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Protein Inhibitor of Activated STAT 1 (PIAS1) Is Identified as the SUMO E3 Ligase of CCAAT/Enhancer-Binding Protein β (C/EBPβ) during Adipogenesis
Liu, Yang
Zhang, Ya-Dong
Guo, Liang
Huang, Hai-Yan
Zhu, Hao
Huang, Jia-Xin
Liu, Yuan
Zhou, Shui-Rong
Dang, Yong-Jun
Li, Xi
Tang, Qi-Qun
Molecular and Cellular Biology
It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.
Related Articles
Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP*
Prigge, Justin R.
Schmidt, Edward E.
The Journal of biological chemistry
Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors.
Related Articles
NF-κB Repression by PIAS3 Mediated RelA SUMOylation
Liu, Yuangang
Bridges, Rebecca
Wortham, Aaron
Kulesz-Martin, Molly
PLoS ONE
Negative regulation of the NF-κB transcription factor is essential for tissue homeostasis in response to stress and inflammation. NF-κB activity is regulated by a variety of biochemical mechanisms including phosphorylation, acetylation, and ubiquitination. In this study, we provide the first experimental evidence that NF-κB is regulated by SUMOylation, where the RelA subunit of NF-κB is SUMOylated by PIAS3, a member of the PIAS (protein inhibitor of activated STAT) protein family with E3 SUMO ligase activity. PIAS3-mediated NF-κB repression was compromised by either RelA mutant resistant to SUMOylation or PIAS3 mutant defective in SUMOylation. PIAS3-mediated SUMOylation of endogenous RelA was induced by NF-κB activation thus forming a negative regulatory loop. The SUMOylation of endogenous RelA was enhanced in IκBα null as compared with wild type fibroblasts. The RelA SUMOylation was induced by TNFα but not leptomycin B mediated RelA nuclear translocation. Furthermore, RelA mutants defective in DNA binding were not SUMOylated by PIAS3, suggesting that RelA DNA binding is a signal for PIAS3-mediated SUMOylation. These results support a novel negative feedback mechanism for NF-κB regulation by PIAS3-mediated RelA SUMOylation.
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PIAS1 Mediates TGFβ-Induced SM α-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation
Kawai-Kowase, Keiko
Ohshima, Takayuki
Matsui, Hiroki
Tanaka, Toru
Shimizu, Takehisa
Iso, Tatsuya
Arai, Masashi
Owens, Gary K.
Kurabayashi, Masahiko
Arteriosclerosis, Thrombosis, and Vascular Biology
TGFβ and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) α-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFβ activates SM α-actin through PIAS1.
Methods and Results
An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFβ-induced expression of SM α-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM α-actin promoter activity in a TGFβ control element (TCE)-dependent manner. Because the TCE within the SM α-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmuno-precipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM α-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation.
These results provide evidence that PIAS1 promotes TGFβ-induced activation of SM α-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.
transforming growth factor β; protein inhibitor of activated STAT1; vascular smooth muscle cells; Krüppel-like factor
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Association and nuclear translocation of PIAS3-STAT3 complex is ligand and time dependent
Dabir, Snehal
Kluge, Amy
Dowlati, Afshin
Molecular cancer research : MCR
The Epidermal Growth Factor Receptor (EGFR) activation of downstream Signal Transducers and Activators of Transcription 3 (STAT3) plays a crucial role in the pathogenesis of lung cancer. STAT3 transcriptional activity can be negatively regulated by Protein Inhibitor of Activated STAT3 (PIAS3). We investigated PIAS3 time-dependent shuffling and binding to STAT3 in an EGF-dependent model in lung cancer by using confocal microscopy, immunoprecipitation, luciferase reporter assay and protein analysis of segregated cellular components. We also explored the role of phosphorylation at Tyr705 of STAT3 in the formation of PIAS3/STAT3 complex and intracellular shuffling. In a growth factor-free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. Upon exposure to EGF, we observed STAT3 phosphorylation and rapid formation of the PIAS3/STAT3 complex. Within 5 minutes there was a progressive translocation of the complex to the nucleus and by 10 minutes PIAS3 was uniquely localized to the nuclear compartment. Thirty minutes after, PIAS3 returned to the cytoplasm. Using site directed mutagenesis, we substituted Tyr705 of STAT3 with a phenylalanine. Despite EGF stimulation, we observed a significant decrease in PIAS3 and STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in PIAS3 capacity to reduce STAT3 mediated gene transcription. In wild type STAT3 cells, increasing concentrations of PIAS3 resulted in a proportional decrease in STAT3 phosphorylation. These data suggest an important role for the negative regulatory effect of PIAS3 on STAT3 in epidermal growth factor driven tumors.
PIAS3; STAT3; non small cell lung cancer
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Combinatorial Recruitment of CREB, C/EBPβ and c-Jun Determines Activation of Promoters upon Keratinocyte Differentiation
Rozenberg, Julian M.
