Solanoeclepin B, a hatching factor for potato cyst nematode.

The potato cyst nematode (PCN) causes extensive crop losses worldwide. Because the hatching of PCN requires host-derived molecules known as hatching factors (HFs), regulating HF production in host plants may help to control this harmful pest. Solanoeclepin A (SEA), isolated from potato, is the most active HF for PCN; however, its biosynthesis is completely unknown. We discovered a HF called solanoeclepin B (SEB) from potato and tomato root exudates and showed that SEB was biosynthesized in the plant and converted to SEA outside the plant by biotic agents. Moreover, we identified five SEB biosynthetic genes encoding three 2-oxoglutarate-dependent dioxygenases and two cytochrome P450 monooxygenases in tomato. Exudates from tomato hairy roots in which each of the genes was disrupted contained no SEB and had low hatch-stimulating activity for PCN. These findings will help to breed crops with a lower risk of PCN infection.

Blimp-1 Functions as a Molecular Switch to Prevent Inflammatory Activity in Foxp3+RORγt+ Regulatory T Cells.

Foxp3+ regulatory T cells (Treg) are essential modulators of immune responses, but the molecular mechanisms underlying their function are not fully understood. Here we show that the transcription factor Blimp-1 is a crucial regulator of the Foxp3+RORγt+ Treg subset. The intrinsic expression of Blimp-1 in these cells is required to prevent production of Th17-associated cytokines. Direct binding of Blimp-1 to the Il17 locus in Treg is associated with inhibitory histone modifications but unaltered binding of RORγt. In the absence of Blimp-1, the Il17 locus is activated, with increased occupancy of the co-activator p300 and abundant binding of the transcriptional regulator IRF4, which is required, along with RORγt, for IL-17 expression in the absence of Blimp-1. We also show that despite their sustained expression of Foxp3, Blimp-1-/- RORγt+IL-17-producing Treg lose suppressor function and can promote intestinal inflammation, indicating that repression of Th17-associated cytokines by Blimp-1 is a crucial requirement for RORγt+ Treg function.

T-Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation.

Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.

Differential regulation of Effector and Regulatory T cell function by Blimp1.

The transcriptional regulator Blimp1 plays crucial roles in controlling terminal differentiation in several lineages. In T cells, Blimp1 is expressed in both effector (Teff) and regulatory (Treg) cells, and mice with T cell-specific deletion of Blimp1 (Blimp1CKO mice) spontaneously develop severe intestinal inflammation, indicating a crucial role for Blimp1 in T cell homeostasis regulation. Blimp1 has been shown to function as a direct activator of the Il10 gene and although its requirement for IL10 expression has been demonstrated in both Treg and Teff cells under inflammatory conditions, the intrinsic requirement of Blimp1 for homeostatic maintenance of these T cell subsets had not been investigated. Using mice with Foxp3+ Treg-cell specific deletion of Blimp1 and other approaches, here we show that Foxp3+ Treg cell-intrinsic expression of Blimp1 is required to control Treg and Teff cells homeostasis but, unexpectedly, it is dispensable to prevent development of severe spontaneous intestinal inflammation. In addition, we show that Blimp1 controls common and unique aspects of Treg and Teff cell function by differentially regulating gene expression in these T cell subsets. These findings document previously unappreciated aspects of Blimp1's role in T cell biology and shed light on the intricate mechanisms regulating Treg and Teff cell function.

O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection.

Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1ß and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1ß, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity.

A Bacterial Artificial Chromosome Reporter System for Expression of the Human FOXP3 Gene in Mouse Regulatory T-Cells.

The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human FOXP3 (hFOXP3) gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate hFOXP3 gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that hFOXP3 gene expression in BAC transgenic mice is solely restricted to CD4+ T-cells, as for mFoxp3 gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-ß. These data suggest that, at least in part, cell type-specific human and mouse foxp3 gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate hFOXP3 gene expression further and to screen potential therapeutics in modulating hFOXP3 gene expression in vitro, we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-ß dependent, and substantially enhanced by an mTOR inhibitor, Torin1. In both the reporter transgenic mice and cell lines, histone H4 molecules in the hFOXP3 promoter and enhancers located in human CNS1 and CNS2 regions were highly acetylated in natural Treg and TCR/TGF-ß-induced Treg, indicating hFOXP3 gene expression is regulated by mechanisms similar to those previously identified for the mFoxp3 gene.

Gene expression in the Gitr locus is regulated by NF-κB and Foxp3 through an enhancer.

