Factor AP-1 Research Articles

Induction of Epstein-Barr Virus Oncoprotein LMP1 by Transcription Factors AP-2 and Early B Cell Factor.

Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-Jκ, NF-κB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1.

Dissecting the role of single nucleotide polymorphism of lymphotoxin beta gene during pig domestication using bioinformatic and experimental approaches

Studies of wild and laboratory animals have revealed a trade-off between reproductive success and immunity. Therefore, it is likely that domestication favored selection of individuals with high reproductive performance but low immunity. The low responsiveness of the immune system could become hereditary through fixation of genes with “unfavorable” mutations in populations. The objectives of this work are: 1) determination of frequencies of genotypes and alleles of the rs340283541 SNP in the gene for the lymphotoxin beta (LTB) cytokine in pigs of domestic breeds and wild boars; 2) investigation of the expression of LTB mRNA in minipigs with different genotypes, and 3) bioinformational analysis of the putative functional role of the SNP. The frequency of the GG genotype in the wild boar sample was significantly lower than in the pooled sample of domestic pigs. The LTB mRNA expression rate in the lymph node of minipigs with genotype GG tended to increase (p < 0.06) in comparison with carriers o allele A. The rs340283541 SNP occurs in a DNA motif highly conservative among 11 mammalian species; thus, it may be of functional significance. Context analysis shows that allele A has putative binding sites for transcription factors BRN-2 and AP-1, whereas allele G has binding sites for transcription factors RFX1, ISGF3 (site ISRE), and USF expressed in cells of the immune system. Thus, pig domestication was accompanied by an increase in the frequency of the GG genotype for the rs340283541 SNP, occurring in the 3’ region of the LTB gene. It is likely that the GG genotype is associated with elevated LTB mRNA expression in the lymph node tissue. This increase may be related to the formation of binding sites for RFX1, ISRE, and USF and/or disruption of binding sites for BRN-2 and AP-1. A linkage disequilibrium between rs340283541 and another functionally significant mutation in LTB is also conceivable.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

CREB, AP-1, ternary complex factors and MAP kinases connect transient receptor potential melastatin-3 (TRPM3) channel stimulation with increased c-Fos expression.

The rise in intracellular Ca(2+) stimulates the expression of the transcription factor c-Fos. Depending on the mode of entry of Ca(2+) into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca(2+) influx via activation of transient receptor potential melastatin-3 (TRPM3) channels with enhanced c-Fos expression. Transcription of c-Fos promoter/reporter genes that were integrated into the chromatin via lentiviral gene transfer was analysed in HEK293 cells overexpressing TRPM3. The transcriptional activation potential of c-Fos was measured using a GAL4-c-Fos fusion protein. The signalling pathway connecting TRPM3 stimulation with enhanced c-Fos expression requires the activation of MAP kinases. On the transcriptional level, three Ca(2+) -responsive elements, the cAMP-response element and the binding sites for the serum response factor (SRF) and AP-1, are essential for the TRPM3-mediated stimulation of the c-Fos promoter. Ternary complex factors are additionally involved in connecting TRPM3 stimulation with the up-regulation of c-Fos expression. Stimulation of TRPM3 channels also increases the transcriptional activation potential of c-Fos. Signalling molecules involved in connecting TRPM3 with the c-Fos gene are MAP kinases and the transcription factors CREB, SRF, AP-1 and ternary complex factors. As c-Fos constitutes, together with other basic region leucine zipper transcription factors, the AP-1 transcription factor complex, the results of this study explain TRPM3-induced activation of AP-1 and connects TRPM3 with the biological functions regulated by AP-1.

BRAFV600E Induces ABCB1/P-Glycoprotein Expression and Drug Resistance in B-Cells Via AP-1 Activation

