Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

(6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population.

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV-positive than non-oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV-positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus-unrelated HNSCC and virus-related HNSCC consisting of nasopharyngeal and HPV-positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus-unrelated HNSCC. A disruptive mutation was never found in virus-related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus-unrelated HNSCC. Moreover, in virus-unrelated HNSCC, G:C to T:A transversions were more frequent in ever-smokers than in never-smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever-smokers than in never-smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus-related and virus-unrelated subgroups. In virus-related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus-unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Estrogen receptor 2b is the major determinant of sex-typical mating behavior and sexual preference in medaka.

Male and female animals typically display innate sex-specific mating behaviors, which, in vertebrates, are highly dependent on sex steroid signaling. While estradiol-17ß (E2) signaling through estrogen receptor 2 (ESR2) serves to defeminize male mating behavior in rodents, the available evidence suggests that E2 signaling is not required in teleosts for either male or female mating behavior. Here, we report that female medaka deficient for Esr2b, a teleost ortholog of ESR2, are not receptive to males but rather court females, despite retaining normal ovarian function with an unaltered sex steroid milieu. Thus, contrary to both prevailing views in rodents and teleosts, E2/Esr2b signaling in the brain plays a decisive role in feminization and demasculinization of female mating behavior and sexual preference in medaka. Further behavioral testing showed that mutual antagonism between E2/Esr2b signaling and androgen receptor-mediated androgen signaling in adulthood induces and actively maintains sex-typical mating behaviors and preference. Our results also revealed that the female-biased sexual dimorphism in esr2b expression in the telencephalic and preoptic nuclei implicated in mating behavior can be reversed between males and females by altering the sex steroid milieu in adulthood, likely via mechanisms involving direct E2-induced transcriptional activation. In addition, Npba, a neuropeptide mediating female sexual receptivity, was found to act downstream of E2/Esr2b signaling in these brain nuclei. Collectively, these functional and regulatory mechanisms of E2/Esr2b signaling presumably underpin the neural mechanism for induction, maintenance, and reversal of sex-typical mating behaviors and sexual preference in teleosts, at least in medaka.

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes).

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light-dependent repair of ultraviolet (UV)-induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6-4 photolyase, which repairs 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs). Although the Cry-DASH protein is classified as a cryptochrome, it also has light-dependent DNA repair activity. To determine the significance of the three light-dependent repair enzymes in recovering from solar UV-induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light-dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light-dependent repair of CPD was lost in the CPD photolyase-deficient mutant, whereas weak repair activity against 6-4PPs persisted in the 6-4 photolyase-deficient mutant. These results suggest the existence of a heretofore unknown 6-4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish.

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner.

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

Loss of follicle-stimulating hormone receptor function causes masculinization and suppression of ovarian development in genetically female medaka.

FSH, a glycoprotein hormone, is circulated from the pituitary and functions by binding to a specific FSH receptor (FSHR). FSHR is a G protein-coupled, seven-transmembrane receptor linked to the adenylyl cyclase or other pathways and is expressed in gonadal somatic cells. In some nonmammalian species, fshr expression is much higher in the ovary than in the testis during gonadal sex differentiation, suggesting that FSHR is involved in ovarian development in nonmammalian vertebrates. However, little is known of FSHR knockout phenotypes in these species. Here we screened for fshr mutations by a medaka (Oryzias latipes) target-induced local lesion in the genomes and identified one nonsense mutation located in the BXXBB motif, which is involved in G protein activation. Next, we used an in vitro reporter gene assay to demonstrate that this mutation prevents FSHR function. We then analyzed the phenotypes of fshr mutant medaka. The fshr mutant male medaka displayed normal testes and were fertile, whereas the mutant female fish displayed small ovaries and were infertile because vitellogenesis was inhibited. The mutant females also have suppressed expression of ovary-type aromatase (cyp19a1a), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, resulting in decreased 17ß-estradiol levels. Moreover, loss of FSHR function caused female-to-male sex reversal in some cases. In addition, the transgenic overexpression of fshr in fshr mutants rescued FSHR function. These findings strongly suggest that in the medaka, FSH regulates the ovarian development and the maintenance mainly by the elevation of estrogen levels. We present the first FSHR knockout phenotype in a nonmammalian species.

The Cryptochrome/Photolyase Family in aquatic organisms.

The Cryptochrome/Photolyase Family (CPF) represents an ancient group of widely distributed UV-A/blue-light sensitive proteins sharing common structures and chromophores. During the course of evolution, different CPFs acquired distinct functions in DNA repair, light perception and circadian clock regulation. Previous phylogenetic analyses of the CPF have allowed reconstruction of the evolution and distribution of the different CPF super-classes in the tree of life. However, so far only limited information is available from the CPF orthologs in aquatic organisms that evolved in environments harboring great diversity of life forms and showing peculiar light distribution and rhythms. To gain new insights into the evolutionary and functional relationships within the CPF family, we performed a detailed study of CPF members from marine (diatoms, sea urchin and annelid) and freshwater organisms (teleost) that populate diverse habitats and exhibit different life strategies. In particular, we first extended the CPF family phylogeny by including genes from aquatic organisms representative of several branches of the tree of life. Our analysis identifies four major super-classes of CPF proteins and importantly singles out the presence of a plant-like CRY in diatoms and in metazoans. Moreover, we show a dynamic evolution of Cpf genes in eukaryotes with various events of gene duplication coupled to functional diversification and gene loss, which have shaped the complex array of Cpf genes in extant aquatic organisms. Second, we uncover clear rhythmic diurnal expression patterns and light-dependent regulation for the majority of the analyzed Cpf genes in our reference species. Our analyses reconstruct the molecular evolution of the CPF family in eukaryotes and provide a solid foundation for a systematic characterization of novel light activated proteins in aquatic environments.

Circadian and circalunar clock interactions in a marine annelid.

Life is controlled by multiple rhythms. Although the interaction of the daily (circadian) clock with environmental stimuli, such as light, is well documented, its relationship to endogenous clocks with other periods is little understood. We establish that the marine worm Platynereis dumerilii possesses endogenous circadian and circalunar (monthly) clocks and characterize their interactions. The RNAs of likely core circadian oscillator genes localize to a distinct nucleus of the worm's forebrain. The worm's forebrain also harbors a circalunar clock entrained by nocturnal light. This monthly clock regulates maturation and persists even when circadian clock oscillations are disrupted by the inhibition of casein kinase 1δ/ε. Both circadian and circalunar clocks converge on the regulation of transcript levels. Furthermore, the circalunar clock changes the period and power of circadian behavior, although the period length of the daily transcriptional oscillations remains unaltered. We conclude that a second endogenous noncircadian clock can influence circadian clock function.

ATF6α/ß-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

ATF6α and ATF6ß are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6ß single-knockout mice develop normally, but ATF6α/ß double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/ß double knockout, but not ATF6α- or ATF6ß single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6ß-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/ß double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/ß-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.