. ZooFISH probes created from its chromosomes provided a strong and clean signal in chromosome painting experiments and were valuable for studying the evolution of carnivoran genome architecture. The research revealed that the stone marten chromosome set is similar to the presumed ancestral karyotype of the Carnivora, which added an additional value for the species. Using linked-read and Hi-C sequencing, we generated a chromosome-length genome assembly of a male stone marten (Gansu province, China) from a primary cell line. The stone marten assembly had a length of 2.42 Gbp, scaffold N50 of 144 Mbp, and a 96.2% BUSCO completeness score. We identified 19 chromosomal scaffolds (2n = 38) and assigned them chromosome ids based on chromosome painting data. Annotation identified 20,087 protein-coding gene models, of which 18,283 were assigned common names. Comparison of the stone marten assembly with the cat, dog, and human genomes revealed several small syntenic blocks absent on the published painting maps. Finally, we assessed the heterozygosity and its distribution over the chromosomes. The detected low heterozygosity level (0.4 hetSNPs/kbp) and the presence of long runs of homozygosity require further research and a new evaluation of the conservation status of the stone marten in China. Combined with available carnivoran genomes in large-scale synteny analysis, the stone marten genome will highlight new features and events in carnivoran evolution, hidden from cytogenetic approaches.

Uta stansburiana are an emerging model system for studying sexual selection, polymorphism, and the evolution of pace-of-life syndromes (POLS) whose distribution covers variable environments and a wide latitudinal gradient. POLS are suites of traits causing variation of life history along a slow maturing-fast maturing continuum. We present a high-quality chromosome-level reference genome for U. stansburiana and pair it with RNA-seq gene expression data to demonstrate, for the first time, the molecular basis for pace-of-life differences between locations with higher and lower climate seasonality and sexual size dimorphism (SSD). Our assembly is 2.1 Gbp, has scaffold N50 of 320 Mbp, includes 104 scaffolds, and has an L50 of 3. The assembly comprises six macrochromosomes and 11 microchromosomes. We annotated 20,350 genes for the assembly and found a repeat element composition of 49.23%, similar to work in other phrynosomatid lizards. RNA-seq data demonstrate differential expression in genes associated with pace-of-life divergence including those related to stress, sexual reproduction, and cell proliferation/carcinogenesis between distinctive environments. Our results provide the first differential gene expression evidence of environmentally mediated pace-of-life processes related to different degrees of SSD in U. stansburiana and demonstrate the utility of RNA-seq gene expression data in detecting POLS.

The Mountain bongo (Tragelaphus eurycerus isaaci), a critically endangered tragelaphine antelope native to the montane forests of Kenya, faces significant threats from habitat loss and hunting. Although the Mountain bongo is a flagship species in Kenya, the majority are found in small, isolated populations of less than 100 animals total, making it a species of high conservation concern. In this report, we present a chromosome-length draft genome assembly for the Mountain bongo, generated using a combination of linked-read and proximity ligation (Hi-C) sequencing techniques. The assembly resulted in a 2.96 Gb sized genome with a contig N50 of 79.5 kb and a scaffold N50 of 192 Mb. Assembly completeness was 95.1% based on 12,234 Benchmarking Universal Single-Copy Orthologs (BUSCO) and annotation revealed 29,820 protein-coding genes, of which 27,761 were functionally annotated, and a repetitive content of 47.31%. Synteny analysis against the domestic cattle (Bos taurus) genome assembly revealed numerous chromosomal rearrangements between the 2 species. Our analysis also revealed insights into the evolutionary and demographic history of the Mountain bongo, offering valuable information for conservation management. We also assembled and annotated the mitochondrial genome which showed <1% differences from the Lowland bongo subspecies, T. e. eurycerus. By integrating genomic data with traditional conservation methods, this reference lays the foundation to evaluate and preserve genetic diversity of both in situ and ex situ populations of the Mountain bongo amidst growing environmental pressures.

Freshwater ecosystems and their biota are under increasing pressure from anthropogenic stressors. In response to declining fish stocks, hatchery and stocking programmes are widely implemented as core components of restoration and management strategies, with positive outcomes for some wild populations. Despite this, stocking remains contentious due to potential genetic and ecological risks to wild populations. Monitoring and evaluation of stocking outcomes are critical to ensuring the long-term sustainability of wild populations, but identification of stocked individuals post-release remains a key challenge, particularly for mobile species. In this study, we combined otolith (natal origin and age) and genomic data to identify stocked individuals and evaluate the genetic implications of stocking for a culturally and socioeconomically important and mobile freshwater fish, golden perch Macquaria ambigua (family: Percichthyidae), across Australia's Murray-Darling Basin (MDB). We also generated a chromosome-level genome assembly. Many close kin were detected across the MDB, increasing in prevalence over recent decades and mostly of hatchery origin. Rivers with many close kin were associated with low effective population sizes (Ne < 100). Genetic signatures of stocking varied according to local context, being most pronounced in but not restricted to rivers considered functionally isolated for management purposes. Where fish are stocked into rivers that are part of the connected metapopulation, there is scope to modify current stocking practices to avoid over-representation of related stocked individuals. Increased focus on the genetic diversity of stocked fish is likely to promote the long-term persistence of golden perch in the wild.

