Tomoaki Fujii

Smyd3 Is Required for the Development of Cardiac and Skeletal Muscle in Zebrafish

Modifications of histone tails are involved in the regulation of a wide range of biological processes including cell cycle, cell survival, cell division, and cell differentiation. Among the modifications, histone methylation plays a critical role in cardiac and skeletal muscle differentiation. In our earlier studies, we found that SMYD3 has methyltransferase activity to histone H3 lysine 4, and that its up-regulation is involved in the tumorigenesis of human colon, liver, and breast. To clarify the role of Smyd3 in development, we have studied its expression patterns in zebrafish embryos and the effect of its suppression on development using Smyd3-specific antisense morpholino-oligonucleotides. We here show that transcripts of smyd3 were expressed in zebrafish embryos at all developmental stages examined and that knockdown of smyd3 in embryos resulted in pericardial edema and defects in the trunk structure. In addition, these phenotypes were associated with abnormal expression of three heart-chamber markers including cmlc2, amhc and vmhc, and abnormal expression of myogenic regulatory factors including myod and myog. These data suggest that Smyd3 plays an important role in the development of heart and skeletal muscle.

A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC.

Layer-by-Layer Growth of InAlN Films on ZnO(000\bar1) Substrates at Room Temperature

We have grown In-rich InxAl1-xN (x = 0.6–0.7) films on nearly lattice-matched ZnO(0001) substrates at various temperatures ranging from room temperature (RT) to 600 °C by the use of pulsed laser deposition and investigated their structural properties. Grazing-incidence X-ray reflection and X-ray diffraction revealed that films grown at RT are composed of single-phase InAlN and possess atomically flat surfaces and abrupt interfaces. In addition, we have found that RT-growth of InAlN films on ZnO(0001) surfaces proceeds in a layer-by-layer mode from the initial stages of film growth.

Room-Temperature Epitaxial Growth of High-Quality m-Plane InAlN Films on Nearly Lattice-Matched ZnO Substrates

We have found that single-phase m-plane In0.24Al0.76N(1100) grows without phase separation in the layer-by-layer mode at room temperature from the initial stages of the growth. The full-width at half-maximum (FWHM) values of the 1100 X-ray rocking curves (XRCs) for the film with X-ray incident azimuths perpendicular to the c- and a-axes are 119 and 102 arcsec, respectively. m-plane In0.24Al0.76N films grew without accommodating misfit dislocations beyond the critical thickness on ZnO(1100) substrates, which is probably due to the large energy barrier for the initiation process of misfit dislocations and is responsible for the small FWHM values for XRCs.

Structural Characteristics of GaN/InN Heterointerfaces Fabricated at Low Temperatures by Pulsed Laser Deposition

We have fabricated c-plane GaN/InN heterostructures on yttria-stabilized zirconia (111) substrates by pulsed laser deposition, and have characterized the abruptness of the interfaces. We have found that GaN can be epitaxially grown on InN even at 300 ?C and that the GaN/InN interface is quite abrupt, although an intermixed layer (In0.15Ga0.85N) is formed during the growth of GaN on InN at 500 ?C. It has also been revealed that the abruptness of GaN/InN interfaces fabricated at 300 ?C remains unchanged even after annealing at 500 ?C. These results indicate that the activation energy for the intermixing reactions between InN and GaN is much larger than that between InN and film precursors during the growth of GaN. These results indicate that the use of the low temperature growth technique is quite promising for the fabrication of high-mobility electronic devices based on GaN/InN heterostructures.

Fabrication and Characterization of AlN/InN Heterostructures

AlN/InN heterostructures have been fabricated on yttria stabilized zirconia (YSZ) substrates using the low temperature pulsed laser deposition (PLD) growth technique, and their structural and electronic properties have been investigated with various characterization techniques. We have found that using the low temperature PLD growth technique allows us to prepare abrupt and high quality AlN/InN heterointerfaces with an interfacial roughness less than 2 nm. Photoemission spectroscopy measurements using hard X-ray around 6 keV have revealed that the conduction band offset at the AlN/InN heterointerface is as large as 3.3 eV.

