Conserved Noncoding Sequences Research Articles

Evolutionary changes of noncoding elements associated with transition of sexual mode in Caenorhabditis nematodes.

The transition of the sexual mode occurs widely in animal evolution. In Caenorhabditis nematodes, androdioecy, a sexual polymorphism composed of males and hermaphrodites having the ability to self-fertilize, has evolved independently multiple times. While the modification of noncoding regulatory elements likely contributed to the evolution of hermaphroditism, little is known about these changes. Here, we conducted a genome-wide analysis of conserved noncoding elements (CNEs) focusing on the evolution of hermaphroditism in Caenorhabditis nematodes. We found that, in androdioecious nematodes, mutations rapidly accumulated in CNEs' neighboring genes associated with sexual traits. Expression analysis indicate that the identified CNEs are involved in spermatogenesis in hermaphrodites and associated with the transition of gene expression from dioecious to androdioecious nematodes. Last, genome editing of a CNE neighboring laf-1 resulted in a change in its expression in the gonadal region undergoing spermatogenesis. Our bioinformatic and experimental analyses highlight the importance of CNEs in gene regulation associated with the development of hermaphrodites.

Genomic insights into adaptation to bipedal saltation and desert-like habitats of jerboas.

Jerboas is a lineage of small rodents displaying atypical mouse-like morphology with elongated strong hindlimbs and short forelimbs. They have evolved obligate bipedal saltation and acute senses, and been well-adapted to vast desert-like habitats. Using a newly sequenced chromosome-scale genome of the Mongolian five-toed jerboa (Orientallactaga sibirica), our comparative genomic analyses and in vitro functional assays showed that the genetic innovations in both protein-coding and non-coding regions played an important role in jerboa morphological and physiological adaptation. Jerboa-specific amino acid substitutions, and segment insertions/deletions (indels) in conserved non-coding elements (CNEs) were found in components of proteoglycan biosynthesis pathway (XYLT1 and CHSY1), which plays an important role in limb development. Meanwhile, we found specific evolutionary changes functionally associated with energy or water metabolism (e.g., specific amino acid substitutions in ND5 and indels in CNEs physically near ROR2) and senses (e.g., expansion of vomeronasal receptors and the FAM136A gene family) in jerboas. Further dual-luciferase reporter assay verified that some of the CNEs with jerboa-specific segment indels exerted a significantly different influence on luciferase activity, suggesting changes in their regulatory function in jerboas. Our results revealed the potential molecular mechanisms underlying jerboa adaptation since the divergence from the Eocene-Oligocene transition, and provided more resources and new insights to enhance our understanding of the molecular basis underlying the phenotypic diversity and the environmental adaptation of mammals.

A RORE-dependent Intronic Enhancer in the IL-7 Receptor-α Locus Controls Glucose Metabolism via Vγ4+ γδT17 Cells.

The IL-7R regulates the homeostasis, activation, and distribution of T cells in peripheral tissues. Although several transcriptional enhancers that regulate IL-7Rα expression in αβ T cells have been identified, enhancers active in γδ T cells remain unknown. In this article, we discovered an evolutionarily conserved noncoding sequence (CNS) in intron 2 of the IL-7Rα-chain (IL-7Rα) locus and named this region CNS9. CNS9 contained a conserved retinoic acid receptor-related orphan receptor (ROR)-responsive element (RORE) and exerted RORγt-dependent enhancer activity invitro. Mice harboring point mutations in the RORE in CNS9 (CNS9-RORmut) showed reduced IL-7Rα expression in IL-17-producing Vγ4+ γδ T cells. In addition, the cell number and IL-17A production of Vγ4+ γδ T cells were reduced in the adipose tissue of CNS9-RORmut mice. Consistent with the reduction in IL-17A, CNS9-RORmut mice exhibited decreased IL-33 expression in the adipose tissue, resulting in fewer regulatory T cells and glucose intolerance. The CNS9-ROR motif was partially responsible for IL-7Rα expression in RORγt+ regulatory T cells, whereas IL-7Rα expression was unaffected in RORγt-expressing Vγ2+ γδ T cells, Th17 cells, type 3 innate lymphoid cells, and invariant NKT cells. Our results indicate that CNS9 is a RORΕ-dependent, Vγ4+ γδ T cell-specific IL-7Rα enhancer that plays a critical role in adipose tissue homeostasis via regulatory T cells, suggesting that the evolutionarily conserved RORΕ in IL-7Rα intron 2 may influence the incidence of type 2 diabetes.