Advances in the field of three-dimensional (3D) genomics have revealed an ever-expanding array of architectural features that were unknown only a few years ago. Just as ribbon diagrams integrate spatial and symbolic representation to communicate the shape of a protein, the representation of genomes in 3D space requires the development and use of new cartographic symbols and visual conventions. Here, we propose a conceptualized grammar that makes it easier to create visual 3D representations of genomes.

Two-thirds of Earth's species have undergone population declines, leaving many vulnerable to genomic erosion and inbreeding depression. Genetic rescue can boost the fitness of small populations, but perceived risks of outbreeding depression can limit its use. We quantified these trade-offs in hundreds of endangered Pacific pocket mice (Perognathus longimembris pacificus) by combining whole-genome sequences with fitness data. The impacts of genomic erosion in remnant populations were reversed in an admixed breeding program, suggesting the potential benefits of genetic rescue. However, differences in chromosome numbers increase the risk of genetic incompatibilities. Fitness analyses suggested that although admixed karyotypes may have reduced fertility, non-admixed mice with low heterozygosity and high genetic load had even lower fitness, pointing to a greater risk of extinction if populations remain isolated.

We present a high-quality assembly and annotation of the periodical cicada species, Magicicada septendecula (Hemiptera: Auchenorrhyncha: Cicadidae). Periodical cicadas have a significant ecological impact, serving as a food source for many mammals, reptiles, and birds. Magicicada are well known for their massive emergences of 1 to 3 species that appear in different locations in the eastern United States nearly every year. These year classes ("broods") emerge dependably every 13 or 17 yr in a given location. Recently, it has become clear that 4-yr early or late emergences of a sizeable portion of a population are an important part of the history of brood formation; however, the biological mechanisms by which they track the passage of time remain a mystery. Using PacBio HiFi reads in conjunction with Hi-C proximity ligation data, we have assembled and annotated the first whole genome for a periodical cicada, an important resource for future phylogenetic and comparative genomic analysis. This also represents the first quality genome assembly and annotation for the Hemipteran superfamily Cicadoidea. With a scaffold N50 of 518.9 Mb and a complete BUSCO score of 96.7%, we are confident that this assembly will serve as a vital resource toward uncovering the genomic basis of periodical cicadas' long, synchronized life cycles and will provide a robust framework for further investigations into these insects.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth’s death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

The three-dimensional organization of genomes plays a crucial role in essential biological processes. The segregation of chromatin into A and B compartments highlights regions of activity and inactivity, providing a window into the genomic activities specific to each cell type. Yet, the steep costs associated with acquiring Hi-C data, necessary for studying this compartmentalization across various cell types, pose a significant barrier in studying cell type specific genome organization. To address this, we present a prediction tool called compartment prediction using recurrent neural networks (CoRNN), which predicts compartmentalization of 3D genome using histone modification enrichment. CoRNN demonstrates robust cross-cell-type prediction of A/B compartments with an average AuROC of 90.9%. Cell-type-specific predictions align well with known functional elements, with H3K27ac and H3K36me3 identified as highly predictive histone marks. We further investigate our mispredictions and found that they are located in regions with ambiguous compartmental status. Furthermore, our model's generalizability is validated by predicting compartments in independent tissue samples, which underscores its broad applicability.

Phenotypic variation among species is a product of evolutionary changes to developmental programs 1,2 . However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.

Background B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. Results Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. Conclusions Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

Pteronarcys californica () is commonly referred to as the giant salmonfly and is the largest species of stonefly (Insecta: Plecoptera) in the western United States. Historically, it was widespread and abundant in western rivers, but populations have experienced a substantial decline in the past few decades, becoming locally extirpated in numerous rivers in Utah, Colorado, and Montana. Although previous research has explored the ecological variables conducive to the survivability of populations of the giant salmonfly, a lack of genomic resources hampers exploration of how genetic variation is spread across extant populations. To accelerate research on this imperiled species, we present a de novo chromosomal-length genome assembly of P. californica generated from PacBio HiFi sequencing and Hi-C chromosome conformation capture. Our assembly includes 14 predicted pseudo chromosomes and 98.8% of Insecta universal core orthologs. At 2.40 gigabases, the P. californica assembly is the largest of available stonefly assemblies, highlighting at least a 9.5-fold variation in assembly size across the order. Repetitive elements account for much of the genome size increase in P. californica relative to other stonefly species, with the content of Class I retroelements alone exceeding the entire assembly size of all but two other species studied. We also observed preliminary suborder-specific trends in genome size that merit testing with more robust taxon sampling.