A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation.Cell line models to study the mechanism of BRAFV600E in B cell leukemia do not exist. Therefore, we utilized the CLL-like B-cell line OSUCLL (PLoS One 2013, 8(10):e76607) to generate cells with doxycycline (dox)-inducible BRAFV600E expression to examine transcriptional and biochemical features induced by this mutation in B-cells. We observed that BRAFV600E induction enhanced proliferation and activation of MAPK signaling in these cells. Microarray using Affymetrix U133 Plus 2.0 GeneChips demonstrated that 235 genes were up-regulated at least 2-fold, and 227 genes were down-regulated at least 2-fold. Several were confirmed by real-time RT-PCR analysis, including ABCB1 (p<0.001, vehicle versus dox-treated). Increased expression of the ABCB1 gene product, P-glycoprotein (P-gp), was also confirmed by immunoblot. This increase resulted in enhanced P-gp function as determined by rhodamine exclusion assays (p<0.005), an effect that was significantly reversed by the P-gp inhibitor verapamil (p<0.005). As an additional demonstration of P-gp function, BRAFV600E induction via dox treatment resulted in a significant increase in resistance to the P-gp substrate vincristine, and the addition of verapamil significantly reduced vincristine resistance. Importantly, pharmacological inhibition of BRAFV600E and MEK, by vemurafenib and CI-1040 respectively, diminished BRAFV600E-induced MAPK pathway activation and enhancement of ABCB1/P-gp expression. To further understand the transcriptional mechanism of BRAFV600E-induced P-gp expression, we performed luciferase assays using a vector containing 1 kb of ABCB1 promoter driving a luciferase reporter, co-transfected into HEK293 cells with either an empty vector or a vector containing BRAFV600E. These assays showed that BRAFV600E expression enhanced luciferase activity (p<0.001), and this effect was down-regulated by vemurafenib and CI-1040 (p<0.001). The role of AP-1 has been reported in ABCB1 regulation. Thus, we conducted electrophoretic mobility shift assays in the BRAFV600E-transfected OSUCLL B-cells, with or without dox treatment, to identify AP-1 factor(s) involved in ABCB1 regulation by BRAFV600E. In these experiments, a supershift in the AP-1 complex was produced by an antibody to JunD in the presence of dox treatment, demonstrating that at least JunD activity is important in this mechanism.Based on these observations, we conclude that BRAFV600E activates AP-1 proteins including JunD to induce ABCB1/P-gp expression and drug resistance in B-cells. This study uncovers a new pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK may be beneficial in delaying disease progression and occurrence of resistance to drugs that are substrates of P-gp. DisclosuresByrd:Acerta Pharma BV: Research Funding.

TRAF6 is required for BLyS-mediated NF-κB signaling in multiple myeloma cells.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the IL-1R/TLR and TNFR superfamilies to the transcription factors NF-κB and AP1. Elevated expression of the TNF family member B-lymphocyte stimulator (BLyS) in multiple myeloma (MM) has been described recently. However, the precise process by which BLyS signals in myeloma cell remains unknown. Here, we identified increased expression of TRAF6 in MM patient cells and the MM cell lines U266, RPMI8226, and KM3. Furthermore, rhBLyS induced TRAF6 up-regulation in these cells in a dose-dependent manner. Both the classical and alternative NF-κB pathways were activated by rhBLyS treatment. Depletion of TRAF6 by siRNA decreased levels of p-p65 and p-p100, even after stimulation with rhBLyS. Down-regulation of TRAF6 also abrogated rhBLyS-mediated cell viability. These findings suggest that TRAF6 is required for BLyS-mediated NF-κB signaling in myeloma cells and is a potential molecular therapeutic target in MM.

Abstract P612: TLR3-Induced Placental miR-155 Contributes to Preeclampsia by Augmenting Inflammation

Hypertensive disorders of pregnancy including preeclampsia (PE) affect ~15% of all pregnancies. We have previously demonstrated that excessive maternal immune system activation via Toll-like receptor 3 (TLR3) contributes to the development of PE-like symptoms such as hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant. Recently, increased miR-155 (an inflammation-related miRNA) expression was reported in placentas from women with PE compared to normotensive women suggesting a pathophysiological role of miR-155 in PE. Whether miRNAs play a role in the etiology of PE by augmenting inflammation is unknown. We confirmed miR-155 expression was up-regulated in placentas of poly I:C (a TLR3 agonist) treated wild type mice by microarray (P-PIC: 1.56 fold, p&lt;0.5 vs controls) and qRT-PCR analyses (P-PIC: 3.15 fold, p&lt;0.05 vs. controls). Based on our data we hypothesized that TLR3 activation induces placental miR-155 expression that augments maternal inflammation leading to PE. Transcription factors NF-κB and AP-1 which are known to bind to the promoter of miR-155 were increased in placentas of P-PIC mice. TargetScan, a target identification algorithm predicted suppressor of cytokine signaling 1 (SOCS1) as a putative target of miR-155. SOCS1 prevents overactivation of immune responses but failed to increase in P-PIC mice. ICAM, a marker of inflammation increased significantly in P-PIC mice. P-PIC TLR3 KO mice did not develop hypertension and proteinuria as well as did not exhibit increased placental levels of NF-κB, AP-1, miR-155 expression and ICAM or decreased levels of SOCS1. To determine the placental etiology, human cytotrphoblasts (CTBs) were treated with poly I:C and NF-κB, AP-1 transcription factor levels and miR-155 expression were increased significantly while SOCS1 levels decreased. Together, these data suggest that TLR3 activation increases placental miR-155 expression which downregulates SOCS1 levels and increases inflammation which may contribute to the development of PE.