Bhattacharya, Paramita
Chatterjee, Raghunath
Glass, Kimberly
Vinson, Charles
PLoS ONE
Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.
Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite "Half and Half" consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.
Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.
Related Articles
Protein Inhibitor of Activated STAT-3 Expression in Lung Cancer
Kluge, Amy
Dabir, Snehal
Vlassenbroeck, Ilse
Eisenberg, Rosana
Dowlati, Afshin
Molecular oncology
Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration-dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non-small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5-azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation-specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post-translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.
Related Articles
Modulation of inducible nitric oxide synthase expression by sumoylation
Akar, Candan A
Feinstein, Douglas L
Journal of Neuroinflammation
In astrocytes, the inflammatory induction of Nitric Oxide Synthase type 2 (NOS2) is inhibited by noradrenaline (NA) at the transcriptional level however its effects on specific transcription factors are not fully known. Recent studies show that the activity of several transcription factors including C/EBPβ, which is needed for maximal NOS2 expression, is modulated by conjugation of the small molecular weight protein SUMO. We examined whether the expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and the protease SENP1) are affected by inflammatory conditions or NA and whether SUMO-1 regulates NOS2 through interaction with C/EBPβ.
Bacterial endotoxin lipopolysaccharide (LPS) was used to induce inflammatory responses including NOS2 expression in primary astrocytes. The mRNA levels of SRGs were determined by QPCR. A functional role for SUMOylation was evaluated by determining effects of over-expressing SRGs on NOS2 promoter and NFκB binding-element reporter constructs. Interactions of SUMO-1 and C/EBPβ with the NOS2 promoter were examined by chromatin immunoprecipitation assays. Interactions of SUMO-1 with C/EBPβ were examined by immunoprecipitation and Western blot analysis and by fluorescence resonance energy transfer (FRET) assays.
LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary astrocytes and a similar decrease occurred during normal aging in brain. NA attenuated the LPS-induced reductions and increased SUMO-1 above basal levels. Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2 promoter, whereas activation of a 4 × NFκB binding-element reporter was only reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPβ with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA. SUMO-1 co-precipitated with C/EBPβ and a close proximity was confirmed by FRET analysis.
Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPβ as a possible mechanism of action. Targeting the SUMOylation pathway may therefore offer a novel means to regulate inflammatory NOS2 expression in neurological conditions and diseases.
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CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation
Ushijima, Takahiro
Okazaki, Ken
Tsushima, Hidetoshi
Ishihara, Kohei
Doi, Toshio
Iwamoto, Yukihide
PLoS ONE
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.
Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.
C/EBPβ and RUNX2 cooperatively stimulate expression of Ihh through direct interactions with a C/EBPβ binding element, which further promotes hypertrophic differentiation of chondrocytes during the chondrocyte differentiation process.
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Regulation of Basal and Induced Expression of C-Reactive Protein through an Overlapping Element for OCT-1 and NF-κB on the Proximal Promoter
Voleti, Bhavya
Agrawal, Alok
Journal of immunology (Baltimore, Md. : 1950)
C-reactive protein (CRP) is an acute phase protein produced by hepatocytes. A minor elevation in the baseline levels of serum CRP is considered an indicator of chronic inflammation. In hepatoma Hep3B cells, IL-6 induces CRP expression by activating transcription factors STAT3 and C/EBPβ. IL-1 synergistically enhances the effects of IL-6. The first 157 bp of the CRP promoter are sufficient for IL-1 synergy. Previously, NF-κB, a transcription factor activated by IL-1β in Hep3B cells, has been shown to increase endogenous CRP expression. The purpose of this study was to investigate the possible action of NF-κB on the 157 bp of the proximal promoter. In this study we show that NF-κB requires and acts synergistically with C/EBPβ on the CRP-proximal promoter to regulate CRP expression. We located the regulatory element that consisted of overlapping binding sites for NF-κB (p50-p50 and p50-p65) and OCT-1. The κB site was responsible for the synergy between NF-κB and C/EBPβ and was also necessary for the CRP transactivation by C/EBPβ through the C/EBP site. Mutation of the κB site decreased the synergistic effect of IL-1β on IL-6-induced CRP expression. Basal CRP expression increased dramatically when binding of both OCT-1 and NF-κB was abolished. Combined data from luciferase transactivation assays and EMSA lead us to conclude that the binding of OCT-1 to the promoter, facilitated by p50-p50 in a novel way, represses, whereas replacement of OCT-1 by p50-p65 induces CRP transcription in cooperation with C/EBPβ. This model for CRP expression favors the variation seen in baseline serum CRP levels in a normal healthy population.
