Hidenori Kaneoka

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPα in the differentiation of hepatocytes. The amount of sumoylated C/EBPα gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPα in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPα-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPα in primary fetal hepatocytes. The enhancement of C/EBPα-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPα blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPα to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPα, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

SUMOylation of damaged DNA-binding protein DDB2

Damaged DNA-binding protein (DDB) is a heterodimer composed of two subunits, p127 and p48, which have been designated DDB1 and DDB2, respectively. DDB2 recognizes and binds to UV-damaged DNA during nucleotide excision repair. Here, we demonstrated that DDB2 was SUMOylated in a UV-dependent manner, and its major SUMO E3 ligase was PIASy as determined by RNA interference-mediated knockdown. The UV-induced physical interaction between DDB2 and PIASy supported this notion. PIASy knockdown reduced the removal of cyclobutane pyrimidine dimers (CPDs) from total genomic DNA, but did not affect that of 6-4 pyrimidine pyrimidone photoproducts (6-4PPs). Thus, DDB2 plays an indispensable role in CPD repair, but not in 6-4PP repair, which is consistent with the observation that DDB2 was SUMOylated by PIASy. These results suggest that the SUMOylation of DDB2 facilitates CPD repair.

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.

Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene.

The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIP assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5kb S/MAR-GRE) and the promoter to the inner nuclear matrix.

YY1 binds to regulatory element of chicken lysozyme and ovalbumin promoters

Chicken lysozyme is highly expressed in the oviduct. The 5′ regulatory region of this gene contains a negative element that represses transcription. To assess the molecular basis underlying the regulation of lysozyme gene expression, we investigated the binding protein to this region. Sequence motif analysis suggested the existence of putative YY1 binding sites in this regulatory region. Electrophoretic mobility shift assay showed the specific binding of YY1 to the negative element. In addition, chromatin immunoprecipitation assay indicated that YY1 specifically bound to the negative element in oviduct cells but not in erythrocytes. It was suggested by electrophoretic mobility shift assay and chromatin immunoprecipitation assay that YY1 also bound to the negative regulatory region in the promoter of the ovalbumin gene which also shows oviduct-specific expression. Western blot analysis showed that YY1 was expressed in relatively high levels in the oviduct and nucleus fractionation experiments showed that YY1 was localized both in chromosome and nuclear matrix fractions. These results suggest that there are some specific roles in the negative regulatory regions of these genes in relation to the multifunctional transcription factor YY1.

Characterization of chicken interferon-inducible transmembrane protein-10

Interferon-inducible transmembrane protein (IFITM) family proteins are antivirus factors. In the present study, we examined the expression pattern of chicken IFITM10 using quantitative reverse transcription-polymerase chain reaction. In adult chickens, IFITM10 levels were markedly lower than those of IFITM3, which exhibits antivirus activity. On the other hand, IFITM10 was expressed in levels similar to those of IFITM3 in embryonic organs. Primordial germ cells in 2.5-d embryos expressed high levels of IFITM10, which gradually decreased with time. The interferon-α stimulation of embryonic fibroblast cells did not enhance the expression of IFITM10. The forced expression of IFITM10 slightly inhibited the infectivity of the VSV-G-pseudotyped lentiviral vector. Furthermore, cell fusion was inhibited by IFITM10 when HeLa cells transfected with the VSV-G expression vector were treated with low pH buffer. Although it remains unclear whether IFITM10 inhibits viral infections under physiological conditions, these results suggest that chicken IFITM10 exhibits antivirus activity. Graphical abstract Explanation for the graphical abstract (Okuzaki et al.) Chicken IFITM10 inhibits VSV-G-pseudotyped lentiviral vector infection and VSV-G-mediated cell fusion.

Analyses of chicken sialyltransferases related to N-glycosylation

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-β1,4-N-acetylglucosamine (Galβ1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.

Constitutive expression of the brg1 gene requires GC-boxes near to the transcriptional start site

We previously reported that BRG1, an ATPase subunit of SWI/SNF chromatin remodelling complexes, is constitutively expressed and that the alternative ATPase subunit (BRM) is inducibly expressed through differentiation in mammalian cells. In the present study, the regulatory elements that confer constitutive expression on brg1 were explored. First, we analysed the promoter proximal region surrounding its transcriptional start site. Using computer-aided analysis, a TATA-less, GC-rich promoter containing four putative binding sites for Sp1/3 was predicted. One of the putative Sp1/3-binding sites (from -21 to -15 bp) overlapped with a putative YY1-binding site. A gel-shift assay showed that YY1 but not Sp1/3 bound to this sequence and that Sp3 but not Sp1 bound to the other three predicted binding sites. Furthermore, chromatin immunoprecipitation analysis showed that Sp3 and YY1 bound to the promoter region together with TATA-binding protein in vivo. In vivo and in vitro binding assays showed that Sp3 and YY1 interacted with each other. Together, these results suggest that Sp3 and YY1 recruit general transcription factors and facilitate the assembly of a preinitiation complex.

Molecular cloning of chicken TET family genes and role of chicken TET1 in erythropoiesis

Ten-eleven translocation (TET) methylcytosine dioxygenase has potential as an active eraser to regulate the genomic DNA methylation status. We herein cloned chicken TET (cTET) family genes, and confirmed their functions. Quantitative reverse-transcription PCR showed that cTET1 was strongly expressed in erythrocytes throughout development. This cTET1 expression pattern, together with the results of methylated or hydroxymethylated DNA immunoprecipitation, suggests that cTET1 contributes to demethylation around the promoter region of the definitive-type β-globin gene βΑ in erythroid cells. The knockdown of cTET1 in T2ECs chicken erythroid progenitor cells suppressed the induction of βΑ expression under differentiation conditions. These results suggest that cTET1 plays an important role in erythroid cell differentiation.

Genetically Manipulated Chickens as Animal Bioreactor

Molecular chaperones are essential for guarding proteins that are indispensable for normal cellular functions. Heat shock protein 90 (Hsp90) is a vital molecular chaperone in eukaryotes that participates in stabilizing and activating approximately 200 target proteins, called “clients,” many of which are involved in signal transduction pathways. Cancer cells however utilize Hsp90 to chaperone an array of mutated and overexpressed oncoproteins to protect them from misfolding and degradation. Therefore, Hsp90 is an attractive target in cancer therapy. Hsp90 chaperone function relies on ATP binding and hydrolysis, which in turn guides its carefully orchestrated conformational changes. This chaperone cycle is fine-tuned by another group of proteins called co-chaperones. They are able to accelerate or decelerate the cycle, allowing Hsp90 to chaperone different clients. Posttranslational modifications (PTMs) can also regulate the chaperone cycle at an epigenetic level thereby tailoring Hsp90 function to suit a specific cell type or environmental condition. Recent evidence suggests that inhibition of the enzymes that catalyze the PTM of Hsp90 can act synergistically with Hsp90 inhibitors, providing a novel therapeutic strategy to enhance the efficacy of Hsp90 inhibitors in cancer cells. A-22016 WORLD CONGRESS ON IN VITRO BIOLOGY ABSTRACT ISSUE : Animal Symposia and Workshops, A-16