Abstract 2061: CHIP functions as tumor suppressor in prostate cancer through suppression of ROS-inflammasome in a chaperone-dependent mechanism

Abstract Prostate Cancer (PCa) is a disease closely associated with aging. Normal aging is driven by oxidative stress, resulting in accumulation of markers of protein and lipid/phospholipid damage with subsequent activation of inflammatory/oncogenic transcription factors NFκB and AP-1. Indeed, the tumor milieu characterized by ROS signaling, and NFκB- and AP1-activation is thought to be important in fueling cancer progression and metastasis. CHIP is a U-box-type E3-ubiquitin ligase that exerts a protective role against oxidative stress by targeting damaged proteins for proteasomal degradation to maintain tissue homeostasis. CHIP induces ubiquitylation and degradation of substrates, including oncogenes and oxidatively damaged, and misfolded proteins. We hypothesized that CHIP suppresses prostate tumor progression by inhibiting ROS generation and downstream oncogenic pathways. CHIP depletion in PCa cells significantly increased their anchorage-independent growth, migration and invasiveness. PCa cells depleted of CHIP exhibited increased ROS generation (H2O2) and expression of markers of oxidative stress 8-isoprostane, NOQ1, NNMT and sulfiredoxin with subsequent activation NFκB and AP-1. The increased activation of NFκB and AP-1 in CHIP-depleted cells was ROS-induced as it was inhibited by N-acetyl cysteine (NAC) or knockdown of NOQ1. Pharmacologic inhibition of Hsp90 inhibited basal and ROS-induced NFκB and AP-1 activation as well as their invasive phenotype suggesting that the effect of CHIP is in part chaperone-dependent. Furthermore, in subcutaneous and orthotopic xenografts, PCa cells depleted of CHIP exhibited significantly accelerated growth compared to controls. Molecular characterization of CHIP-depleted tumors revealed significantly increased proliferation, increased oxidative stress and inflammation with increased nuclear localization of NFκB and AP-1. Collectively, our data suggest a novel tumor suppressor role of CHIP in PCa mediated through inhibition of ROS-induced inflammation. Citation Format: Neveen A. Said, James Larner. CHIP functions as tumor suppressor in prostate cancer through suppression of ROS-inflammasome in a chaperone-dependent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2061. doi:10.1158/1538-7445.AM2015-2061

Abstract 169: c-fos and Caspase 8 Mediate Palmitate-induced Cardiomyocyte Apoptosis

