Yujin Inayoshi

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system.

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.

Transcription Factor YY1 Interacts with Retroviral Integrases and Facilitates Integration of Moloney Murine Leukemia Virus cDNA into the Host Chromosomes

ABSTRACT Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.

Galactosylation of human erythropoietin produced by chimeric chickens expressing galactosyltransferase.

Human erythropoietin produced in the egg white of chimeric chicken contains N-glycan with lower amounts of terminal galactose and sialic acid; therefore, the chicken galactosyltransferase gene was introduced together with the human erythropoietin gene by a retroviral vector. We found that erythropoietin accumulated in the egg white was partially galactosylated.

Moloney murine leukemia virus integrase and reverse transcriptase interact with PML proteins

Pull-down assay and co-immunoprecipitation of cell extracts in which the integrase or reverse transcriptase of Moloney murine leukemia virus was transiently expressed showed that both enzymes interacted with PML proteins. In infected cells, interaction between the integrase and PML was also observed. Transient expression of PIASy and SUMO proteins facilitated SUMOylation of the integrase but had no apparent effects on the interaction with PML. A FLAG-tagged integrase co-localized with PML protein possibly in the PML body. Knockdown of PML by small interfering RNA resulted in reduced viral cDNA levels and integration efficiency. This suggested that PML proteins activated reverse transcription.

Transcriptional regulation of the α-fetoprotein gene in hepatocytes

α-Fetoprotein (AFP) is a fetal serum protein enriched in fetal liver whose expression is downregulated during development. The proximal region of the AFP promoter contains two binding sites for CCAAT/enhancerbinding protein α (C/EBPα), two for hepatocyte nuclear factor-1α (HNF1α) and one for glucocorticoid receptor (GR) in addition to a nuclear factor-1 (NF-1) binding site, which partly overlaps with the distal C/EBPα binding site. Luciferase reporter assays showed that a combination of C/EBPα, HNF1α, NF-1 and coactivator p300 gave the maximal activity. Mutation in either HNF1α binding site diminished the expression completely but mutation in either or both of the C/EBPα binding sites did not severely reduce the expression level. Chromatin immunoprecipitation assays showed that GR, HNF1α, C/EBPα, NF-1, and p300 bound to the AFP promoter in adult liver.

Transcriptional Regulation of the α-fetoprotein Gene by SWI/SNF Chromatin Remodeling Complex

Abstractα-Fetoprotein (AFP) is a fetal serum protein abundant in fetal liver whose expression is downregulated in adult liver. The promoter–proximal region of the AFP gene contains two binding sites for CCAAT/enhancer-binding protein α (C/EBPα), two for hepatocyte nuclear factor-1α (HNF-1α) and one for nuclear factor-1 (NF-1). Luciferase reporter assays showed that a combination of C/EBPα, HNF-1α, NF-1 and coactivator p300 gave the maximal activity but the BRG1 and BRM, ATPase subunit of the chromatin remodeling complex SWI/SNF, repressed the transactivation of the AFP gene by these transcription factors. Deletion analyses of C/EBPα binding sites suggested that C/EBPα recruited SWI/SNF complex and caused the repression. This repression may also play important roles in the downregulation of the AFP gene in adult hepatocytes.

A Chromatin Remodeling Factor, BRM has Important Roles in Cell Differentiation

The modification of chromatin structure is increasingly recognized to be an important factor for transcriptional regulation. So far, it is known that the state of chromatin compaction is controlled by two major mechanisms. One is a histone acetyltransferase and deacetylase system and the other is a regulatory system containing chromatin remodeling complexes (2). The former enzymes acetylate or deacetylate N-terminal tail structures of histones and control chromatin opening and compaction. The chromatin remodeling complex was characterized first in Saccharomyces cerevisiae as a multi-protein complex which controls mating type switch (SWI) and sucrose non-fermenting (SNF) genes. ATP-dependent chromatin remodeling activity of the complex was recently identified (3). This SWI/SNF complex has an ATPase subunit called SWI2/SNF2, and its activity is essential for chromatin remodeling (3). The homologs of SWI2/SNF2 have been identified in higher eukaryotes, and they also form multi-protein complexes of chromatin remodeling factors (1). Mammalian SWI/SNF complexes are composed of more than 8 subunits. Until now, two ATPase subunits have been identified and named BRM and BRG1 (4, 5). The SWI/SNF complex containing either BRG1 or BRM shows chromatin remodeling activity, and activates or inactivates gene expression. In this article, we analyzed BRM and BRG1 expression during neural differentiation of NPCs and P19 embryonal carcinoma cells and liver differentiation to study its role in neural differentiation.