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Regulation of Id2 expression by CCAAT/enhancer binding protein β
Karaya, Kazuhiro
Mori, Seiichi
Kimoto, Hisashi
Shima, Yoko
Tsuji, Yoshihito
Kurooka, Hisanori
Akira, Shizuo
Yokota, Yoshifumi
Nucleic Acids Research
Mice deficient for Id2, a negative regulator of basic helix–loop–helix (bHLH) transcription factors, exhibit a defect in lactation due to impaired lobuloalveolar development during pregnancy, similar to the mice lacking the CCAAT enhancer binding protein (C/EBP) β. Here, we show that Id2 is a direct target of C/EBPβ. Translocation of C/EBPβ into the nucleus, which was achieved by using a system utilizing the fusion protein between C/EBPβ and the ligand-binding domain of the human estrogen receptor (C/EBPβ-ERT), demonstrated the rapid induction of endogenous Id2 expression. In reporter assays, transactivation of the Id2 promoter by C/EBPβ was observed and, among three potential C/EBPβ binding sites found in the 2.3 kb Id2 promoter region, the most proximal element was responsible for the transactivation. Electrophoretic mobility shift assay (EMSA) identified this element as a core sequence to which C/EBPβ binds. Chromatin immunoprecipitation (ChIP) furthermore confirmed the presence of C/EBPβ in the Id2 promoter region. Northern blotting showed that Id2 expression in C/EBPβ-deficient mammary glands was reduced at 10 days post coitus (d.p.c.), compared with that in wild-type mammary glands. Thus, our data demonstrate that Id2 is a direct target of C/EBPβ and provide insight into molecular mechanisms underlying mammary gland development during pregnancy.
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PIAS3 Interacts with ATF1 and Regulates the Human Ferritin H Gene through an Antioxidant-responsive Element*s
Iwasaki, Kenta
Hailemariam, Kiros
Tsuji, Yoshiaki
The Journal of biological chemistry
Gene transcription is coordinately regulated by the balance between activation and repression mechanisms in response to various external stimuli. Ferritin, composed of H and L subunits, is the major intracellular iron storage protein involved in iron homeostasis. We previously identified an enhancer, termed antioxidant-responsive element (ARE), in the human ferritin H gene and its respective transcriptional activators including Nrf2 and JunD. Here we found that ATF1 (activating transcription factor 1) is a transcriptional repressor of the ferritin H ARE. Subsequent yeast two-hybrid screening identified PIAS3 (protein inhibitor of activated STAT3) as an ATF1-binding protein. Further investigation of the human ferritin H ARE regulation showed that 1) PIAS3 reversed ATF1-mediated repression of the ferritin H ARE; 2) ATF1 was sumoylated, but PIAS3, a SUMO E3 ligase, did not appear to play a major role in SUMO1-mediated ATF1 sumoylation or ATF1 transcription activating function; 3) PIAS3 decreased ATF1 binding to the ARE; and 4) ATF1 knockdown with siRNA increased ferritin H expression, whereas PIAS3 knockdown decreased basal expression and oxidative stress-mediated induction of ferritin H. These results suggest that PIAS3 antagonizes the repressor function of ATF1, at least in part by blocking its DNA binding, and ultimately activates the ARE. Collectively our results suggest that PIAS3 is a new regulator of ATF1 that regulates the ARE-mediated transcription of the ferritin H gene.
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Negative Regulation of NF-κB Signaling by PIAS1†
Liu, Bin
Yang, Randy
Wong, Kelly A.
Getman, Crescent
Stein, Natalie
Teitell, Michael A.
Cheng, Genhong
Wu, Hong
Shuai, Ke
Molecular and Cellular Biology
The NF-κB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-κB activity is critical, since abnormal NF-κB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-κB. Upon cytokine stimulation, the p65 subunit of NF-κB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-κB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-κB-regulated genes is significantly enhanced in Pias1−/− cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-κB-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-κB.
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Inhibition of Corticosteroid-Binding Globulin Gene Expression by Glucocorticoids Involves C/EBPβ
Verhoog, Nicolette
Allie-Reid, Fatima
Vanden Berghe, Wim
Smith, Carine
Haegeman, Guy
Hapgood, Janet
Louw, Ann
PLoS ONE
Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ's involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ.
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Pias3-dependent SUMOylation directs rod photoreceptor development
Onishi, Akishi
Peng, Guang-Hua
Hsu, Chengda
Alexis, Uel
Chen, Shiming
Blackshaw, Seth
The vertebrate retina is a tractable system in which to address the question of neuronal cell fate specification. Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 directly interacts with the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identifies Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.
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Protein Inhibitor of Activated STAT1 Interacts with and Up-regulates Activities of the Pro-proliferative Transcription Factor Krüppel-like Factor 5*
Du, James X.
Yun, C. Chris
Bialkowska, Agnieszka
Yang, Vincent W.
