De novo transcriptome assembly of the midgut glands of herbivorous land crabs, Chiromantes haematocheir, and identification of laccase genes involved in lignin degradation.

Herbivorous land crabs such as Chiromantes haematocheir and C. dehaani show biomass-degrading activities. In this study, we performed RNA-seq analysis to detect biomass-degrading enzymes. A de novo transcriptome assembly in the midgut glands of molting and non-molting C. haematocheir crabs was constructed using RNA sequencing. Illumina sequencing generated 44,937,002 and 44,394,310 reads from the two midgut glands. In total, 178,710 contigs with an average length of 750 bp and an N50 value of 1,235 bp were assembled, of which 37,890 contigs were annotated using BLASTx search against the NCBI database. We identified 22 contigs (11 genes) belonging to the laccase family and 44 contigs (22 genes) belonging to the peroxidase family. Sixteen contigs (three genes) belonging to the GH9 cellulase family were also detected. We selected the gene accounting for the majority of expressed laccase and analyzed its properties. The 24131-laccase transcript (2465 bp) had one complete open reading frame, nt 149-1987, encoding a protein of 613 amino acids with a deduced molecular mass of 67.708 kDa. The enzyme was shown to belong to the multicopper oxidase family. The 24131-laccase protein was confirmed to have oxidation activity against 2,6-dimethoxyphenol by ectopic expression in Escherichia coli. Laccase activity was significantly enhanced by feeding land crabs with plant diets. These data suggest that the enzyme plays an important role in the digestion of lignin in the guts of land crabs.

Bicarbonate and unsaturated fatty acids enhance capsular polysaccharide synthesis gene expression in oral streptococci, Streptococcus anginosus.

We recently reported on the capsular polysaccharide (CP) synthesis (cps) genes of the oral streptococci, Streptococcus anginosus. In this study, we investigate the effects of carbon dioxide (CO2), bicarbonate (HCO3-) and unsaturated fatty acids (UFAs) on CP synthesis of S. anginosus. We found that CP production increased when bacteria were exposed to high concentrations of CO2. This increase was similarly observed in the presence of sodium bicarbonate (NaHCO3) under atmospheric condition. Since ectopic expression of carbonic anhydrase, which converts CO2 to HCO3-, eliminated the requirement for CO2 in CP production and growth of S. anginosus lacking this enzyme, it seemed that HCO3- is an essential factor for CP production. Furthermore, UFAs also stimulated the CP production. Promoter-reporter assay and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that stimulation of CP production occurs at the transcription level. The results of the promoter assays suggest that the expression and stimulation of cps genes by HCO3- or UFAs require the cpsA gene, which is located in the first position of the cps operon. With respect to the relationship between HCO3-and UFAs, HCO3- may stimulate UFA synthesis pathway at transcription level. Therefore, it is possible that UFAs are definitive signals for the CP production in S. anginosus.

Guaiacol oxidation activity of herbivorous land crabs, Chiromantes haematocheir and Chiromantes dehaani.

The land crabs, Chiromantes haematocheir (Akate-gani) and Chiromantes dehaani (Kurobenkei-gani) inhabit seaside forests in Japan. The crabs mainly consume plant material and its detritus. Therefore, they are expected to possess the ability to degrade the major components of biomass, cellulose and lignin in order to digest plant materials. In this study, we analyzed biomass-degrading activities of the land crabs, especially guaiacol oxidation activity, which seems to be related to lignin degradation. Cellulase activity was detected from almost all gut samples including the stomach, midgut gland and intestine of all dissected crabs. Conversely, high guaiacol oxidation activity was detected in the midgut gland of all C. dehaani and several female C. haematocheir crabs. This is consistent with a previous study showing that female crabs were more herbivorous than male crabs were and observation that C. dehaani crabs are more herbivorous than C. haematocheir. Guaiacol oxidation activity might play an important role in the herbivorous behavior of land crabs.

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-ß1,4-N-acetylglucosamine (Galß1,4GlcNAc) 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.

Implication of SUMO E3 ligases in nucleotide excision repair.

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

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.

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.

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 ß1,4-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.

Organization of the capsule biosynthesis gene locus of the oral streptococcus Streptococcus anginosus.

