Activator Protein-1 Transcription Research Articles

Major Histocompatibility Complex Class II HLA-DRα Is Downregulated by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Lytic Transactivator RTA and MARCH8.

Kaposi's sarcoma-associated herpesvirus (KSHV) maintains two modes of life cycle, the latent and lytic phases. To evade the attack of the cell host's immune system, KSHV switches from the lytic to the latent phase, a phase in which only a few of viral proteins are expressed. The mechanism by which KSHV evades the attack of the immune system and establishes latency has not been fully understood. Major histocompatibility complex class II (MHC-II) molecules are key components of the immune system defense mechanism against viral infections. Here we report that HLA-DRα, a member of the MHC-II molecules, was downregulated by the replication and transcription activator (RTA) protein encoded by KSHV ORF50, an important regulator of the viral life cycle. RTA not only downregulated HLA-DRα at the protein level through direct binding and degradation through the proteasome pathway but also indirectly downregulated the protein level of HLA-DRα by enhancing the expression of MARCH8, a member of the membrane-associated RING-CH (MARCH) proteins. Our findings indicate that KSHV RTA facilitates evasion of the virus from the immune system through manipulation of HLA-DRα. Kaposi's sarcoma-associated herpesvirus (KSHV) has a causal role in a number of human cancers, and its persistence in infected cells is controlled by the host's immune system. The mechanism by which KSHV evades an attack by the immune system has not been well understood. This work represents studies which identify a novel mechanism by which the virus can facilitate evasion of an immune system. We now show that RTA, the replication and transcription activator encoded by KSHV (ORF50), can function as an E3 ligase to degrade HLA-DRα. It can directly bind and induce degradation of HLA-DRα through the ubiquitin-proteasome degradation pathway. In addition to the direct regulation of HLA-DRα, RTA can also indirectly downregulate the level of HLA-DRα protein by upregulating transcription of MARCH8. Increased MARCH8 results in the downregulation of HLA-DRα. Furthermore, we also demonstrate that expression of HLA-DRα was impaired in KSHV de novo infection.

Transcription activation complex analyzed

To better understand how DNA is transcribed into RNA, scientists have long been trying to obtain a detailed structure of a protein-DNA complex that initiates and regulates transcription of a specific gene. But such complexes have been hard to crystallize. Richard H. Ebright and coworkers at Rutgers University have now found a thermophilic bacterial complex that forms crystals readily and have determined its 4.4-Å structure (Science 2016, DOI: 10.1126/science.aaf4417). The complex includes a transcription activator protein, an initiation factor, RNA polymerase, a DNA template, and an RNA primer. The crystal structure reveals that a first set of protein-protein interactions between the activator and RNA polymerase helps the enzyme bind DNA and a second set of protein-protein interactions helps the enzyme unwind DNA so it can be transcribed. “It’s a lovely picture that you can tell is right” from decades of earlier biochemistry and genetics experiments on similar transcription activation complexes,

Structural basis of transcription activation.

Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.

Transcription activation all about timing

Transcription Regulating transcription by RNA polymerase (RNAP) is central to controlling gene expression. Transcription factors influence the activity of the RNAP. Feng et al. determined the crystal structure of a bacterial transcription activation complex. The transcription activator protein (TAP) converts the closed RNAP-promoter complex into an open complex through simple stabilizing protein-protein interactions with RNAP. The critical contacts did not go through the RNAP active center or the RNAP clamp. Instead, it seems that the timing of the interaction during transcription complex formation is critical for activation. Science , this issue p. [1330][1] [1]: /lookup/doi/10.1126/science.aaf4417

The calmodulin-binding transcription activator CAMTA1 is required for long-term memory formation in mice.