Convergent accelerated evolution of mammal-specific conserved non-coding elements in hibernators

Mammals maintain their body temperature, yet hibernators can temporarily lower their metabolic rate as an energy-saving strategy. It has been proposed that hibernators evolved independently from homeotherms, and it is possible that the convergent evolution of hibernation involved common genomic changes among hibernator-lineages. Since hibernation is a seasonal trait, the evolution of gene regulatory regions in response to changes in season may have been important for the acquisition of hibernation traits. High-frequency accumulation of mutations in conserved non-coding elements (CNEs) could, in principle, alter the expression of neighboring genes and thereby contribute to the acquisition of new traits. To address this possibility, we performed a comparative genomic analysis of mammals to identify accelerated CNEs commonly associated with hibernation. We found that accelerated CNEs are common to hibernator-lineages and could be involved with hibernation. We also found that common factors of genes that located near accelerated CNEs and are differentially expressed between normal and hibernation periods related to gene regulation and cell-fate determination. It suggests that the molecular mechanisms controlling hibernation have undergone convergent evolution. These results help broaden our understanding of the genetic adaptations that facilitated hibernation in mammals and may offer insights pertaining to stress responses and energy conservation.

Eomesodermin spatiotemporally orchestrates the early and late stages of NK cell development by targeting KLF2 and T-bet, respectively.

Eomesodermin (Eomes) is a critical factor in the development of natural killer (NK) cells, but its precise role in temporal and spatial coordination during this process remains unclear. Our study revealed that Eomes plays distinct roles during the early and late stages of NK cell development. Specifically, the early deletion of Eomes via the CD122-Cre transgene resulted in significant blockade at the progenitor stage due to the downregulation of KLF2, another important transcription factor. ChIP-seq revealed direct binding of Eomes to the conserved noncoding sequence (CNS) of Klf2. Utilizing the CHimeric IMmune Editing (CHIME) technique, we found that deletion of the CNS region of Klf2 via CRISPRi led to a reduction in the NK cell population and developmental arrest. Moreover, constitutive activation of this specific CNS region through CRISPRa significantly reversed the severe defects in NK cell development caused by Eomes deficiency. Conversely, Ncr1-Cre-mediated terminal deletion of Eomes expedited the transition of NK cell subsets from the CD27+CD11b+ phenotype to the CD27-CD11b+ phenotype. Late-stage deficiency of Eomes led to a significant increase in T-bet expression, which subsequently increased the expression of the transcription factor Zeb2. Genetic deletion of one allele of Tbx21, encoding T-bet, effectively reversed the aberrant differentiation of Eomes-deficient NK cells. In summary, we utilized two innovative genetic models to elucidate the intricate mechanisms underlying Eomes-mediated NK cell commitment and differentiation.

Elucidating inflammation-induced cell fate decisions in primary human Tregs

Abstract Adoptive regulatory T-cell (Treg) therapy has remarkable efficacy in promoting immune tolerance in preclinical models of transplantation and autoimmune disease. However, lineage-tracing studies have revealed that Tregs can undergo epigenetic reprogramming in chronically inflamed tissue environments, resulting in the acquisition of proinflammatory functions and the capacity to exacerbate autoimmunity. Despite intense interests in developing Treg therapy for humans, inflammation-induced human Treg lineage-decommitment remains poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven human Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)-2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. Single-cell transcriptomic and chromatin accessibility analyses of human Tregs undergoing destabilization revealed a gradual closing of a Treg-specific element (A) and a reciprocal opening of a competing element (B) at the IRF4 locus. CRISPR-mediated excision of the B element (IRF4ΔB) preserved Treg identity and enhanced in vitro suppressive activity following prolonged inflammatory exposure. Ongoing experiments aim to evaluate the in vivo function and stability of IRF4ΔB Tregs in a xenogeneic graft-versus-host model. These findings may facilitate the design of Treg therapeutics with greater potency and improved safety.