The Journal of biological chemistry
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates proliferation of various cell types, including fibroblasts, smooth muscle cells, and intestinal epithelial cells. To identify proteins that interact with KLF5, we performed a yeast two-hybrid screen of a 17-day mouse embryo cDNA library with KLF5 as bait. The screen revealed 21 preys clustered in four groups as follows: proteins mediating gene expression, metabolism, trafficking, and signaling. Among them was protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) ligase that regulates transcription factors through SUMOylation or physical interaction. Association between PIAS1 and KLF5 was verified by co-immunoprecipitation. Structural determination showed that the acidic domain of PIAS1 bound to both the amino- and carboxyl-terminal regions of KLF5 and that this interaction was inhibited by the amino terminus of PIAS1. Indirect immunofluorescence demonstrated that PIAS1 and KLF5 co-localized to the nucleus. Furthermore, the PIAS1-KLF5 complex was co-localized with the TATA-binding protein and was enriched in RNA polymerase II foci. Transient transfection of COS-7 cells by PIAS1 and KLF5 significantly increased the steady-state protein levels of each other. Luciferase reporter and chromatin immunoprecipitation assays showed that PIAS1 significantly activated the promoters of KLF5 and PIAS1 and synergistically increased the transcriptional activity of KLF5 in activating the cyclin D1 and Cdc2 promoters. Importantly, PIAS1 increased the ability of KLF5 to enhance cell proliferation in transfected cells. These results indicate that PIAS1 is a functional partner of KLF5 and enhances the ability of KLF5 to promote proliferation.
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CCAAT/Enhancer-binding Protein β Inhibits Proliferation in Monocytic Cells by Affecting the Retinoblastoma Protein/E2F/Cyclin E Pathway but Is Not Directly Required for Macrophage Morphology*
Gutsch, Romina
Kandemir, Judith D.
Pietsch, Daniel
Cappello, Christian
Meyer, Johann
Simanowski, Kathrin
Huber, René
Brand, Korbinian
The Journal of Biological Chemistry
Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβWT macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβKO. The typical macrophage morphology was only observed in C/EBPβWT, whereas C/EBPβKO stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβKO macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβWT cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.
C/EBP Transcription Factor; Cell Differentiation; E2F Transcription Factor; Macrophage; Retinoblastoma (Rb); C/EBP beta; Cyclin E; Monocyte; Morphology; Proliferation
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Type I Interferon Induces Inhibitory 16-kD CCAAT/ Enhancer Binding Protein (C/EBP)β, Repressing the HIV-1 Long Terminal Repeat in Macrophages: Pulmonary Tuberculosis Alters C/EBP Expression, Enhancing HIV-1 Replication
Honda, Yoshihiro
Rogers, Linda
Nakata, Koh
Zhao, Ben-Yang
Pine, Richard
Nakai, Yushi
Kurosu, Katsushi
Rom, William N.
Weiden, Michael
The Journal of Experimental Medicine
We have previously observed that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro THP-1 cell–derived macrophages inhibited HIV-1 replication after infection with Mycobacterium tuberculosis. Suppression of HIV-1 replication was associated with inhibition of the HIV-1 long terminal repeat (LTR) and induction of ISGF-3, a type I interferon (IFN)–specific transcription factor. Repression of the HIV-1 LTR required intact CCAAT/enhancer binding protein (C/EBP) sites. THP-1 cell–derived macrophages infected with M. tuberculosis, lipopolysaccharide, or IFN-β induced the 16-kD inhibitory C/EBPβ isoform and coincidentally repressed HIV-1 LTR transcription. C/EBPβ was the predominant C/EBP family member produced in THP-1 macrophages during HIV-1 LTR repression. In vivo, alveolar macrophages from uninflamed lung strongly expressed inhibitory 16-kD C/EBPβ, but pulmonary tuberculosis abolished inhibitory C/EBPβ expression and induced a novel C/EBP DNA binding protein. Therefore, in vitro, proinflammatory stimulation produces an IFN response inhibiting viral replication by induction of a C/EBPβ transcriptional repressor. THP-1 cell–derived macrophages stimulated with type I IFN are similar to alveolar macrophages in the uninflamed lung in vivo. In contrast, the cellular immune response in active pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression and allowing high level viral replication.
interferon β; CCAAT/enhancer binding protein β; HIV-1 long terminal repeat; tuberculosis; repression
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A Novel RBP-Jκ-Dependent Switch from C/EBPβ to C/EBPζ at the C/EBP Binding Site on the C-Reactive Protein Promoter1
Singh, Prem Prakash
Voleti, Bhavya
Agrawal, Alok
Journal of immunology (Baltimore, Md. : 1950)
Regulation of basal and cytokine (IL-6 and IL-1β)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-κB p50 binding site. In response to IL-6, C/EBPβ and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPζ (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPζ. EMSA using this extract revealed formation of a C/EBPζ-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-Jκ, a transcription factor known to interact with κB sites. RBP-Jκ was required for the formation of C/EBPζ-containing complex. The RBP-Jκ-dependent C/EBPζ-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBPζ abolished both C/EBPβ-induced and (IL-6 + IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPζ occupies the C/EBP site, an action that requires RBP-Jκ. Under induced conditions, C/EBPζ is replaced by C/EBPβ and p50. We conclude that the switch between C/EBPβ and C/EBPζ participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-Jκ into C/EBPζ complexes solely to support the binding of C/EBPζ to the C/EBP site.