Background: We previously demonstrated that p38α activation mediates palmitate (PA)-induced cardiomyocyte apoptosis, and that specific p38α siRNA markedly and dose-dependently attenuates apoptosis rates in the presence of PA. We tested the hypothesis that c-fos and caspase 8 would be up-regulated concurrently with p38a. Methods and Results: Immortalized human adult ventricular cardiomyocytes ( AC16 cells) were exposed to high physiological levels of PA. The p38α-dependent pathway was evaluated using p38α siRNA knockdown. PA dose-dependently increased transcription of an AP-1 factor, c-fos, in AC16 cardiomyocytes (control:0.34±0.11, 150 μM PA:0.83±0.19, 300 μM PA:1.58±0.45, n=5, p&lt;0.05). c-fos protein expression increased dose-dependently with PA treatment (control:0±0%, 150 μM PA:26%±28%, 300 μM PA:258%±103%, n=4, p&lt;0.05). Phospho-c-fos levels increased dose-dependently as well (control:0±0%, 150 μM PA:73%±41%, 300 μM PA:302%±62%, n=4, p&lt;0.05). Transcription of a putative downstream effector, caspase 8, also increased (control:0.94±0.10, 150 μM PA:1.40±0.10, 300 μM PA:1.42±0.11, n=3, p&lt;0.05) but protein levels did not. p38α knockdown dose-dependently blocked the PA-induced increase in phospho-c-fos protein level (control siRNA:0±0%, 300 μM PA:206±47%, 300 μM PA+30 pmol siRNA:98±31%, 300 μM PA+60 pmol siRNA:58±40%, 300 μM PA+120 pmol siRNA:-21±19%, n=4, p&lt;0.05), without a clear reduction in total c-fos. Interestingly, caspase 8 level was also dose-dependently reduced by p38α knockdown (control siRNA:0±0%, 300 μM PA:-25±3%, 300 μM PA+30 pmol p38α siRNA:-38±13%, 300 μM PA+60 pmol p38α siRNA:-54±7%, 300 μM PA+120 pmol p38α siRNA:-46±11%, n=3, p&lt;0.05). Conclusions: This study in cardiomyocytes demonstrated that 1) PA dose-dependently increases gene expression of c-fos and protein levels of total c-fos and phospho-c-fos, 2) PA increases transcription of caspase 8, and 3) reduced p38α expression dose-dependently attenuates PA-induced increases in c-fos and phospho-c-fos protein levels, and decreases caspase 8. Our results suggest that p38α activated by PA up-regulates the downstream transcription factor, c-fos, and maintains caspase 8 level. These molecules may underlie PA-induced cardiomyocyte apoptosis.

Inflammation in Parkinson's disease: role of glucocorticoids.

Chronic inflammation is a major characteristic feature of Parkinson’s disease (PD). Studies in PD patients show evidence of augmented levels of potent pro-inflammatory molecules e.g., TNF-α, iNOS, IL-1β whereas in experimental Parkinsonism it has been consistently demonstrated that dopaminergic neurons are particularly vulnerable to activated glia releasing these toxic factors. Recent genetic studies point to the role of immune system in the etiology of PD, thus in combination with environmental factors, both peripheral and CNS-mediated immune responses could play important roles in onset and progression of PD. Whereas microglia, astrocytes and infiltrating T cells are known to mediate chronic inflammation, the roles of other immune-competent cells are less well understood. Inflammation is a tightly controlled process. One major effector system of regulation is HPA axis. Glucocorticoids (GCs) released from adrenal glands upon stimulation of HPA axis, in response to either cell injury or presence of pathogen, activate their receptor, GR. GR regulates inflammation both through direct transcriptional action on target genes and by indirectly inhibiting transcriptional activities of transcriptional factors such as NF-κB, AP-1 or interferon regulatory factors. In PD patients, the HPA axis is unbalanced and the cortisol levels are significantly increased, implying a deregulation of GR function in immune cells. In experimental Parkinsonism, the activation of microglial GR has a crucial effect in diminishing microglial cell activation and reducing dopaminergic degeneration. Moreover, GCs are also known to regulate human brain vasculature as well as blood brain barrier (BBB) permeability, any dysfunction in their actions may influence infiltration of cytotoxic molecules resulting in increased vulnerability of dopamine neurons in PD. Overall, deregulation of glucocorticoid receptor actions is likely important in dopamine neuron degeneration through establishment of chronic inflammation.

STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer.

BackgroundTransient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.ResultsWe show that during the transformation process, nucleosome-depleted regions (defined by formaldehyde-assisted isolation of regulatory elements (FAIRE)) are largely unchanged and that both before and during transformation, STAT3 binds almost exclusively to previously open chromatin regions. Roughly, a third of the transformation-inducible genes require STAT3 for the induction. STAT3 and NF-κB appear to drive the regulation of different gene sets during the transformation process. Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity. Lastly, many STAT3 binding sites are also bound by FOS and the expression of several AP-1 factors is altered during transformation in a STAT3-dependent manner, suggesting that STAT3 may cooperate with AP-1 proteins.ConclusionsThese observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch. In addition, gene expression changes during transformation, whether driven by pre-existing or induced transcription factors, occur largely through pre-existing nucleosome-depleted regions.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-8935-8-7) contains supplementary material, which is available to authorized users.

