Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells.

Polydom is an extracellular matrix protein involved in lymphatic vessel development. Polydom-deficient mice die immediately after birth due to defects in lymphatic vessel remodeling, but the mechanism involved is poorly understood. Here, we report that Polydom directly binds to Tie1, an orphan receptor in the Angiopoietin-Tie axis, and facilitates migration of lymphatic endothelial cells (LECs) in a Tie1-dependent manner. Polydom-induced LEC migration is diminished by PI3K inhibitors but not by an ERK inhibitor, suggesting that the PI3K/Akt signaling pathway is involved in Polydom-induced LEC migration. In line with this possibility, Akt phosphorylation in LECs is enhanced by Polydom although no significant Tie1 phosphorylation is induced by Polydom. LECs also exhibited nuclear exclusion of Foxo1, a signaling event downstream of Akt activation, which was impaired in Polydom-deficient mice. These findings indicate that Polydom is a physiological ligand for Tie1 and participates in lymphatic vessel development through activation of the PI3K/Akt pathway.

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation.

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation.

In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.

Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator.

Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa.

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.

Molecular cloning of a new member of TEKTIN family, Tektin4, located to the flagella of rat spermatozoa.

Tektins are composed of a family of filament-forming proteins associated with ciliary and flagellar microtubules. A new member of the TEKTIN gene family, which was designated as rat Tektin4, was obtained by PCR technique combined with yeast two-hybrid screening. Rat Tektin4 cDNA consists of 1,341 bp encoding a 52 kDa protein of 447 amino acids. Tektin4 protein contains a Tektin domain including a nonapeptide signature sequence (RPNVELCRD), which is a prominent feature of Tektins. Its amino acid sequence showed 29% approximately 58% identities to that of other Tektin family proteins registered in the public databases. Tektin4 gene, which was mapped to rat chromosome 10q12, is composed of six exons and spanning 5 kb. Reverse-transcriptional-PCR (RT-PCR) analysis indicated that Tektin4 was predominantly expressed in testis and its expression was upregulated during testis development. In situ hybridization analysis showed that Tektin4 mRNA was localized in round spermatids in the seminiferous tubules of the rat testis. Tektin4 protein was predominantly localized in the flagella of spermatozoa, suggesting that it might works as a flagellar component requisite for flagellar stability or sperm motility.

Identification of rab12 as a vesicle-associated small GTPase highly expressed in Sertoli cells of rat testis.

We examined the expression and the localization of a small GTPase, rab12, in rat testis. Northern blot analysis showed that 2.3 kb transcript of rab12 was expressed in rat testis. RT-PCR analysis indicated constant expression of rab12 throughout testis development. Immunohistochemical studies revealed that rab12 protein was highly expressed in Sertoli cells in the seminiferous tubules, while both spermatogenic germ cells and interstitial cells exhibited faint or no immunosignal for rab12. The expression pattern of rab12 in Sertoli cells varied between the tubules: its immunostaining appeared as a wheel-like pattern at stage I approximately III and as a luminal staining pattern at stage IV approximately VI, whereas the immunostaining signals were only rudimentary detected at stage VIII and thereafter (approximately stage XIV). The diversified staining pattern of rab12 in the tubules seemed to reflect either the different shape of Sertoli cells during the cycle of the seminiferous epithelium or the variant expression levels of rab12 in Sertoli cells at each stage of the tubules. In cultured rat Sertoli cells and normal rat kidney (NRK) cells, rab12 was found to be associated with small vesicles distributed throughout the cytoplasm, but not with the Golgi apparatus. When overexpressed in NRK cells, rab12-associated small vesicles were not only distributed throughout the cytoplasm but also accumulated in the perinuclear cytoplasm around centrosome. We interrupt these data as a potential role of rab12 in acceleration of vesicular transport from the cell periphery to the perinuclear centrosome region.

Molecular cloning of rat Spergen-3, a spermatogenic cell-specific gene-3, encoding a novel 75-kDa protein bearing EF-hand motifs.