Genomic insights into the seawater adaptation in Cyprinidae

BackgroundCyprinidae, the largest fish family, encompasses approximately 367 genera and 3006 species. While they exhibit remarkable adaptability to diverse aquatic environments, it is exceptionally rare to find them in seawater, with the Far Eastern daces being of few exceptions. Therefore, the Far Eastern daces serve as a valuable model for studying the genetic mechanisms underlying seawater adaptation in Cyprinidae.ResultsHere, we sequenced the chromosome-level genomes of two Far Eastern daces (Pseudaspius brandtii and P. hakonensis), the two known cyprinid fishes found in seawater, and performed comparative genomic analyses to investigate their genetic mechanism of seawater adaptation. Demographic history reconstruction of the two species reveals that their population dynamics are correlated with the glacial-interglacial cycles and sea level changes. Genomic analyses identified Pseudaspius-specific genetic innovations related to seawater adaptation, including positively selected genes, rapidly evolving genes, and conserved non-coding elements (CNEs). Functional assays of Pseudaspius-specific variants of the prolactin (prl) gene showed enhanced cell adaptation to greater osmolarity. Functional assays of Pseudaspius specific CNEs near atg7 and usp45 genes suggest that they exhibit higher promoter activity and significantly induced at high osmolarity.ConclusionsOur results reveal the genome-wide evidence for the evolutionary adaptation of cyprinid fishes to seawater, offering valuable insights into the molecular mechanisms supporting the survival of migratory fish in marine environments. These findings are significant as they contribute to our understanding of how cyprinid fishes navigate and thrive in diverse aquatic habitats, providing useful implications for the conservation and management of marine ecosystems.

Conserved Noncoding Elements Evolve Around the Same Genes Throughout Metazoan Evolution.

Conserved noncoding elements (CNEs) are DNA sequences located outside of protein-coding genes that can remain under purifying selection for up to hundreds of millions of years. Studies in vertebrate genomes have revealed that most CNEs carry out regulatory functions. Notably, many of them are enhancers that control the expression of homeodomain transcription factors and other genes that play crucial roles in embryonic development. To further our knowledge of CNEs in other parts of the animal tree, we conducted a large-scale characterization of CNEs in more than 50 genomes from three of the main branches of the metazoan tree: Cnidaria, Mollusca, and Arthropoda. We identified hundreds of thousands of CNEs and reconstructed the temporal dynamics of their appearance in each lineage, as well as determining their spatial distribution across genomes. We show that CNEs evolve repeatedly around the same genes across the Metazoa, including around homeodomain genes and other transcription factors; they also evolve repeatedly around genes involved in neural development. We also show that transposons are a major source of CNEs, confirming previous observations from vertebrates and suggesting that they have played a major role in wiring developmental gene regulatory mechanisms since the dawn of animal evolution.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten–eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Identification and functional characterization of conserved cis-regulatory elements responsible for early fruit development in cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved noncoding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865 to 43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2, and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

Cis-regulatory control of mammalian Trps1 gene expression.

TRPS1 serves as the causative gene for tricho-rhino phalangeal syndrome, known for its craniofacial and skeletal abnormalities. The Trps1 gene encodes a protein that represses Wnt signaling through strong interactions with Wnt signaling inhibitors. The identification of genomic cis-acting regulatory sequences governing Trps1 expression is crucial for understanding its role in embryogenesis. Nevertheless, to date, no investigations have been conducted concerning these aspects of Trps1.To identify deeply conserved noncoding elements (CNEs) within the Trps1 locus, we employed a comparative genomics approach, utilizing slowly evolving fish such as coelacanth and spotted gar. These analyses resulted in the identification of eight CNEs in the intronic region of the Trps1 gene. Functional characterization of these CNEs in zebrafish revealed their regulatory potential in various tissues, including pectoral fins, heart, and pharyngeal arches. RNA in-situ hybridization experiments revealed concordance between the reporter expression pattern induced by the identified set of CNEs and the spatial expression pattern of the trps1 gene in zebrafish. Comparative in vivo data from zebrafish and mice for CNE7/hs919 revealed conserved functions of these enhancers. Each of these eight CNEs was further investigated in cell line-based reporter assays, revealing their repressive potential. Taken together, in vivo and in vitro assays suggest a context-dependent dual functionality for the identified set of Trps1-associated CNE enhancers.This functionally characterized set of CNE-enhancers will contribute to a more comprehensive understanding of the developmental roles of Trps1 and can aid in the identification of noncoding DNA variants associated with human diseases.