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Contribution of C/EBP Proteins to Epstein-Barr Virus Lytic Gene Expression and Replication in Epithelial Cells
Huang, Jian
Liao, Gangling
Chen, Honglin
Wu, Frederick Y.
Hutt-Fletcher, Lindsey
Hayward, Gary S.
Hayward, S. Diane
Journal of Virology
The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPβ and had limited C/EBPα expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPα and C/EBPβ. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPβ expression and a prolonged increase in C/EBPα expression. In AGS/BX1 cells, endogenous C/EBPα and C/EBPβ proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPα and C/EBPβ proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPβ and C/EBPα expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.
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Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2.
Rapp, B
Pawellek, A
Kraetzer, F
Schaefer, M
May, C
Purdie, K
Grassmann, K
Iftner, T
Journal of Virology
Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.
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Essential Role for the Major Autolysin in the Fibronectin-Binding Protein-Mediated Staphylococcus aureus Biofilm Phenotype▿
Houston, Patrick
Rowe, Sarah E.
Pozzi, Clarissa
Waters, Elaine M.
O'Gara, James P.
Infection and Immunity
Staphylococcus aureus clinical isolates are capable of producing at least two distinct types of biofilm mediated by the fibronectin-binding proteins (FnBPs) or the icaADBC-encoded polysaccharide intercellular adhesin (PIA). Deletion of the major autolysin gene atl reduced primary attachment rates and impaired FnBP-dependent biofilm production on hydrophilic polystyrene in 12 clinical methicillin-resistant S. aureus (MRSA) isolates but had no effect on PIA-dependent biofilm production by 9 methicillin-susceptible S. aureus (MSSA) isolates. In contrast, Atl was required for both FnBP- and PIA-mediated biofilm development on hydrophobic polystyrene. Here we investigated the role of Atl in biofilm production on hydrophilic polystyrene. The alternative sigma factor σB, which represses RNAIII expression and extracellular protease production, was required for FnBP- but not PIA-dependent biofilm development. Furthermore, mutation of the agr locus enhanced FnBP-dependent biofilm development, whereas a sarA mutation, which increases protease production, blocked FnBP-mediated biofilm development. Mutation of sigB in MRSA isolate BH1CC lowered primary attachment rates, in part via reduced atl transcription. Posttranslational activation or inhibition of Atl activity with phenylmethylsulfonyl fluoride and polyanethole sodium sulfonate or mutation of the Atl amidase active site interfered with lytic activity and biofilm development. Consistent with these observations, extracellular DNA was important for the early stages of Atl/FnBP-dependent biofilm development. Further analysis of atl regulation revealed that atlR encodes a transcriptional repressor of the major autolysin and that an atlR::Tcr mutation in BH1CC enhanced biofilm-forming capacity. These data reveal an essential role for the major autolysin in the early events of the FnBP-dependent S. aureus biofilm phenotype.
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Transcriptional activation of histone H4 by C/EBPβ during the mitotic clonal expansion of 3T3-L1 adipocyte differentiation
Zhang, You-You
Li, Xi
Qian, Shu-Wen
Guo, Liang
Huang, Hai-Yan
He, Qun
Liu, Yuan
Ma, Chun-Gu
Tang, Qi-Qun
Molecular Biology of the Cell
Histone H4 is activated by C/EBPβ in mitotic clonal expansion during adipogenesis. C/EBP-binding sites are identified in histone H4 promoters, and H4 expression is suppressed when C/EBPβ is knocked down or its DNA-binding activity is inhibited by A-C/EBP. These results help in our understanding of how C/EBPβ plays important roles in the proliferation of other cells.
CCAAT enhancer binding protein β (C/EBPβ) is required for both mitotic clonal expansion (MCE) and terminal differentiation during the 3T3-L1 adipocyte differentiation program. Whereas the mechanism of C/EBPβ during terminal differentiation is well understood, the mechanism of C/EBPβ in MCE is not. We provide evidence that histone H4, the most conserved cell cycle–related histone, the change of which is strictly correlated with DNA content change during the cell cycle, is transcriptionally activated by C/EBPβ during MCE. Expression of histone H4 is increased at 16 h after induction when 3T3-L1 preadipocytes synchronously reenter S phase, which is correlated with the sequential phosphorylation and activation of C/EBPβ, and expression was partially suppressed when A-C/EBP (dominant negative for C/EBP protein) was overexpressed. One C/EBP-binding site was identified in one of the histone H4 gene promoters (hist4h4), confirmed by both electrophoretic mobility shift assay and chromatin immunoprecipitation assay. C/EBP-binding sites were also found in 9 of 11 other histone H4 promoters, which can also be transactivated by C/EBPβ. Knockdown of C/EBPβ by stealth small interfering RNA partially decreased H4 gene expression and arrested cells in G1 phase as indicated by bromodeoxyuridine incorporation and fluorescence-activated cell sorting analysis of DNA content. This study provides new insights into why C/EBPβ is required for MCE during 3T3-L1 adipocyte differentiation and why C/EBPβ plays important roles in the proliferation of other cell types.