Water extract of Magnolia officinalis cortex inhibits osteoclastogenesis and bone resorption by downregulation of nuclear factor of activated T cells cytoplasmic 1

BackgroundMagnolia officinalis cortex has been traditionally used to treat stomach and intestine diseases in traditional Korean medicine. In this study, we investigated the effect of water extract of M. officinalis cortex (WEMC) on osteoclast differentiation and function. MethodsPhytochemical characterization of WEMC was performed by high-performance liquid chromatography analysis. Osteoclast differentiation of bone marrow-derived macrophages was determined by tartrate-resistant acid phosphatase activity assay. Receptor activator of nuclear factor-κB ligand (RANKL) signaling factors and transcription factors regulating osteoclast differentiation were analyzed by Western blot and real-time polymerase chain reaction. Bone resorption function of mature osteoclasts was examined by using culture plate coated with inorganic crystalline calcium phosphate. Furthermore, the in vivo effect of WEMC on osteoporosis was examined using RANKL-induced bone loss model, characterized by micro-computed tomography and bone metabolism marker analysis. ResultsWEMC inhibited RANKL-induced osteoclast differentiation and the bone resorbing activity of mature osteoclasts. WEMC contains gallic acid and honokiol as active constituents contributing to the inhibitory effect of WEMC on osteoclast differentiation. Further, WEMC suppressed RANKL-induced activation of p38 and nuclear factor-κB pathways and expression of osteoclastogenic transcription factors such as c-Fos for AP-1 and nuclear factor of activated T cells cytoplasmic 1. Ectopic overexpression of a constitutive active form of nuclear factor of activated T cells cytoplasmic 1 rescued the antiosteoclastogenic effect of WEMC. Consistent with the in vitro results, WEMC suppressed RANKL-induced trabecular bone loss in mice. ConclusionWEMC might have a therapeutic potential to treat pathological bone diseases due to increased osteoclast differentiation and function.

Suppressing AP1 Factor Signaling in the Suprabasal Epidermis Produces a Keratoderma Phenotype

Keratodermas comprise a heterogeneous group of highly debilitating and painful disorders characterized by thickening of the skin with marked hyperkeratosis. Some of these diseases are caused by genetic mutation, whereas other forms are acquired in response to environmental factors. Our understanding of signaling changes that underlie these diseases is limited. In the present study, we describe a keratoderma phenotype in mice in response to suprabasal epidermis-specific inhibition of activator protein 1 transcription factor signaling. These mice develop a severe phenotype characterized by hyperplasia, hyperkeratosis, parakeratosis, and impaired epidermal barrier function. The skin is scaled, constricting bands encircle the tail and digits, the footpads are thickened and scaled, and loricrin staining is markedly reduced in the cornified layers and increased in the nucleus. Features of this phenotype, including nuclear loricrin localization and pseudoainhum (autoamputation), are characteristic of the Vohwinkel syndrome. We confirm that the phenotype develops in a loricrin-null genetic background, indicating that suppressed suprabasal AP1 factor function is sufficient to drive this disease. We also show that the phenotype regresses when suprabasal AP1 factor signaling is restored. Our findings suggest that suppression of AP1 factor signaling in the suprabasal epidermis is a key event in the pathogenesis of keratoderma.

Genetic deletion of Wdr13 improves the metabolic phenotype of Lepr (db/db) mice by modulating AP1 and PPARγ target genes.

Type 2 diabetes is a complex disease characterised by hyperglycaemia, hyperinsulinaemia, dyslipidaemia and insulin resistance accompanied by inflammation. Previously, we showed that mice lacking the Wdr13 gene had increased islet mass due to enhanced beta cell proliferation. We hypothesised that introgression of a Wdr13-null mutation, a beta cell-proliferative phenotype, into Lepr(db/db) mice, a beta cell-destructive phenotype, might rescue the diabetic phenotype of the latter. Wdr13-deficient mice were crossed with Lepr(db/db) mice to generate mice with the double mutation. We measured various serum metabolic variables of Wdr13(+/0)Lepr(db/db) and Wdr13(-/0) Lepr(db/db) mice. Further, we analysed the histopathology and gene expression of peroxisome proliferator-activated receptor (PPAR)γ and, activator protein (AP)1 targets in various metabolic tissues. Lepr(db/db) mice with the Wdr13 deletion had a massively increased islet mass, hyperinsulinaemia and adipocyte hypertrophy. The increase in beta cell mass in Wdr13(-/0)Lepr(db/db) mice was due to an increase in beta cell proliferation. Hypertrophy of adipocytes may be the result of increase in transcription of Pparg and its target genes, leading in turn to increased expression of several lipogenic genes. We also observed a significant decrease in the expression of AP1 and nuclear factor κ light chain enhancer of activated B cells (NFκB) target genes involved in inflammation. This study provides evidence that loss of WD repeat domain 13 (WDR13) protein in the Lepr (db/db) mouse model of diabetes is beneficial. Based on these findings, we suggest that WDR13 may be a potential drug target for ameliorating hyperglycaemia and inflammation in diabetic conditions.

