Hiromi Kanda

A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein.

SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.

Rainbow trout SOX9: cDNA cloning, gene structure and expression.

We have isolated and characterized rainbow trout SOX9 cDNA and genomic clones. The cDNA encodes a protein of 488 amino acids (aa) with an HMG box and has 70% aa sequence identity with human SOX9. The rainbow trout and human SOX9 genes show a similar gene structure, and the two introns in the coding region are located at conserved positions. In rainbow trout, the SOX9 mRNA was prominent in testis and brain.

cDNA cloning of a new member of the FTZ-F1 subfamily from a rainbow trout

We describe here cDNA cloning of an orphan nuclear receptor family member, tFZR1, which has a FTZ-F1 box. The amino acid sequences of the zinc finger domain and the FTZ-F1 box has 92.8% and 100% identity, respectively, with those of zebrafish FTZ-F1. On the other hand, the overall homology between tFZR1 and zebrafish FTZ-F1 is low (33.0%). The results indicate that tFZR1 is a new member of fushitarazu factor 1 (FTZ-F1) subfamily.

Xenopus W-linked DM-W induces Foxl2 and Cyp19 expression during ovary formation.

The molecular mechanisms of vertebrate ZZ/ZW-type sex-determining systems remain unclear. We recently indicated that a W-linked gene, DM-W is a likely ovary-determining gene in Xenopus laevis. We first examined whether Cyp19 for estrogen-synthesizing enzyme P450 aromatase and Foxl2 showed female-specific expression in developing gonads. Both genes showed much higher expression in ZW than in ZZ gonads during and after sex determination. Importantly, transgenic ZZ gonads expressing exogenous DM-W at the sex-determining stage showed a ZW-type pattern of Cyp19 and Foxl2 expression. These results suggest that DM-W up-regulates Cyp19 and Foxl2 expression to guide primary ovary development in X. laevis.

Expression and promoter analysis of Xenopus DMRT1 and functional characterization of the transactivation property of its protein

The doublesex and mab‐3‐related transcription factor 1 (DMRT1) is involved in testis formation in a variety of vertebrates. In the teleost fish, Medaka, DMY/DMRT1Y on the Y chromosome, a duplicate of the autosomal DMRT1 gene, is characterized as a sex‐determining gene. We report here the characterization of the Xenopus DMRT1 genes. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed that X. laevis DMRT1 was expressed throughout the embryo during early development and was restricted to the primordial gonads after embryogenesis. Whole‐mount in situ hybridization analysis of the gene confirmed its specific expression in the primordial gonads. To study the transcriptional control of DMRT1 gene expression, we isolated the predicted promoter region of X. tropicalis DMRT1 using databases for this species. Analysis of transgenic tadpoles with a green fluorescence protein (GFP) reporter showed that approximately 3 kb of the 5′‐flanking sequence of the DMRT1 gene was implicated in DMRT1 expression in the primordial gonads. We also showed that the C‐terminal region of DMRT1 functioned as a transactivation domain in cultured cells, by a luciferase reporter assay using fusion proteins with the DNA‐binding domain of GAL4. These findings suggest that DMRT1 functions as an activator of one or more genes involved in sex determination or gonadal differentiation.

RAINBOW TROUT SOX24, A NOVEL MEMBER OF THE SOX FAMILY, IS A TRANSCRIPTIONAL REGULATOR DURING OOGENESIS

We isolated a cDNA clone encoding a novel SRY-type HMG box (Sox) protein, designated Sox24, from a rainbow trout ovary cDNA library. On the basis of the HMG box amino acid sequence, Sox24 can be categorized into the same subgroup of Sox proteins as SOX4, SOX11, and SOX22. The proteins in this group also share a highly conserved sequence at the C-terminus. The Sox24 mRNA is expressed at high levels in the ovary, and in-situ hybridization localized its expression to oocytes. The recombinant protein containing the Sox24 HMG box region bound to an AACAAT sequence strongly in a gel retardation assay. Upon co-transfection into CHO cells, the full-length Sox24 transactivated transcription from a reporter plasmid through the AACAAT binding motif. We used GAL4/Sox24 chimeras with the DNA binding domain of yeast GAL4 at the N-terminus to map the transactivation function to the C-terminal region, which included the conserved sequence. These results suggest that Sox24 plays a role as a transcriptional regulator during oogenesis.

cDNA cloning of a novel rainbow trout SRY-type HMG box protein, rtSox23, and its functional analysis.