By use of differential display in combination with cDNA cloning approach, we isolated a novel rat gene designated as Spergen-3, which has an open reading frame of 2055-length nucleotides encoding a protein of 685 amino acids. Spergen-3 gene is composed of 15 exons and mapped on chromosome 5q36, and its mouse counterpart, which shares 85.5% identity to rat Spergen-3 at the amino acid level, is mapped on chromosome 4E1. Spergen-3 encodes a 75-kDa soluble protein bearing putative 2 EF-hand motifs, proline-repeat, and a putative nuclear localization signal. Of the 2 EF-hand motifs in Spergen-3, the second one seems to match the consensus sequence. Reverse transcription-polymerase chain reaction analysis showed that the expression of Spergen-3 is developmentally up-regulated and that it is exclusively expressed in testis. In situ hybridization revealed that Spergen-3 mRNA was exclusively expressed in haploid spermatids, but its signal was weak or undetectable in spermatogonia, spermatocytes, and Sertoli cells as well as in interstitial cells. We interpreted these data as a potential role of Spergen-3, a new member of EF-hand family, in differentiation of haploid spermatids in testis.

Bisphenol A-induced apoptosis of cultured rat Sertoli cells.

Bisphenol A (BPA) was examined for its effects on cultured Sertoli cells established from 18-day-old rat testes. We demonstrated that exposure of cultured Sertoli cells to BPA decreased the cell viability in a dose- and a time-dependent manner and that exposure to BPA brought about morphologic changes of the cells, such as membrane blebs, cell rounding, cytoskeletal collapse, and chromatin condensation or fragmentation, all of which conform to the morphologic criteria for apoptosis. Immunocytochemistry showed that active caspase-3, a major execution caspase, was expressed in round Sertoli cells positively labeled by the TUNEL method. Co-localization of active caspase-3 and aggregated actin fragments was also observed in the round Sertoli cells. Theses results suggest that BPA induces cell death of Sertoli cells by promoting apoptosis. Apoptosis-inducing cell death was observed in cells exposed to 150-200 microM BPA, while BPA at <100 microM had only slight cytotoxic effects on the cells.

Complementary DNA cloning and characterization of rat spergen-2, a spermatogenic cell-specific gene 2 encoding a 56-kilodalton nuclear protein bearing ankyrin repeat motifs.

Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1-6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX-XIV) and spermatids (steps 1-11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.

Spergen-1 might be an adhesive molecule associated with mitochondria in the middle piece of spermatozoa.

Spergen-1, a recently identified molecule specifically expressed in haploid spermatids in testis, is a small protein of 154 amino acids with a mitochondria-targeting signal at the N terminus. To examine the localization of spergen-1 protein in germ cells, we performed immunocytochemistry with the anti-spergen-1 antibody on frozen sections of rat testis and purified spermatozoa. Immunolabeling for spergen-1 was detected in mitochondria of elongating spermatids and of the middle pieces of matured spermatozoa. Immunoelectron microscopy revealed that spergen-1 was localized to the surface of mitochondria in the middle piece of spermatozoa. To investigate the properties of spergen-1, COS-7 cells were transfected with vectors encoding various spergen-1 mutants. The transfection experiments showed that spergen-1 expressed in the cells tended to agglutinate mitochondria and assemble them into aggregations and that the C-terminal region of spergen-1 as well as the N-terminal mitochondrial targeting signal was requisite for induction of mitochondrial aggregation. These results suggest that spergen-1, a mitochondria-associated molecule in spermatozoa, has a property to induce mitochondrial aggregation at least in cultured cells. We hypothesize that spergen-1 might function as an adhesive molecule to assemble mitochondria into the mitochondrial sheath around the outer dense fibers during spermiogenesis.

Complementary DNA cloning and characterization of rat spergen-1, a spermatogenic cell-specific gene-1, containing a mitochondria-targeting signal.

To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.