Tomoko Ishikawa-Fujiwara

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.

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

The endoplasmic reticulum (ER) membrane-bound transcription factors ATF6α and ATF6β mediate adjustment of chaperone levels to increased demands in the ER, which is essential for development of the notochord; the latter synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

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.

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.

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 17β-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.

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.

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.

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.

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

Abstract (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.

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.