The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by calcium/calmodulin signaling in the nucleus. Recent studies have drawn attention to a new family of transcriptional regulators, the so-called calmodulin-binding transcription activator (CAMTA) proteins. CAMTAs are expressed at particularly high levels in the mouse and human brain, and we reasoned that, as calmodulin-binding transcription factors, CAMTAs may regulate the formation of long-term memory by coupling synaptic activity and calcium/calmodulin signaling to memory-related transcriptional responses. This hypothesis is supported by genetic studies that reported a correlation between Camta gene polymorphisms or mutations and cognitive capability in humans. Here, we show that acute knockdown of CAMTA1, but not CAMTA2, in the hippocampus of adult mice results in impaired performance in two memory tests, contextual fear conditioning and object–place recognition test. Short-term memory and neuronal morphology were not affected by CAMTA knockdown. Gene expression profiling in the hippocampus of control and CAMTA knockdown mice revealed a number of putative CAMTA1 target genes related to synaptic transmission and neuronal excitability. Patch clamp recordings in organotypic hippocampal slice cultures provided further evidence for CAMTA1-dependent changes in electrophysiological properties. In summary, our study provides experimental evidence that confirms previous human genetic studies and establishes CAMTA1 as a regulator of long-term memory formation.

‐Altered DNA‐Binding Specificity Transcription Activator Variant Provides Key To Unlocking Gene Activation Mechanism

Transcription activator proteins confer target selective, gene‐specific activation in response to appropriate stimuli. In order to dissect the molecular mechanisms of gene transcription activation, it is imperative to understand the role(s) of the transcription activator. Unfortunately, a subset of DNA‐binding transcriptional activator proteins are resistant to study by the conventional means of fusing putative Activation Domains (ADs) to heterologous DNA Binding Domains (DBDs) to identify key transcription factor functional elements. The budding yeast Saccharomyces cerevisiae Repressor Activator Protein 1 (Rap1) is one such activator protein. Rap1 specifically binds over 500 DNA sites in the yeast genome, serving key roles in multiple cellular processes including: (a) telomere stabilization; (b) transcription repression of silent mating type loci; and (c) activation of two of the most vigorously transcribed classes of yeast genes: the SAGA‐dominated glycolytic enzyme‐encoding genes (GEs) and the TFIID‐dominated ribosomal protein genes (RPGs). However, fusions of Rap1 or Rap1‐domains with heterologous DBDs reportedly fail to robustly drive reporter gene expression.To enable study of the role of Rap1 in activation, an altered DNA‐binding specificity Rap1 variant (Rap1 AS) was generated using a direct screening/selection strategy in yeast wherein activation of a selectable HIS3 reporter gene depended upon the ability of DBD‐mutagenized variant forms of Rap1 to efficiently drive expression from a mutant Rap1 enhancer (UASRAP1). Multiple candidate putative Rap1 AS variants scored in this screen. One has been characterized in detail. In both biochemical (DNA gel mobility shift) and functional (HIS3 reporter gene expression) assays, Rap1 AS preferentially bound the selecting mutant UASRAP1, displaying a significant discrimination for mutant vs. wild‐type (WT) UASRAP1 sequences. Yeast pseudodiploid for Rap1 (Rap1 WT and Rap1 AS) grow like WT, showing that Rap1 AS does not interfere with the many essential functions of Rap1 WT. Subsequent mutagenesis experiments with Rap1 AS identified a putative activation domain that mapped to a ~40 amino acid region within the C‐terminal 1/3 of Rap1. Systematic point mutagenesis of this mapped AD has identified eight amino acids that contribute critically to AD function.The collection of Rap1 AD mutants will be used to investigate the transcription activation mechanisms used to drive the TFIID‐dominated RPGs. Since we have previously shown that Rap1 interacts directly with TFIID, and is the only RPG activator absolutely required for RPG transcription, mechanistic analyses performed with the Rap1 AD mutants should unlock the key step(s) to RPG activation. Because few TFIID‐dependent activation mechanisms have been carefully dissected, these key steps will likely reveal general principles of TFIID‐dependent gene activation applicable to all organisms.Support or Funding InformationThis research was supported by the NIH Molecular Endocrinology Training Program (METP) training grant (T32 DK07563) and the NIH R01 grants GM52461 and GM115892.

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA- or Virus-Induced Proinflammatory Response.

Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP(-/-)) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)β-liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPβ binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPβ. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP(-/-) mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response.

Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid.

