A regulatory network for coordinated flower maturation.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.

A mutant cell line partially responsive to both IFN-a and IFN-g

A recessive mutant cell line, B7, which is partially responsive to both interferon (IFN)- a and IFN-g is described. B7 was FACS sorted from a cellular pool, which was obtained from the parental cell line 2C4, after several rounds of mutagenesis. The partial responsiveness to IFN was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (9-27; 6-16; 2'-5' OAS; GBP and HLA-DRa). A genetic cross with the U4 mutant (JAK1-, a member of the Janus family of nonreceptor tyrosine kinase) did not restore full IFN-responsiveness to B7, and JAK1 cDNA transfection into B7 restored the wild phenotype of the cell line, defining B7 as a member of the U4 complementation group. Nevertheless, JAK1 mRNA was not detected in this mutant. Transcriptional regulator complexes such as IRF1/2 (IFN-regulatory factor) and ISGF3-g (IFN-stimulated gene factor) were constitutively formed in the B7 mutant and co-migrated with the IFN-induced complexes expressed in the parental cell line 2C4. Thus, this cell line seems to be useful for understanding cis-acting elements governing JAK1 mRNA expression.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

Herpes simplex virus 1 α regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a promiscuous transactivator shown to enhance the expression of gene introduced into cells by infection or transfection. At the molecular level, ICP0 is a 775-aa ring finger protein localized initially in the nucleus and late in infection in the cytoplasm and mediates the degradation of several proteins and stabilization of others. None of the known functions at the molecular level account for the apparent activity of ICP0 as a transactivator. Here we report that ICP0 functionally interacts with cellular transcription factor BMAL1, a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) super family of transcriptional regulators. Specifically, sequences mapped to the exon II of ICP0 interacted with BMAL1 in the yeast two-hybrid system and in reciprocal pull-down experiments in vitro. Moreover, the enhancement of transcription of a luciferase reporter construct whose promoter contained multiple BMAL1-binding sites by ICP0 and BMAL1 was significantly greater than that observed by ICP0 or BMAL1 alone. Although the level of BMAL1 present in nuclei of infected cells remained unchanged between 3 and 8 h after infection, the level of cytoplasmic BMAL1 was reduced at 8 h after infection. The reduction of cytoplasmic BMAL1 was significantly greater in cells infected with the ICP0-null mutant than in the wild-type virus-infected cells, suggesting that ICP0 mediates partial stabilization of the protein. These results indicate that ICP0 interacts physically and functionally with at least one cellular transcription-regulatory factor.

Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.

Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.

Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins. responsible for this inhibition.

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation.

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

The systemic lupus erythematosus IRF5 risk haplotype is associated with systemic sclerosis

Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10(-8), OR  = 1.22, CI 95%  = 1.14-1.30; rs2004640: P  = 4.60×10(-7), OR  = 0.84, CI 95%  = 0.78-0.90; rs10488631: P  = 7.53×10(-20), OR  = 1.63, CI 95%  = 1.47-1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10(-22), OR  = 1.75, CI 95%  = 1.56-1.97) better explained the observed association (likelihood P-value  = 1.48×10(-4)), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.

Whole-rat protein content estimation: applicability of the Nx6.25 factor

The amino acid composition of the protein from three strains of rat (Wistar, Zucker lean and Zucker obese), subjected to reference and high-fat diets has been used to determine the mean empirical formula, molecular weight and N content of whole-rat protein. The combined whole protein of the rat was uniform for the six experimental groups, containing an estimate of 17.3% N and a mean aminoacyl residue molecular weight of 103.7. This suggests that the appropriate protein factor for the calculation of rat protein from its N content should be 5.77 instead of the classical 6.25. In addition, an estimate of the size of the non-protein N mass in the whole rat gave a figure in the range of 5.5 % of all N. The combination of the two calculations gives a protein factor of 5.5 for the conversion of total N into rat protein.

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED: Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in W64;15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity. (Hepatology 2015;62:1375-1387).

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Quantitative expression of myogenic regulatory factors MyoD and myogenin in pacu (Piaractus mesopotamicus) skeletal muscle during growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)