The reference genome of Macropodus opercularis (the paradise fish).

Amongst fishes, zebrafish (Danio rerio) has gained popularity as a model system over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species has a highly complex behavioral repertoire and has been the subject of many ethological investigations but lacks genomic resources. Here we report the reference genome assembly of M. opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of 483,077,705 base pairs (~483 Mb) on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned ~90% of them to orthogroups.

The reference genome of the paradise fish (Macropodus opercularis).

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of ≈483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned ≈90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

Somatic mutations in facial skin from countries of contrasting skin cancer risk.

The incidence of keratinocyte cancer (basal cell and squamous cell carcinomas of the skin) is 17-fold lower in Singapore than the UK1-3, despite Singapore receiving 2-3 times more ultraviolet (UV) radiation4,5. Aging skin contains somatic mutant clones from which such cancers develop6,7. We hypothesized that differences in keratinocyte cancer incidence may be reflected in the normal skin mutational landscape. Here we show that, compared to Singapore, aging facial skin from populations in the UK has a fourfold greater mutational burden, a predominant UV mutational signature, increased copy number aberrations and increased mutant TP53 selection. These features are shared by keratinocyte cancers from high-incidence and low-incidence populations8-13. In Singaporean skin, most mutations result from cell-intrinsic processes; mutant NOTCH1 and NOTCH2 are more strongly selected than in the UK. Aging skin in a high-incidence country has multiple features convergent with cancer that are not found in a low-risk country. These differences may reflect germline variation in UV-protective genes.

Modeling the Structure of Keratin 1 and 10 Terminal Domains and their Misassembly in Keratoderma.

The terminal domains of suprabasal keratins of the skin epithelium are very resistant to evidence-based structural analysis because of their inherent flexibility and lack of predictable structure. We present a model for the structure and interactions of the head and tail domains of epidermal keratins 1 and 10, based on all-atom 3D simulations of keratin primary amino acid sequences, and tyrosine phosphorylation predictions, extracted from published databases. We observed that keratin 1 and 10 end domains are likely to form a tetrameric terminal domain complex incorporating a reversibly extendable region potentially acting as a molecular spring. This structure is formed by intermolecular stacking of aromatic residues, which would spatially constrain the keratin 1/keratin 10 end domains to allow filament compaction and bundling, whilst also retaining extensibility to ensure flexibility of the keratin filament network in the differentiating epidermis. The tetrameric terminal domain complex model may also help to elucidate the effects of mutations in the end domains of suprabasal keratins and so contribute to understanding of the mechanisms leading to keratinopathies such as striate palmoplantar keratoderma, as reported in this study.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

Assays to Study Consequences of Cytoplasmic Intermediate Filament Mutations: The Case of Epidermal Keratins.

The discovery of the causative link between keratin mutations and a growing number of human diseases opened the way for a better understanding of the function of the whole intermediate filament families of cytoskeleton proteins. This chapter describes analytical approaches to identification and interpretation of the consequences of keratin mutations, from the clinical and diagnostic level to cells in tissue culture. Intermediate filament pathologies can be accurately diagnosed from skin biopsies and DNA samples. The Human Intermediate Filament Database collates reported mutations in intermediate filament genes and their diseases, and can help clinicians to establish accurate diagnoses, leading to disease stratification for genetic counseling, optimal care delivery, and future mutation-aligned new therapies. Looking at the best-studied keratinopathy, epidermolysis bullosa simplex, the generation of cell lines mimicking keratinopathies is described, in which tagged mutant keratins facilitate live-cell imaging to make use of today's powerful enhanced light microscopy modalities. Cell stress assays such as cell spreading and cell migration in scratch wound assays can interrogate the consequences of the compromised cytoskeletal network. Application of extrinsic stresses, such as heat, osmotic, or mechanical stress, can enhance the differentiation of mutant keratin cells from wild-type cells. To bring the experiments to the next level, 3D organotypic human cultures can be generated, and even grafted onto the backs of immunodeficient mice for greater in vivo relevance. While development of these assays has focused on mutant K5/K14 cells, the approaches are often applicable to mutations in other intermediate filaments, reinforcing fundamental commonalities in spite of diverse clinical pathologies.

Late-Onset Multiple Self-Healing Squamous Epithelioma Ferguson-Smith Recurrence Induced by Radiotherapy.

We report the case of a woman in her 60s with confirmed multiple self-healing squamous epitheliomas (MSSE) Ferguson-Smith. After recurrences following surgery and radiotherapy, the patient was successfully treated with minimal surgical intervention combined with intralesional injections of triamcinolone acetate. The histological comparison between mature and regressed keratoacanthomas (KA) revealed an increased inflammatory infiltrate with numerous plasmacytoid dendritic cells in the regressed KA in comparison to the mature one, speaking in favor of an inflammation-mediated regression process. Corticosteroids injection in MSSE may have paradoxical effects with action on the proliferation phase rather than the inflammatory regression phase of the KA. Our case confirms previous reports showing that radiotherapy may exacerbate MSSE and should be avoided. Intralesional triamcinolone acetate injection is a safe and easy to use method also effective for multiple lesions. Our case underlines the difference between squamous cell carcinoma and KA, responding differently to therapies like imiquimod or radiotherapy.

Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.

Multiple self-healing squamous epithelioma is caused by a disease-specific spectrum of mutations in TGFBR1.

Multiple self-healing squamous epithelioma (MSSE), also known as Ferguson-Smith disease (FSD), is an autosomal-dominant skin cancer condition characterized by multiple squamous-carcinoma-like locally invasive skin tumors that grow rapidly for a few weeks before spontaneously regressing, leaving scars. High-throughput genomic sequencing of a conservative estimate (24.2 Mb) of the disease locus on chromosome 9 using exon array capture identified independent mutations in TGFBR1 in three unrelated families. Subsequent dideoxy sequencing of TGFBR1 identified 11 distinct monoallelic mutations in 18 affected families, firmly establishing TGFBR1 as the causative gene. The nature of the sequence variants, which include mutations in the extracellular ligand-binding domain and a series of truncating mutations in the kinase domain, indicates a clear genotype-phenotype correlation between loss-of-function TGFBR1 mutations and MSSE. This distinguishes MSSE from the Marfan syndrome-related disorders in which missense mutations in TGFBR1 lead to developmental defects with vascular involvement but no reported predisposition to cancer.

Association of skin barrier genes within the PSORS4 locus is enriched in Singaporean Chinese with early-onset psoriasis.

Psoriasis (OMIM#177900) is a common polygenic skin disorder affecting approximately 2% of the northern European population and 0.1% of the Han Chinese. Psoriasis patients suffer from chronic skin inflammation, manifested by erythematous scaly lesions. PSORS1-PSORS9 have been confirmed as psoriasis susceptibility loci in independent genetic studies on predominantly Caucasian populations, with psoriasis susceptibility loci (PSORS1, PSORS9) and additional loci at 9q33-34 and 2p22.3-11.2 reported in Han Chinese patients. In this study, we show the association of PSORS4 with psoriasis in Singaporean Chinese. Dense genotyping of single-nucleotide polymorphism-tagging candidate genes within the epidermal differentiation complex revealed significant association in the proximity of the involucrin gene (IVL); the strongest association was seen in early-onset psoriasis patients (P=0.0014). A follow-up genome-wide association screen localized the psoriasis susceptibility region to approximately 360 kb along chromosome 1 in the vicinity of IVL, small proline-rich region (SPRR) and proline-rich region 9 (PRR9) genes. The study of interactions between the causative variant(s) in this locus will provide insights into a possible role for epidermal barrier formation in the pathogenesis of psoriasis.

The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases.

We describe a revised and expanded database on human intermediate filament proteins, a major component of the eukaryotic cytoskeleton. The family of 70 intermediate filament genes (including those encoding keratins, desmins, and lamins) is now known to be associated with a wide range of diverse diseases, at least 72 distinct human pathologies, including skin blistering, muscular dystrophy, cardiomyopathy, premature aging syndromes, neurodegenerative disorders, and cataract. To date, the database catalogs 1,274 manually-curated pathogenic sequence variants and 170 allelic variants in intermediate filament genes from over 459 peer-reviewed research articles. Unrelated cases were collected from all of the six sequence homology groups and the sequence variations were described at cDNA and protein levels with links to the related diseases and reference articles. The mutations and polymorphisms are presented in parallel with data on protein structure, gene, and chromosomal location and basic information on associated diseases. Detailed statistics relating to the variants records in the database are displayed by homology group, mutation type, affected domain, associated diseases, and nucleic and amino acid substitutions. Multiple sequence alignment algorithms can be run from queries to determine DNA or protein sequence conservation. Literature sources can be interrogated within the database and external links are provided to public databases. The database is freely and publicly accessible online at www.interfil.org (last accessed 13 September 2007). Users can query the database by various keywords and the search results can be downloaded. It is anticipated that the Human Intermediate Filament Database (HIFD) will provide a useful resource to study human genome variations for basic scientists, clinicians, and students alike.

Large-scale systematic study on stability of the Ds element and timing of transposition in rice.

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.

Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences.

A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice.

Detection and analysis of transpositionally active head-to-tail dimers in three additional Escherichia coli IS elements.

This study demonstrates that Escherichia coli insertion elements IS3, IS150 and IS186 are able to form transpositionally active head-to-tail dimers which show similar structure and transpositional activity to the dimers of IS2, IS21 and IS30. These structures arise by joining of the left and right inverted repeats (IRs) of two elements. The resulting junction includes a spacer region (SR) of a few base pairs derived from the flanking sequence of one of the reacting IRs. Head-to-tail dimers of IS3, IS150 and IS186 are unstable due to their transpositional activity. They can be resolved in two ways that seem to form a general rule for those elements reported to form dimers. One way is a site-specific process (dimer dissolution) which is accompanied by the loss of one IS copy along with the SR. The other is 'classical' transposition where the joined ends integrate into the target DNA. In intramolecular transposition this often gives rise to deletion formation, whereas in intermolecular transposition it gives rise to replicon fusion. The results presented for IS3, IS150 and IS186 are in accordance with the IS dimer model, which is in turn consistent with models based on covalently closed minicircles.