Gastroesophageal reflux disease is considered to be a major risk in the development of esophageal adenocarcinoma. Nuclear factor-kappa B (NF-κB) plays important roles in the regulation of several genes coding for cytokines, cell proliferation, and apoptosis. To understand the role of bile and acid in the causation of esophageal cancer, we have examined the effects of bile acids and acid on NF-κB activation in the esophageal epithelial cells OE33 and SKGT-4 qualitatively and quantitatively. Analysis of NF-κB activation in esophageal epithelial cells in response to bile acids and acid was performed by electrophoretic mobility shift assay, Western blotting and the translocation NF-κB was assessed by high content analysis (HCA). Cyclooxygenase-2 (COX-2) promoter activity was assessed by transient transfection assays. This study demonstrated that bile acids and acid activated NF-κB in a dose- and time-dependent manner. HCA analysis was an invaluable method in quantifying NF-κB translocation at the single cell population level following bile or acid treatment. Furthermore, deoxycholic acid (DCA) and acid-induced COX-2 promoter activity, and a mutation in the NF-κB and activator protein-1 (AP-1) binding sites remarkably reduced the reporter gene activity induced by DCA or acid. Our data demonstrate that bile and acid induce NF-κB activation in esophageal cells qualitatively and quantitatively. The induction of COX-2 promoter activity by DCA and acid was mediated via NF-κB and AP-1 transcription. The activation of NF-κB signaling pathway in esophageal cells may contribute to the development of esophageal cancer, and, therefore, modulating of NF-κB pathway may uncover new therapeutic strategies.

Angiotensin III increases monocyte chemoattractant protein-1 expression in cultured human proximal tubular epithelial cells.

Background/Aims:We investigated whether angiotensin III (Ang III) is involved in monocyte recruitment through regulation of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal tubular epithelial cells (HK-2 cells).Methods:We measured MCP-1 levels in HK-2 cells that had been treated with various concentrations of Ang III and Ang II type-1 (AT1) receptor antagonists at various time points. The phosphorylation states of p38, c-Jun N-terminal kinases (JNK), and extracellular-signal-regulated kinases were measured in Ang III-treated cells to explore the mitogen-activated protein kinase (MAPK) pathway. MCP-1 levels in HK-2 cell-conditioned media were measured after pre-treatment with the transcription factor inhibitors curcumin or pyrrolidine dithiocarbamate.Results:Ang III increased MCP-1 protein production in dose- and time-dependent manners in HK-2 cells, which was inhibited by the AT1 receptor blocker losartan. p38 MAPK activity increased significantly in HK-2 cells exposed to Ang III for 30 minutes, and was sustained at higher levels after 60 minutes (p < 0.05). Total phosphorylated JNK protein levels tended to increase 20 minutes after stimulation with Ang III. Pre-treatment with a p38 inhibitor, a JNK inhibitor, or curcumin significantly inhibited Ang III-induced MCP-1 production.Conclusions:Ang III increases MCP-1 synthesis via stimulation of intracellular p38 and JNK MAPK signaling activity and subsequent activated protein-1 transcriptional activity in HK-2 cells.

In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd88.

Toll-like receptors (TLRs) are evolutionarily conserved receptors with trimodular structure to respond to endogenous ligands and exogenous ligands from microbial pathogens. The highly conserved cytoplasmic C-terminal Toll/interleukin-1 receptor (TIR) domain of TLRs plays a crucial role in inflammatory reactions. In myeloid differentiation primary-response protein 88 (MyD88)- dependent signaling pathway, the interaction of TLRsTIR with cytosolic adaptor protein, MyD88TIR recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor kB (NF-kB) and activation protein 1 (AP-1) and other effector molecules. In the present investigation, TLR5TIR, TLR6TIR and MyD88TIR genes were subcloned and overexpressed in bacterium Escherichia coli strain BL- 21 (DE3). The purification and biochemical characterization of TLR5TIR and TLR6TIR&, and MyD88TIR proteins were also performed. The protein-protein interactions between TIR domains of TLR5 and TLR6 with MyD88, respectively, were evaluated in vitro at physiological pH and salt concentration. The in vitro reconstitution results showed that under physiological pH and salt concentration, MyD88TIR interacted with TLR5TIR, and did not interact with TLR6TIR protein. Both TIR domain-containing TLR5 and TLR6 proteins were prone to aggregation in a temperature-dependent manner at room temperature. At normal physiological pH and salt concentration, with the addition of binding partner MyD88TIR to TLR5/6TIR, time-dependent aggregation was not observed in both TLRsTIR at both room temperature and 4 ºC for 2 d, influencing the solubility of TLR5/6TIR. Moreover, TLR5TIR alone exhibited increase in solubility of the protein with increase in the salt concentration of the buffered solution from 0.025 M to 1.25 M at room temperature.