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Identification of a New Site of Sumoylation on Tel (ETV6) Uncovers a PIAS-Dependent Mode of Regulating Tel Function▿
Roukens, M. Guy
Alloul-Ramdhani, Mariam
Vertegaal, Alfred C. O.
Anvarian, Zeinab
Balog, Crina I. A.
Deelder, André M.
Hensbergen, Paul J.
Baker, David A.
Molecular and Cellular Biology
Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.
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The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells
Campion, Carole G.
Labrie, Marilyne
Grosset, Andrée-Anne
St-Pierre, Yves
PLoS ONE
Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.
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Fly Tyer
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Fly Tyer is an American magazine dedicated to the subject of fly tying, the art of tying materials to a hook for the purposes of fly fishing. Published four times a year, Fly Tyer is currently the largest fly-tying magazine in terms of circulation. It employs "perfect binding" instead of stapled pages, and usually features a close-up image of a fly on its cover.
The magazine is edited by David Klausmeyer, author of Unnaturals: A Practical Guide to Tying with Synthetics, Trout Streams of Northern New England and Tying Classic Freshwater Streamers: An Illustrated Step-by-Step Guide, among other works. Fly Tyer is an asset of Morris Communications, which also owns publications such as Gray's Sporting Journal, American Angler, and Saltwater Fly Fishing.
Fly Tyer was spun off of American Angler magazine in 1995, when it became clear that the market would support a fly-tying specific publication. Its success has proven the desire for fly tying content. Fly Tyer was also the original name of the publication that became American Angler.
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Alan Miller, 53, claims he is Jesus Christ and his wife, Mary Luck, is Mary Magdalene, because they have memories of their lives together from 2,000 years ago.
7/26/2016 Reads: 124 Submitted By: 0x6a656666 Source: www.dailymail.co.uk
Have you ever experienced a déjà vu? If so, I would like you to imagine how odd it would feel if you could clearly remember things that happened thousands of years before you were born. That is exactly what happened to Dorothy Louise Eady, a British Egyptologist who could vividly recollect her past life.
7/4/2016 Reads: 190 Submitted By: 0x6a656666 Source: www.sott.net
Was Daniel Radcliffe a stern old lady in his past life, or perhaps a soldier going off to war? On Reddit, people have sent in old photos of people (men and women) who look very much like Daniel Radcliffe. It's pretty funny how his face keeps coming up in old black and white pictures.
6/12/2016 Reads: 207 Submitted By: 0x6a656666 Source: sixseeds.patheos.com
You might not know it, but children remembering their past lives is nothing new. Reincarnation is no doubt a fascinating subject, even within the scientific community. Carl Sagan, American astronomer and astrobiologist, even acknowledged the fact that reincarnation deserves serious study, stating that "children sometimes report details of a previous life, which, upon checking, turn out to be accurate and which they could not have known about in any other way than reincarnation."
5/18/2016 Reads: 355 Submitted By: Ohiomstr2 Source: www.collective-evolution.com
AS CONSPIRACY theories go it is pretty crackpot, but some people actually believe the Russian president can time travel or is an immortal vampire.
4/27/2016 Reads: 360 Submitted By: 0x6a656666 Source: www.express.co.uk
Verifying the purported past-life memories of children against the records of people who have died is part of a day's work for Dr. Erlendur Haraldsson.
2/2/2016 Reads: 271 Submitted By: 0x6a656666 Source: www.theepochtimes.com
After a group of men pull the fish from a freezer, they drop it into a metal tank where it lays motionless for about a minute - before stirring its fins then gliding around the tank.
1/23/2016 Reads: 326 Submitted By: 0x6a656666 Source: www.dailymail.co.uk
In an effort to eradicate swindling fakers, China's State Administration for Religious Affairs published its first list of verified buddhas online on Monday.
1/20/2016 Reads: 316 Submitted By: 0x6a656666 Source: shanghaiist.com
New Delhi: Birthmarks that may occur anywhere on the skin are signs of complexities present at the time of birth. They can also develop soon after birth, usually in the first month. Some old wives tales and few scientists have claimed that birthmarks are roadmap to past lives. They indicate the deeply interwoven relationship of our past and present life.
1/19/2016 Reads: 478 Submitted By: 0x6a656666 Source: post.jagran.com
A YOUNG boy was just four when he first claimed to be the reincarnation of a girl who cooked food for the first US president, George Washington.
12/28/2015 Reads: 383 Submitted By: 0x6a656666 Source: www.couriermail.com.au
Reincarnation is not something that is often believed to exist in the West, but in many cultures around the world, this is something of a commonplace belief. A 3-year-old-boy from the Golan Heights region, which is near the border of Syria and Israel, shared with his parents that he was murdered with an ax in his past life.