Glucocorticoid-induced TNFR (Gitr) and Ox40, two members of the TNFR superfamily, play important roles in regulating activities of effector and regulatory T cells (Treg). Their gene expression is induced by T cell activation and further upregulated in Foxp3+ Treg. Although the role of Foxp3 as a transcriptional repressor in Treg is well established, the mechanisms underlying Foxp3-mediated transcriptional upregulation remain poorly understood. This transcription factor seems to upregulate expression not only of Gitr and Ox40, but also other genes, including Ctla4, Il35, Cd25, all critical to Treg function. To investigate how Foxp3 achieves such upregulation, we analyzed its activity on Gitr and Ox40 genes located within a 15.1-kb region. We identified an enhancer located downstream of the Gitr gene, and both Gitr and Ox40 promoter activities were shown to be upregulated by the NF-κB-mediated enhancer activity. We also show, using the Gitr promoter, that the enhancer activity was further upregulated in conjunction with Foxp3. Foxp3 appears to stabilize NF-κB p50 binding by anchoring it to the enhancer, thereby enabling local accumulation of transcriptional complexes containing other members of the NF-κB and IκB families. These findings may explain how Foxp3 can activate expression of certain genes while suppressing others.

TGF-ß-mediated Foxp3 gene expression is cooperatively regulated by Stat5, Creb, and AP-1 through CNS2.

Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3(-) T cells. These sequences are, however, demethylated in Foxp3(+) Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect and shown that transcription factors AP-1, Stat5, and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-ß signaling through Smad3-mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during induced Treg induction, whereas AP-1 and Creb maintain Enhancer 2 activity.

Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic.

BACKGROUND: Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. METHODOLOGY: A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. RESULTS AND CONCLUSIONS: Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.

An atlas of combinatorial transcriptional regulation in mouse and man.

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

Role of survival motor neuron complex components in small nuclear ribonucleoprotein assembly.

Survival motor neuron (SMN) complex is essential for the biogenesis of the small nuclear ribonucleoprotein (snRNP) complex, although the complete role of each SMN complex component for the snRNP synthesis is largely unclear. We have identified an interaction between the two components Gemin2-Gemin7 using the mammalian two-hybrid system. In vitro stability assay revealed that the known SMN-Gemin7 interaction becomes stable in the presence of Gemin2 possibly via the identified Gemin2-Gemin7 interaction. Gemin7 knockdown revealed a decrease in snRNP assembly activity and a decrease in SmE protein, a component of snRNP, in the SMN complex, which was consistent with a previous discussion that the Gemin6-Gemin7 heterodimer may serve as a surrogate for the SmD3-SmB particle in forming a subcore, the intermediate complex for snRNP. Interestingly, we found that Unrip, but not Gemin8, can remove Gemin7 from the stable SMN-Gemin2-Gemin7 ternary complex. In an in vitro snRNP assembly assay using the Unrip knockdown and the untreated cell lysates, we revealed that there was a decrease in Gemin7 and increase in SmB/B' in the SMN complex observed in untreated cells during the assay, suggesting that the Gemin6-Gemin7 heterodimer in the subcore is exchanged by the SmD3-SmB particle to form snRNP. Surprisingly, these changes were not observed in the assay using the Unrip knockdown cell extracts, indicating the importance of Unrip in the formation of snRNP likely via removal of the Gemin6-Gemin7 from the SMN complex. Taken together, these results indicate that snRNP is synthesized by harmonization of the SMN complex components.

Gemin2 plays an important role in stabilizing the survival of motor neuron complex.

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.

Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

BACKGROUND: Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to determine the function of the hypothetical proteins. RESULTS: We examined 960 soluble proteins from Pyrococcus and selected 107 interactions based on luciferase reporter activity, which was then evaluated using a computational approach to assess the reliability of the interactions. We also analyzed the expression of the assay samples by western blot, and a few interactions by in vitro pull-down assays. We identified 11 hetero-interactions that we considered to be located at the same operon, as observed in Helicobacter pylori. We annotated and classified proteins in the selected interactions according to their orthologous proteins. Many enzyme proteins showed self-interactions, similar to those seen in other organisms. CONCLUSION: We found 13 unannotated proteins that interacted with annotated proteins; this information is useful for predicting the functions of the hypothetical Pyrococcus proteins from the annotations of their interacting partners. Among the heterogeneous interactions, proteins were more likely to interact with proteins within the same ortholog class than with proteins of different classes. The analysis described here can provide global insights into the biological features of the protein-protein interactions in P. horikoshii.