We have isolated a cDNA clone for a new member of Sox genes, termed rtSox23, from a rainbow trout ovary cDNA library. rtSox23 mRNA was notably expressed in ovary and brain. rtSox23 contains a leucine zipper in addition to an SRY-type HMG box. Although the recombinant HMG box region protein of rtSox23 could bind to an AACAAT sequence, the full-length rtSox23 could form a homodimer and did not bind to the sequence. Furthermore, using a two-hybrid system, we have isolated a cDNA clone encoding a protein that bound to the leucine zipper region of rtSox23. This protein was the rainbow trout homologue of mouse nucleoporin p62, which is a component of the nuclear pore complex in nuclear envelope. The rainbow trout p62 mRNA was also prominent in ovary and brain. Taken together, these results suggest that the rainbow trout p62 associates with rtSox23 in vivo and modulates the function of rtSox23.

Quantitative Analysis of fushi tarazu Factor 1 Homolog Messenger Ribonucleic Acids in the Pituitary of Salmon at Different Prespawning Stages

Abstract Steroidogenic factor 1 (SF-1) or Ad4BP is a member of the fushi tarazu factor 1 (FTZ-F1) family and an orphan nuclear receptor that plays an important role in the hypothalamus-pituitary-gonadal axis and the adrenal cortex. Although its critical role in the differentiation of adrenals, gonads, and pituitary gonadotropes has been well demonstrated, regulatory function of SF-1 during sexual maturation is yet to be examined. To investigate the potential role of SF-1 in sexual maturation, expression of two salmon FTZ-F1 homolog genes, sFF1-I and sFF1-II, was examined in the pituitaries of chum and sockeye salmons, using specific and sensitive RNase protection assays. Only sFF1-I mRNA was found in the pituitary and other organs, such as the ovary, spleen, liver, brain, and skeletal muscle. In chum salmon during upstream migration from the bay to the hatchery, the level of sFF1-I mRNA in the male fish was increased on the midway in the river, where the levels of gonadotropin α- and IIβ-subunit mRNAs were increased. In maturing sockeye salmon, the expression of the sFF1-I gene was elevated in the mature male fish, but the administration of GnRH analog did not further enhance the expression. These results indicate that sFF1-I gene expression in the pituitary is upregulated in maturing salmon, and this upregulation may not depend on GnRH.

JNK-binding protein 1 regulates NF-κB activation through TRAF2 and TAK1

The mitogen‐activated protein kinase (MAPK) cascades, including c‐Jun N‐terminal kinase (JNK), are composed of a MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Previously, we reported that JNK‐binding protein 1 (JNKBP1) enhances JNK activation induced by the TGF‐β‐activated kinase1 (TAK1) MAPKKK in transfected cells. We have investigated whether JNKBP1 functions as an adaptor protein for nuclear factor (NF)‐κB activation mediated by TAK1 in COS‐7 cells. Co‐expression experiments showed that JNKBP1 interacted with not only TAK1, but also with its upstream regulators, TNF‐receptor associated factors 2 and 6 (TRAF2 and TRAF6). An endogenous interaction between JNKBP1 and TRAF2 or TAK1 was confirmed by immunoprecipitation analysis. We also found that JNKBP1 could enhance the NF‐κB activation induced by TAK1 and TRAF2, and could promote TRAF2 polyubiquitination. These results suggest a scaffolding role for JNKBP1 in the TRAF2–TAK1–NF‐κB signaling pathway.

Mammalian Sox15 Gene: Promoter Analysis and Implications for Placental Evolution

Abstract Sox15 belongs to the Sox (Sry-type HMG box) protein family, which is involved in placental development and muscle regeneration. Previously, we showed that the Sox15 gene is highly expressed in the trophoblast giant cells of the mouse placenta. To elucidate the molecular mechanisms of the tissue-dependent transcription of the gene, we isolated approximately 2.2 kb of the 5′-flanking sequence upstream of the transcription initiation site and used it to construct luciferase reporter plasmids. A variety of cell lines, including trophoblast stem (TS) cells, placenta-derived Rcho-1 cells, and myoblast C2C12 cells, required the same 5′-flanking sequence, from −109 to −8, for basal promoter activity. In contrast, the sequences from −297 to −149 and from −148 to −110 were required for cell-type-specific promoter activity in myoblast-derived C2C12 cells and placenta-derived Rcho-1 and TS cells, respectively. These results suggest that the region from −297 to −8 of the Sox15 gene contains three distinct cis-elements that respectively control placenta-specific, myoblast-specific, and common basal expression. We also searched for Sox15 ortholog(s) in the genome databases of various vertebrate species. The results indicated that the three regulatory promoter sequences of the Sox15 genes were conserved among eutherian mammals during vertebrate evolution. Interestingly, the marsupial opossum gene that is closest to Sox15 appeared to be a pseudogene. These findings indicate that Sox15 may have been involved in placental evolution.