Identification and Functional Study of Enhancers of EYA1: The Causative Gene of Branchio-Oto-Renal Syndrome

Introduction: Branchio-oto-renal syndrome (BOR syndrome) is a rare genetic disorder with an incidence of 1 in 40,000, affecting the development of multiple organs, including the branchio, ear, and kidney. It is responsible for 2% of childhood deafness. Currently, variants in the coding regions of the main causative genes, such as EYA1, SIX1, and SIX5, explain only half of the disease’s etiology. Therefore, there is a need to explore the non-coding regions, which constitute the majority of the genome, especially the regulatory regions, as potential new causative factors. Method: In this study, we focused on the EYA1 gene, which accounts for over 40% of BOR syndrome cases, and conducted a screening of candidate enhancers within a 250-kb region upstream and downstream of the gene using comparative genomics. We characterized the enhancer activities of these candidates in zebrafish using the Tol2 transposon system. Results: Our findings revealed that out of the 11 conserved non-coding elements (CNEs) examined, four exhibited enhancer activity. Notably, CNE16.39 and CNE16.45 displayed tissue-specific enhancer activity in the ear. CNE16.39 required the full-length 206 bp sequence for inner-ear-specific expression, while the core functional region of CNE16.45 was identified as 136 bp. Confocal microscopy results demonstrated that both CNE16.39 and CNE16.45 drove the expression of GFP in the sensory region of the crista of the inner ear in zebrafish, consistent with the expression pattern of eya1. Conclusion: This study contributes to the understanding of the regulatory network governing EYA1 expression and offers new insights to further clarify the pathogenic role of EYA1 in BOR syndrome.

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals.

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15-21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. These results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

Evolutionary analysis of conserved non-coding elements subsequent to whole-genome duplication in opium poppy.

Whole-genome duplication (WGD) leads to the duplication of both coding and non-coding sequences within an organism's genome, providing an abundant supply of genetic material that can drive evolution, ultimately contributing to plant adaptation and speciation. Although non-coding sequences contain numerous regulatory elements, they have been understudied compared to coding sequences. In order to address this gap, we explored the evolutionary patterns of regulatory sequences, coding sequences and transcriptomes using conserved non-coding elements (CNEs) as regulatory element proxies following the recent WGD event in opium poppy (Papaver somniferum). Our results showed similar evolutionary patterns in subgenomes of regulatory and coding sequences. Specifically, the biased or unbiased retention of coding sequences reflected the same pattern as retention levels in regulatory sequences. Further, the divergence of gene expression patterns mediated by regulatory element variations occurred at a more rapid pace than that of gene coding sequences. However, gene losses were purportedly dependent on relaxed selection pressure in coding sequences. Specifically, the rapid evolution of tissue-specific benzylisoquinoline alkaloid production in P. somniferum was associated with regulatory element changes. The origin of a novel stem-specific ACR, which utilized ancestral cis-elements as templates, is likely to be linked to the evolutionary trajectory behind the transition of the PSMT1-CYP719A21 cluster from high levels of expression solely in P. rhoeas root tissue to its elevated expression in P. somniferum stem tissue. Our findings demonstrate that rapid regulatory element evolution can contribute to the emergence of new phenotypes and provide valuable insights into the high evolvability of regulatory elements.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation—what must be acquired or cannot be lost for a functional symbiosis—and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Developmental regulation of conserved non-coding element evolution provides insights into limb loss in squamates.