The capsular polysaccharide (CPS) of the important oral streptococcus Streptococcus anginosus, which causes endocarditis, and the genes for its synthesis have not been clarified. In this study, we investigated the gene locus required for CPS synthesis in S. anginosus. Southern hybridization using the cpsE gene of the well-characterized bacterium S. agalactiae revealed that there is a similar gene in the genome of S. anginosus. By using the colony hybridization technique and inverse PCR, we isolated the CPS synthesis (cps) genes of S. anginosus. This gene cluster consisted of genes containing typical regulatory genes, cpsA-D, and glycosyltransferase genes coding for glucose, rhamnose, N-acetylgalactosamine, and galactofuranose transferases. Furthermore, we confirmed that the cps locus is required for CPS synthesis using a mutant strain with a defective cpsE gene. The cps cluster was found to be located downstream the nrdG gene, which encodes ribonucleoside triphosphate reductase activator, as is the case in other oral streptococci such as S. gordonii and S. sanguinis. However, the location of the gene cluster was different from those of S. pneumonia and S. agalactiae.

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.

Transcription factor YY1 interacts with retroviral integrases and facilitates integration of moloney murine leukemia virus cDNA into the host chromosomes.

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.

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.

GATA4 inhibits expression of the tryptophan oxygenase gene by binding to the TATA box in fetal hepatocytes.

The glucocorticoid receptor regulates liver-specific expression of the tryptophan oxygenase gene through glucocorticoid responsive elements located -0.45 and -1.2 kb from the transcription start site. However, the hormone-mediated induction is restricted to adult hepatocytes, and fetal hepatocytes are unable to express the gene even in the presence of the receptor and glucocorticoid hormone. The difference in sensitivity to the hormone between adult and fetal hepatocytes has not been well understood. In this study, we analyzed the structure of the tryptophan oxygenase gene's promoter. The promoter has two TATA boxes, and transcription starts from the downstream TATA box. We found that a transcription factor GATA4 bound to the downstream TATA box and may inhibit the binding of TATA-binding protein, resulting in transcriptional repression even in the presence of glucocorticoid in fetal hepatocytes.

Differentiation-specific expression of chromatin remodeling factor BRM.

The chromatin remodeling complex SWI/SNF exclusively contains BRG1 or BRM as an ATPase subunit, but the physiological relevance of these ATPases is not yet clear. Here, we studied the developmental regulation of the brm gene. A differentiation-specific up-regulation of BRM expression was observed with P19, F9 and C2C12 cells as with neural cells and hepatocytes. The promoter region of brm contains two putative binding sites for CCAAT enhancer binding protein beta (C/EBPbeta). Each site partially overlapped with binding sites for GATAs. C/EBPbeta stimulated the transcriptional activity of the brm promoter but GATA2 and 3 down-regulated the expression. C/EBPbeta bound to the proximal sequences of the promoter in differentiated cells and was replaced by GATAs which bound to the overlapping sites in the undifferentiated state. C/EBPbeta and GATAs may developmentally regulate the expression of brm by mutually exclusive binding.

Isolation and quantitative detection of tetrachloroethene (PCE)-dechlorinating bacteria in unsaturated subsurface soils contaminated with chloroethenes.