6/22/2015 Reads: 532 Submitted By: 0x6a656666 Source: www.inquisitr.com
Since the age of 3, Carl Edon had spoken of a previous life as a Nazi airman. His parents didn't believe he was truly remembering a past life, but decades later evidence emerged that made them wonder. Stunning, isn't it? Do you think this is just a coincidence?
11/26/2014 Reads: 1087 Submitted By: dsuchin Source: www.theepochtimes.com
It might come as a surprise to many people, but Indians actually explained Darwin's theory of Evolution Thousands of years before Darwin! Evolutionary theory, when viewed from a certain perspective, neatly intersects with the Dashavatar or 10 incarnations of Lord Vishnu. Man's origins in the waters is evoked by the pisciform nature of Matsya. Then come the tortoise and the boar, taking us from amphibians to land animals. This is followed by a therianthropic form (Narasimha), and then homo sapiens proper, and so on. All this was recorded by Hindu sages thousands of years before Christ.
10/24/2014 Reads: 705 Submitted By: josan420 Source: www.mysteryofindia.com
This article explores a variety of eerie stories in which children seem to be able to recall past lives with uncanny detail.
5/11/2013 Reads: 1536 Submitted By: GhostDiaries Source: theghostdiaries.com
Two families in Transkei are fighting over a psychiatric patient they both say is their son following claims last year that the man had come back from the dead.
2/16/2010 Reads: 1028 Submitted By: Medieval Source: naturalplane.blogspot.com
A youth who was believed to have been stabbed to death and buried in 2005 has resurfaced in yet another mysterious return of a man from the dead in the Transkei, South Africa.
1/13/2010 Reads: 868 Submitted By: Medieval Source: naturalplane.blogspot.com
Miyuki Hatoyama, wife of Japan's newly elected Prime Minister, who has stunned the world with her comments about UFO abduction and her past-life relationship with Tom Cruise.
10/3/2009 Reads: 929 Submitted By: 0x6a656666 Source: naturalplane.blogspot.com
Stories that help prove the case for reincarnation are rare. The better stories seem to come from children - unique cases where it would be hard to understand how the child came by these memories on their own.
6/13/2009 Reads: 829 Submitted By: tremontavenue Source: www.examiner.com
BEIJING, August 16 - The Beijing-backed 11th Panchen Lama, Tibet's second highest spiritual figure whose credibility is questioned by many devoted Tibetan Buddhists, has vowed to maintain national and ethnic unity with China, state media reported Monday.
8/19/2004 Reads: 608 Submitted By: clayroy Source: www.phayul.com
This pleasant young man from Colorado in a short-sleeved white shirt has been identified by the Dalai Lama of Tibet as the latest of many reincarnations of a Buddhist saint who lived in northern India 400 or 500 years ago.
6/12/2004 Reads: 841 Submitted By: clayroy Source: www.nytimes.com


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It's a love story, but not like you think. There's no boy meets girl or star-crossed soul mates. Love is a billion-dollar industry. Cupid's the megalomaniac CEO. And Kali Monroe gets paid to break your heart. Love is all around us. These are the pricks who control it.
Marvel Assembles an Official Title for Third "Avengers" Movie
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Social Calendar
Holly Eve Gala - 30th annual event with entertainment, epicurean delights and silent and live auctions to benefit The Festival at Sandpoint, The Panida Theater, Pend Oreille Arts Council, Community Cancer Services and Bonner Community Hospice. Today, 6 p.m., Sandpoint Events Center, Sandpoint. $25, available at Eve's Leaves, Panhandle State Bank and The Festival at Sandpoint office. (208) 263-9191
Indo-Tibetan Benefit Dinner - Dinner prepared by a professional Tibetan chef, followed by a presentation about efforts to support Tibetan refugee children in India. Proceeds benefit the Tibetan Children's Education Fund. Co-sponsored by One Peace Many Paths and the Unitarian Universalist Church. Today, 6-8 p.m., Unitarian Universalist Church, 4340 W. Fort George Wright Drive. $30/person, $150/table of six. (509) 536-2811, firstname.lastname@example.com
Starlight Children's Foundation-Northwest Benefit - Make a minimum donation of $5 and enjoy free appetizers and drink specials. All proceeds to benefit seriously ill children and their families. Wednesday, 3-6 p.m., Applebee's South Hill, 2107 E. 29th Ave. (509) 321-0799
Festival of Trees - Kootenai Medical Center fundraiser. Friday, Friday delights, 5-8 p.m., $2/at the door. Saturday, Senior Social, 9-11 a.m., $20; Benefit Gala and Auction with master of ceremonies Grant Goodeve, host of Seattle-based outdoor travel show "Northwest Backroads" and best known for his role as David Bradford on "Eight is Enough," 5 p.m.-midnight, $200/person. Nov. 27, Family Day, children's workshop with performances from local youth and crafts and face painting, 11 a.m.-4 p.m., $2/at the door; children's workshop, 11 a.m.-3 p.m., $1/craft. Nov. 28, Luncheon Fashion Show, 11 a.m.-1:30 p.m., $50, and Dinner Fashion Show, 5-7:30 p.m., $55. Coeur d'Alene Resort Convention Center, 115 S. Second St., Coeur d'Alene. (208) 666-2345
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GarageBand is part of the applications which receive an update with the arrival of Apple Music. Both services are now interconnected.