Squamate reptiles are a highly diverse and intriguing group of tetrapods, offering valuable insights into the evolution of amniotes. The Australian water dragon (Intellagama lesueurii) is a member of the Agamidae and sister to the core mesic Australian endemic radiation (Amphibolurinae). The species is renowned for its urban adaptability and complex social systems. We report a 1.8 Gb chromosome-length genome assembly together with the annotation of 23,675 protein-coding genes. Comparative analysis with other squamate genomes highlights gene family expansions associated with immune function, energetic homeostasis, and wound healing. This reference genome will serve as a valuable re-source for studies of evolution and environmental resilience in lizards.

Phlebotomine sand flies are the vectors of leishmaniasis, a neglected tropical disease. High-quality reference genomes are an important tool for understanding the biology and eco-evolutionary dynamics underpinning disease epidemiology. Previous leishmaniasis vector reference sequences were limited by sequencing technologies available at the time and inadequate for high-resolution genomic inquiry. Here, we present updated reference assemblies of two sand flies, Phlebotomus papatasi and Lutzomyia longipalpis. These chromosome-level assemblies were generated using an ultra-low input library protocol, PacBio HiFi long reads, and Hi-C technology. The new P. papatasi reference has a final assembly span of 351.6 Mb and contig and scaffold N50s of 926 kb and 111.8 Mb, respectively. The new Lu. longipalpis reference has a final assembly span of 147.8 Mb and contig and scaffold N50s of 1.09 Mb and 40.6 Mb, respectively. Benchmarking Universal Single-Copy Orthologue (BUSCO) assessments indicated 94.5% and 95.6% complete single copy insecta orthologs for P. papatasi and Lu. longipalpis. These improved assemblies will serve as an invaluable resource for future genomic work on phlebotomine sandflies.

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

Chickpea (Cicer arietinum L.)-an important legume crop cultivated in arid and semiarid regions-has limited genetic diversity. Efforts are being undertaken to broaden its diversity by utilizing its wild relatives, which remain largely unexplored. Here, we present the Cicer super-pangenome based on the de novo genome assemblies of eight annual Cicer wild species. We identified 24,827 gene families, including 14,748 core, 2,958 softcore, 6,212 dispensable and 909 species-specific gene families. The dispensable genome was enriched for genes related to key agronomic traits. Structural variations between cultivated and wild genomes were used to construct a graph-based genome, revealing variations in genes affecting traits such as flowering time, vernalization and disease resistance. These variations will facilitate the transfer of valuable traits from wild Cicer species into elite chickpea varieties through marker-assisted selection or gene-editing. This study offers valuable insights into the genetic diversity and potential avenues for crop improvement in chickpea.

Abstract Genome organization in humans is governed by two major mechanisms: loop extrusion by cohesin and CTCF and the spatial compartmentalization of the chromosomes. These folding mechanisms have been shown to regulate several genomic functions, including gene expression. However, how transcription might regulate the chromatin structure remains a subject of many ongoing investigations. In this study, we use the Integrator protein complex as a tool to understand the impact of aberrant transcription on the 3D structure of the genome. During transcription initiation, RNA Polymerase-II (Pol-II) often pauses proximally to the promoter before further elongating transcripts. The Integrator protein complex regulates this checkpoint by its endonuclease and phosphatase activity. This protein complex is essential in premature transcription termination in hundreds of protein-coding genes. It also facilitates the synthesis of non-coding (ncRNA), such as enhancer RNAs (eRNAs) and long non-coding RNAs (lncRNAs), which have been shown to support loop extrusion domains, and their absence may disrupt these domains. Using HiC sequencing, we found that depleting Integrator proteins can interfere with forming and maintaining loop domains. This study aims to be the first to decipher the relationship between chromosome structure and Integrator protein activity and address our very little knowledge of how misregulation of transcription termination may influence the folding of chromosomes. Citation Format: Naiju Thomas, Timothy E. Reznicek, Erez Lieberman Aiden, M. Jordan Rowley, Eric Wagner, Guy Nir. Defining the impact of aberrant transcription on the chromatin structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1699.

Cartilaginous fishes (chondrichthyans: chimeras and elasmobranchs -sharks, skates, and rays) hold a key phylogenetic position to explore the origin and diversifications of jawed vertebrates. Here, we report and integrate reference genomic, transcriptomic, and morphological data in the small-spotted catshark Scyliorhinus canicula to shed light on the evolution of sensory organs. We first characterize general aspects of the catshark genome, confirming the high conservation of genome organization across cartilaginous fishes, and investigate population genomic signatures. Taking advantage of a dense sampling of transcriptomic data, we also identify gene signatures for all major organs, including chondrichthyan specializations, and evaluate expression diversifications between paralogs within major gene families involved in sensory functions. Finally, we combine these data with 3D synchrotron imaging and in situ gene expression analyses to explore chondrichthyan-specific traits and more general evolutionary trends of sensory systems. This approach brings to light, among others, novel markers of the ampullae of Lorenzini electrosensory cells, a duplication hotspot for crystallin genes conserved in jawed vertebrates, and a new metazoan clade of the transient-receptor potential (TRP) family. These resources and results, obtained in an experimentally tractable chondrichthyan model, open new avenues to integrate multiomics analyses for the study of elasmobranchs and jawed vertebrates.