The estimation of tetrachloethene (PCE) dechlorinating-activity and identification of PCE-dechlorinating bacteria were performed in 65 unsaturated subsurface soils (at a depth 30-60 cm) that were collected from 21 noncontaminated soils and 44 chloroethene-contaminated soils including four soils that dechlorinated PCE to 1,2-cis-dichloroethene (cisDCE) in situ. Sixteen out of the 44 PCE-contaminated soils and three out of the 21 noncontaminated soils dechlorinated PCE to trichloroethene and cisDCE but not vinyl chloride or ethene after a month of incubation with 0.1% yeast extract at 30 degrees C. Desulfitobacterium sp. strain B31e3 that can dechlorinate PCE to cisDCE was isolated from a soil that dechlorinated PCE to cisDCE in situ. 16S rRNA gene of this strain showed the closest similarity of 99.1% with that of Desulfitobacterium hafniense (formally frappieri) strain DP7. Real-time PCR using specific primer sets targeted to the 16S rRNA genes of the representative PCE-dechlorinating bacteria, Dehalococcoides spp., Desulfitobacterium spp., and Dehalobacter spp. were performed using five unsaturated subsurface soils that dechlorinated PCE and three that did not dechlorinate PCE. In two out of the five soils that dechlorinated PCE, Desulfitobacterium spp. (0.12, 0.38% of total bacteria) and Dehalobacter spp. (0.0045, 0.0061% of total bacteria) were detected, and in one of the five soils, only Desulfitobacterium spp. (0.042% of total bacteria) was detected. None of these representative PCE-dechlorinating bacteria were detected in two out of the five soils that dechlorinated PCE and in all of the three soils that did not dechlorinate PCE. Dehalococcoides spp. were not detected in any unsaturated subsurface soils used in this study. These results suggested the involvement of Desulfitobacterium spp. and probably Dehalobacter spp. rather than Dehalococcoides spp. in the dechlorination of PCE to cisDCE in unsaturated subsurface soils.

Anaerobic biodegradation of 4-alkylphenols in a paddy soil microcosm supplemented with nitrate.

Anaerobic degradation of phenol, p-cresol, 4-n-propylphenol (n-PP), 4-i-propylphenol (i-PP), 4-n-butylphenol (n-BP) and 4-sec-butylphenol (sec-BP) was observed in a paddy soil supplemented with nitrate. We detected the metabolites 4'-hydroxypropiophenone (HPP) from n-PP, 4-i-propenylphenol from i-PP, and 4-(1-butenyl)phenol and 4'-hydroxybutyrophenone (HBP) from n-BP. Compared with the original soils, Betaproteobacteria became predominant in the microcosm during the degradation of phenol and p-cresol whereas no remarkable change was observed in the community degrading propylphenols and butylphenols. The microcosm, however, did not degrade 4-t-butylphenol (t-BP), 4-t-octylphenol (t-OP) and 4-n-octylphenol (n-OP). Paddy soil supplemented with sulfate or iron (III) as electron acceptors did not degrade phenol and 4-alkylphenols with the exception of the degradation of p-cresol in sulfate-reducing conditions. It was demonstrated for the first time that anaerobic microbial degradation of alkylphenols, in a paddy soil supplemented with nitrate as an electron acceptor, occurred via oxidation of the alpha carbon in the alkyl chain.

BRG1 chromatin remodeling activity is required for efficient chromatin binding by repressor element 1-silencing transcription factor (REST) and facilitates REST-mediated repression.

Chromatin remodeling enzymes such as SWI/SNF use the hydrolysis of ATP to power the movement of nucleosomes with respect to DNA. BRG1, one of the ATPases of the SWI/SNF complex, can be recruited by both activators and repressors, although the precise role of BRG1 in mechanisms of repression has thus far remained unclear. One transcription factor that recruits BRG1 as a corepressor is the repressor element 1-silencing transcription factor (REST). Here we address for the first time the mechanism of BRG1 activity in gene repression. We found that BRG1 enhanced REST-mediated repression at some REST target genes by increasing the interaction of REST with the local chromatin at its binding sites. Furthermore, REST-chromatin interactions, mediated by BRG1, were enhanced following an increase in histone acetylation in a manner dependent on the BRG1 bromodomain. Our data suggest that BRG1 facilitates REST repression by increasing the interaction between REST and chromatin. Such a mechanism may be applicable to other transcriptional repressors that utilize BRG1.

Sumoylation of CCAAT/enhancer-binding protein alpha 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/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha 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/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-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/EBPalpha 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/EBPalpha 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/EBPalpha, 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.

Identification of cis-acting regions that contribute to neuron-specific expression of the GAP-43 gene.

There are two transcription start sites in the growth-associated protein 43 (GAP-43) promoter, and several repressive elements have been reported in the control region. But the repressive effects have been analyzed only for the distal transcription start site. Among the repressive elements reported, we found that modulator I repressed GAP-43 gene expression from the proximal promoter in non-neuronal cells. We also found a novel stimulative element immediately downstream of modulator I.