Functional Analysis of Cotton Leaf Curl Kokhran Virus/Cotton Leaf Curl Multan Betasatellite RNA Silencing Suppressors.

In South Asia, Cotton leaf curl disease (CLCuD) is caused by a complex of phylogenetically-related begomovirus species and a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). The post-transcriptional gene silencing (PTGS) suppression activities of the transcriptional activator protein (TrAP), C4, V2 and βC1 proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV)/CLCuMuB were assessed in Nicotiana benthamiana. A variable degree of local silencing suppression was observed for each viral protein tested, with V2 protein exhibiting the strongest suppression activity and only the C4 protein preventing the spread of systemic silencing. The CLCuKoV-encoded TrAP, C4, V2 and CLCuMuB-encoded βC1 proteins were expressed in Escherichia coli and purified. TrAP was shown to bind various small and long nucleic acids including single-stranded (ss) and double-stranded (ds) RNA and DNA molecules. C4, V2, and βC1 bound ssDNA and dsDNA with varying affinities. Transgenic expression of C4 under the constitutive 35S Cauliflower mosaic virus promoter and βC1 under a dexamethasone inducible promoter induced severe developmental abnormalities in N. benthamiana. The results indicate that homologous proteins from even quite closely related begomoviruses may differ in their suppressor activity and mechanism of action. The significance of these findings is discussed.

Crystallization and X-ray analysis of the transcription-activator protein C1 of bacteriophage P22 in complex with the PRE promoter element.

The transcription-activator protein C1 of the temperate phage P22 of Salmonella typhimurium plays a key role in the lytic versus lysogenic switch of the phage. A homotetramer of 92-residue polypeptides, C1 binds to an approximate direct repeat similar to the transcription activator CII of coliphage λ. Despite this and several other similarities, including 57% sequence identity to coliphage CII, many biochemical observations on P22 C1 cannot be explained based on the structure of CII. To understand the molecular basis of these differences, C1 was overexpressed and purified and subjected to crystallization trials. Although no successful hits were obtained for the apoprotein, crystals could be obtained when the protein was subjected to crystallization trials in complex with a 23-mer promoter DNA fragment (PRE). These crystals diffracted very well at the home source, allowing the collection of a 2.2 Å resolution data set. The C1-DNA crystals belonged to space group P21, with unit-cell parameters a = 87.27, b = 93.58, c = 111.16 Å, β = 94.51°. Solvent-content analysis suggests that the asymmetric unit contains three tetramer-DNA complexes. The three-dimensional structure is expected to shed light on the mechanism of activation by C1 and the molecular basis of its specificity.

C-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21.

The non-receptor-type tyrosine kinase c-Abl functions as a cytoplasmic signal transducer upon activation of cell-surface receptors. c-Abl is also involved in DDR (DNA-damage response), which is initiated in the nucleus, whereas its molecular functions in DDR are not fully understood. In the present study, we found that c-Abl phosphorylates JunB, a member of the AP-1 (activator protein 1) transcription factor family. Because JunB was suggested to be involved in DDR, we analysed the role of c-Abl-mediated phosphorylation of JunB in DDR. We first analysed phosphorylation sites of JunB and found that c-Abl majorly phosphorylates JunB at Tyr(173), Tyr(182) and Tyr(188). Because c-Abl promotes expression of the cyclin-dependent kinase inhibitor p21 upon stimulation with the DNA-damaging agent Adriamycin (doxorubicin), we analysed the involvement of JunB in Adriamycin-induced p21 expression. We found that JunB suppresses p21 induction through inhibition of its promoter activity. The phosphomimetic JunB, which was generated by glutamic acid substitutions at the phosphorylation sites, failed to repress p21 induction. Recruitment of JunB to the p21 promoter was promoted by Adriamycin stimulation and was further enhanced by co-treatment with the c-Abl inhibitor imatinib. The phosphomimetic glutamic acid substitutions in JunB or Adriamycin treatment impaired the JunB-c-Fos transcription factor complex formation. Taken together, these results suggest that, although JunB represses p21 promoter activity, c-Abl phosphorylates JunB and conversely inhibits its suppressive role on p21 promoter activity upon Adriamycin stimulation. Therefore JunB is likely to be a key target of c-Abl in expression of p21in Adriamycin-induced DDR.