NF-κB factors control the induction of NFATc1 in B lymphocytes.

In peripheral lymphocytes, the transcription factors (TFs) NF-κB, NFAT, and AP-1 are the prime targets of signals that emerge from immune receptors. Upon activation, these TFs induce gene networks that orchestrate the growth, expansion, and effector function of peripheral lymphocytes. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA-binding domain, and there is a subgroup of κB-like DNA promoter motifs that are bound by both types of TFs. However, unlike NFAT and AP-1 factors that interact and collaborate in binding to DNA, NFAT, and NF-κB seem neither to interact nor to collaborate. We show here that NF-κB1/p50 and c-Rel, the most prominent NF-κB proteins in BCR-induced splenic B cells, control the induction of NFATc1/αA, a prominent short NFATc1 isoform. In part, this is mediated through two composite κB/NFAT-binding sites in the inducible Nfatc1 P1 promoter that directs the induction of NFATc1/αA by BCR signals. In concert with coreceptor signals that induce NF-κB factors, BCR signaling induces a persistent generation of NFATc1/αA. These data suggest a tight connection between NFATc1 and NF-κB induction in B lymphocytes contributing to the effector function of peripheral B cells.

Anti-diabetic rosiglitazone remodels the adipocyte transcriptome by redistributing transcription to PPARγ-driven enhancers.

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.

Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380bp in length and consisted of five exons and four introns. The 5′-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection.

Oxidative Stress and Inflammation Modulate Rev-erbα Signaling in the Neonatal Lung and Affect Circadian Rhythmicity

The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erbα, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model. A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFκB) consensus sequences was identified on the mouse Rev-erbα promoter. This sequence mediates Rev-erbα promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erbα, as well as an NFκB-binding site to repress Rev-erbα with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erbα was altered by pre-exposure to hyperoxia or disruption of NFκB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFκB binding on Rev-erbα transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxia-induced lung Rev-erbα transcription was further increased with NFκB disruption. Interestingly, this effect was not observed in similarly exposed adult mice. These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung. Rev-erbα transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erbα's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress.

Lysine Acetyltransferase GCN5b Interacts with AP2 Factors and Is Required for Toxoplasma gondii Proliferation

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

Deregulation of RNAi silencing pathway by Human T-cell leukemia virus type 1 bZIP factor (HBZ)

Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of a non-Hodgkin leukemia known as adult T cell leukemia (ATL). MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate the expression of target genes at posttranscriptional level. Biogenesis of miRNAs is tightly regulated and starts with the production of pri-miRNAs by RNA polymerase II. The pri-miRNAs are then cleaved by Drosha in conjunction with DGCR8 in the nucleus, generating pre-miRNAs, which are then transferred to the cytoplasm where Dicer1 further processes the hairpin into the mature miRNAs. With exception of few types of neoplasm, a general downregulation of miRNAs expression has been observed in cancers cells, mainly due to chromosomal abnormalities, epigenetic changes or aberrant expression of the miRNA biogenesis factors. Involvement of miRNAs alteration in the HTLV-1 life cycle has recently come to light. However, the mechanisms that regulate the expression of miRNAs biogenesis factors in ATL cells are still largely unknown. In this study, we assessed Dicer1 expression levels in ATL patients and investigated its transcriptional regulation in HTLV infected cell lines. We reported a significant correlation between reduced miRNA biogenesis and expression of HTLV-1 basic leucine zipper factor (HBZ). We showed that Dicer1 expression was downregulated in ATL patients vs. asymptomatic patients by qRT-PCR and western blot analysis. We also demonstrated that Dicer1 was regulated by AP-1 factors, through luciferase and Chip assays. Our data suggest that Dicer1 expression may be used as a promising prognostic marker and therapeutic target for ATL.