Identification of Two Wnt-Responsive Elements in the Intron of RING Finger Protein 43 (RNF43) Gene

RING finger protein 43 (RNF43), an E3-type ubiquitin ligase, is frequently up-regulated in human colorectal cancer. It has been shown that expression of RNF43 is regulated by the Wnt-signaling pathway. However the regulatory region(s) for its transcriptional activation has not been clarified. In this study, we have shown for the first time that RNF43 is a direct target of TCF4/β-catenin complex, and that its expression is regulated by a regulatory region containing two Wnt-responsive elements (WREs) in intron2. A reporter gene assay revealed that nucleotide substitutions in the WREs decreased the reporter activity in colon cancer cells, suggesting that both WREs are involved in the transcriptional activation. Knockdown of β-catenin by siRNA suppressed the reporter activity. In addition, ChIP assay showed that both elements associate with TCF4/β-catenin complex in colon cancer cells. These data indicate that expression of RNF43 is regulated by the canonical Wnt/β-catenin pathway through binding of the WREs with TCF4/β-catenin complex. These findings should be useful for the understanding of the regulatory mechanism of RNF43 and may contribute to the clarification of signaling pathways regulated by RNF43.

Growth Orientation Control of Semipolar InN Films Using Yttria-Stabilized Zirconia Substrates

We have investigated the epitaxial growth of InN films on yttria-stabilized zirconia (YSZ) substrates with various surface orientations. Through systematic crystallographic investigations, we have found that the epitaxial relationship of InN[1120] ∥YSZ [110] and InN[0001] ∥YSZ [111] holds in the InN/YSZ system. This enables us to grow semipolar InN films with arbitrary orientations.

Smyd5 plays pivotal roles in both primitive and definitive hematopoiesis during zebrafish embryogenesis

Methylation of histone tails plays a pivotal role in the regulation of a wide range of biological processes. SET and MYND domain-containing protein (SMYD) is a methyltransferase, five family members of which have been identified in humans. SMYD1, SMYD2, SMYD3, and SMYD4 have been found to play critical roles in carcinogenesis and/or the development of heart and skeletal muscle. However, the physiological functions of SMYD5 remain unknown. To investigate the function of Smyd5 in vivo, zebrafish were utilised as a model system. We first examined smyd5 expression patterns in developing zebrafish embryos. Smyd5 transcripts were abundantly expressed at early developmental stages and then gradually decreased. Smyd5 was expressed in all adult tissues examined. Loss-of-function analysis of Smyd5 was then performed in zebrafish embryos using smyd5 morpholino oligonucleotide (MO). Embryos injected with smyd5-MO showed normal gross morphological development, including of heart and skeletal muscle. However, increased expression of both primitive and definitive hematopoietic markers, including pu.1, mpx, l-plastin, and cmyb, were observed. These phenotypes of smyd5-MO zebrafish embryos were also observed when we introduced mutations in smyd5 gene with the CRISPR/Cas9 system. As the expression of myeloid markers was elevated in smyd5 loss-of-function zebrafish, we propose that Smyd5 plays critical roles in hematopoiesis.

Overexpression of cohesion establishment factor DSCC1 through E2F in colorectal cancer.

Ctf18-replication factor C complex including Dscc1 (DNA replication and sister chromatid cohesion 1) is implicated in sister chromatid cohesion, DNA replication, and genome stability in S. cerevisiae and C. elegans. We previously performed gene expression profiling in primary colorectal cancer cells in order to identify novel molecular targets for the treatment of colorectal cancer. A feature of the cancer-associated transcriptional signature revealed from this effort is the elevated expression of the proto-oncogene DSCC1. Here, we have interrogated the molecular basis for deviant expression of human DSCC1 in colorectal cancer and its ability to promote survival of cancer cells. Quantitative PCR and immunohistochemical analyses corroborated that the expression level of DSCC1 is elevated in 60–70% of colorectal tumors compared to their matched noncancerous colonic mucosa. An in silico evaluation of the presumptive DSCC1 promoter region for consensus DNA transcriptional regulatory elements revealed a potential role for the E2F family of DNA-binding proteins in controlling DSCC1 expression. RNAi-mediated reduction of E2F1 reduced expression of DSCC1 in colorectal cancer cells. Gain- and loss-of-function experiments demonstrated that DSCC1 is involved in the viability of cancer cells in response to genotoxic stimuli. We reveal that E2F-dependent expression of DSCC1 confers anti-apoptotic properties in colorectal cancer cells, and that its suppression may be a useful option for the treatment of colorectal cancer.