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domain of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.

Fluorescent Calcium Indicator Protein Expression in the Brain Using Tetracycline-Responsive Transgenic Mice.

To achieve robust long-term fluorescent calcium indicator protein (FCIP) expression in mammalian neurons in vivo, classical mouse transgenesis by pronuclear DNA injection using tetracycline (Tet)-controlled genetic switches can be deployed. This protocol describes methods for regulated expression of FCIP using Tet-responsive transgenic mice. The Tet-inducible system requires three components for inducible and reversible control of gene expression: (1) a potent transcriptional activator protein, either Tet transactivator (tTA) or reverse tTA (rtTA); (2) a minimal Tet-promoter (P(tet)) or a bidirectional Tet-promoter (P(tet)bi) to express one or more responder genes; and (3) Tet or one of its derivatives such as doxycycline (Dox) as an inducer. To ensure a high level of FCIP expression in neurons, transgenic founder mice are screened using an ear fibroblast culture method to identify those that are responsive to Dox treatment before use in experiments. The protocol describes the use of Dox to regulate gene expression and provides a short description of in vivo recording of luciferase activity.

Identification of a gene cluster responsible for hydrogen evolution in Vibrio tritonius strain AM2 with transcriptional analyses

Vibrio tritonius strain AM2 shows high-yield hydrogen production even under saline conditions (1.7 mol hydrogen/mol mannitol). However, the molecular mechanism of efficient hydrogen production has never been studied in the genus Vibrio. The aim of this study is to identify the genes responsible for hydrogen evolution in V. tritonius and the gene expression pattern. Complete genome analysis revealed an existence of a single 24-kb gene cluster containing 21 genes, which are essential for the formation of an energy-conserving formate hydrogen lyase (FHL) complex, to be more specific the vibrio FHL was structurally rather similar to the hyf (hydrogenase four) gene cluster found in Escherichia coli. Moreover, genes responsible to the formate dehydrogenase (FDH-H), fhlA-type transcriptional activator and hydrogenase maturation proteins (hyp) were also located downstream of the vibrio hyf gene cluster to form a “super-gene-set” of the FHL complex gene cluster. The vibrio gene for the large subunit of the FHL complex hyfG possessed typical motifs coordinating the [NiFe] center at the active site, which indicates the V. tritonius hydrogenase was able to be classified as a [NiFe]-hydrogenase. Furthermore, transcriptional analysis revealed that the expression level of the hyfG gene slightly increased upon pH decrease, which correlates to the pH-dependent hydrogen production of V. tritonius. Therefore, we can conclude that the FHL complex of V. tritonius is key enzyme in the hydrogen production under acidic conditions. Moreover hyfABCDEFGHIJ-hycI-hydN-fdhF and hyp genes could be co-transcribed respectively during the efficient hydrogen production state. Details of the gene cluster are discussed here.

A Novel Synthetic Compound, YH-1118, Inhibited LPS-Induced Inflammatory Response by Suppressing I��B Kinase/NF-��B Pathway in Raw 264.7 Cells

For the search of a potent first-in-class compound to inactivate macrophages responsible for inflammatory responses, in the present study, we investigated the anti-inflammatory effects of YH-1118, a novel synthetic compound, in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, Raw 264.7. YH-1118 inhibited LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression at both the protein and mRNA levels. The suppression of LPS-induced iNOS expression by YH-1118 was mediated via nuclear factor kappa B (NF-κB), but not activator protein-1 (AP-1) transcription factor. This was supported by the finding that YH-1118 attenuated the phosphorylation of inhibitor of κBα (IκBα) and nuclear translocation and DNA binding activity of NF-kappaB. Through the mechanisms that YH- 1118 inhibited the activation of IkappaB kinases (IKKs), upstream activators of NF-κB, or p38 MAPK, YH-1118 significantly suppressed LPS-induced production of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 (p < 0.05). In conclusion, our results suggest that YH-1118 inhibits LPS-induced inflammatory responses by blocking IKK and NF-κB activation in macrophages, and may be a therapeutic candidate for the treatment of various inflammatory diseases.

Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth.

BackgroundEndothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection.ResultsIn this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo.ConclusionsWe find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0069-6) contains supplementary material, which is available to authorized users.

Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.