Limb loss shows recurrent phenotypic evolution across squamate lineages. Here, based on three de novo-assembled genomes of limbless lizards from different lineages, we showed that divergence of conserved non-coding elements (CNEs) played an important role in limb development. These CNEs were associated with genes required for limb initiation and outgrowth, and with regulatory signals in the early stage of limb development. Importantly, we identified the extensive existence of insertions and deletions (InDels) in the CNEs, with the numbers ranging from 111 to 756. Most of these CNEs with InDels were lineage-specific in the limbless squamates. Nearby genes of these InDel CNEs were important to early limb formation, such as Tbx4, Fgf10, and Gli3. Based on functional experiments, we found that nucleotide mutations and InDels both affected the regulatory function of the CNEs. Our study provides molecular evidence underlying limb loss in squamate reptiles from a developmental perspective and sheds light on the importance of regulatory element InDels in phenotypic evolution.

TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis.

Methotrexate (MTX) is a standard, first-line therapy for rheumatoid arthritis (RA); however, its precise mechanisms of action other than antifolate activity are largely unknown. We performed DNA microarray analyses of CD4+ T cells in patients with RA before and after MTX treatment and found that TP63 was the most significantly downregulated gene after MTX treatment. TAp63, an isoform of TP63, was highly expressed in human IL-17-producing Th (Th17) cells and was suppressed by MTX in vitro. Murine TAp63 was expressed at high levels in Th cells and at lower levels in thymus-derived Treg cells. Importantly, TAp63 knockdown in murine Th17 cells ameliorated the adoptive transfer arthritis model. RNA-Seq analyses of human Th17 cells overexpressing TAp63 and those with TAp63 knockdown identified FOXP3 as a possible TAp63 target gene. TAp63 knockdown in CD4+ T cells cultured under Th17 conditions with low-dose IL-6 increased Foxp3 expression, suggesting that TAp63 balances Th17 cells and Treg cells. Mechanistically, TAp63 knockdown in murine induced Treg (iTreg) cells promoted hypomethylation of conserved noncoding sequence 2 (CNS2) of the Foxp3 gene and enhanced the suppressive function of iTreg cells. Reporter analyses revealed that TAp63 suppressed the activation of the Foxp3 CNS2 enhancer. Collectively, TAp63 suppresses Foxp3 expression and exacerbates autoimmune arthritis.

Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and Brassica.

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions. To investigate the potential importance of distant contacts, we performed high-throughput chromatin conformation capture (Hi-C) experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea. We then combined our results with published Hi-C data from Arabidopsis thaliana. We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene. Genes with higher numbers of conserved noncoding sequences (CNSs) nearby were more likely to have contact with distant genes. With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity. In addition, for the genes we studied, distant contacting genes with CNSs were more likely to be transcriptionally coordinated. These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors, correlating with distant contacts to ensure coordinated expression. This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

Identification of clade-wide putative cis-regulatory elements from conserved non-coding sequences in Cucurbitaceae genomes.

Cis-regulatory elements regulate gene expression and play an essential role in the development and physiology of organisms. Many conserved non-coding sequences (CNSs) function as cis-regulatory elements. They control the development of various lineages. However, predicting clade-wide cis-regulatory elements across several closely related species remains challenging. Based on the relationship between CNSs and cis-regulatory elements, we present a computational approach that predicts the clade-wide putative cis-regulatory elements in 12 Cucurbitaceae genomes. Using 12-way whole-genome alignment, we first obtained 632 112 CNSs in Cucurbitaceae. Next, we identified 16 552 Cucurbitaceae-wide cis-regulatory elements based on collinearity among all 12 Cucurbitaceae plants. Furthermore, we predicted 3271 potential regulatory pairs in the cucumber genome, of which 98 were verified using integrative RNA sequencing and ChIP sequencing datasets from samples collected during various fruit development stages. The CNSs, Cucurbitaceae-wide cis-regulatory elements, and their target genes are accessible at http://cmb.bnu.edu.cn/cisRCNEs_cucurbit/. These elements are valuable resources for functionally annotating CNSs and their regulatory roles in Cucurbitaceae genomes.

Chromosome-level genome assembly of the Muscovy duck provides insight into fatty liver susceptibility

The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak lipid catabolism capabilities (fatty acid β-oxidation and lipolysis). Furthermore, conserved noncoding elements (CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the susceptibility of Muscovy duck to fatty liver.