The release of Apple Music on June 30 is accompanied by a series of updates in the Apple universe. Besides iOS and iTunes that provide streaming audio service , others like GarageBand also benefit from development.
The versions for OS X 10.1 and iOS 2.0.7 for GarageBand application recording and music creation Apple now lets you share items directly to Apple Music Connect, the "Social" tab of the new music online Apple .
This interconnection with Apple Music is not the only novelty of the update of GarageBand. The app is enriched with ten new drummers with different styles (house, trap, techno, dubstep …), registration of Smart Controls movements of the arrival of 1000 new loops, support the new touchpad Force touch …
The update is free via the App Store for those who already have GarageBand for pc and are otherwise in the latest version of the operating system OS X 10.9 (Yosemite) . The others will be charged 4.99 euros.


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Marvel Assembles an Official Title for Third "Avengers" Movie
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Jane Wiedlin (The Go-Go's) and Bill Morrison (Bongo Comics) blast off this July with the out-of-this-world space adventure "Lady Robotika" from Shadowline and Image Comics.
ROBOT 6's Tom Bondurant lets you know what he'll be doing in July, as he takes a look at DC's solicitations for "Atom Special," "Mighty Crusaders" and more.
Courtesy of DC Comics, CBR brings you an advance look at "Detective Comics" #864 by David Hine & Jeremy Haun and Greg Rucka & Cully Hamner with a cover by Cliff Chiang, on sale next week.
What's up with "Runaways?" How about Iron Fist and Norman Osborn? Marvel's philosophy about digital distribution? Joe Quesada answers your questions and more in CBR TV's exclusive CUP O' VIDEO Q&A!
Courtesy of Image Comics, CBR brings you an exclusive preview of "Random Acts of Violence," the new graphic novel by Jimmy Palmiotti & Justin Gray and Giancarlo Caracuzzo & Paul Mounts, on sale next week.
Some our most beloved fairy tales get a fresh perspective this July with the release of "Fractured Fables," an all-new anthology from Shadowline and Image Comics.
With Deadman's crucial role in "Brightest Day," Comic Book Legends Revealed spotlights turths and rumors about the late, great Arnold Drake, creator of Deadman and the Doom Patrol!
Even the Doctor couldn't help Karen Gillan avoid the fallout from the volcanic eruption in Iceland, giving the actress time to talk to Spinoff Online about "Doctor Who" and her visit to the U.S.
CBR's Chad Nevett says the new "DV8" is off to a promising start, with Brian Wood and Rebekah Isaacs honoring the WildStorm cult classic while giving new readers all they need to enjoy this edgy new series.
Courtesy of DC Comics, CBR brings you an advance look at "Justice Society of America" #38 by Bill Willingham and Jesus Merino & Jesse Delperdang, on sale April 28.
Courtesy of Marvel, CBR presents an exclusive look at "Deadpool" #23, the first issue of Daniel Way and Carlo Barberi's new storyline, "Tricky," featuring covers by Jason Pearson. The issue goes on sale Wednesday, May 19.
Courtesy of Marvel Comics, CBR brings you advance looks at new comics on sale next Wednesday, April 28, including "Captain America" #605, "Amazing Spider-Man" #629, "Invincible Iron Man" #25, and many more!
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Crerar Craiglynne Hotel
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
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- Breakfast Available
- Restaurant
- Room service
- Laundry
- Free parking
- Pets allowed
Photos of Crerar Craiglynne Hotel
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TripAdvisor Reviews Crerar Craiglynne Hotel Grantown-on-Spey
Travel Blogs from Grantown-on-Spey
... Scots. The battle itself was fought on 15 April 1746, was over in less than an hour and resulted in the deaths of about 50 English soldiers and between 1210 and 1500 Jacobites. (The Jacobites were Scottish men who banded together as an army over many years to fight the English and reinstate a Stuart King on the English throne.).
After we had looked at the exhibitions in the visitor centre we walked around the battlefield ...
Epic Hike
... open and there were sort of ice canyons of a couple of metres deep in places, lots with a brilliant blue ice colour. Some really cool and interesting shapes and formations, we were entertained for some time with our icy playground.
Then we continued on to climb up Cairn Gorm mountain itself, the last slog of which was very tiring. On top we were again rewarded with stunning views, and a cool little automatic weather station. Now it was ...
It was a peculiar affair with Ricardo and, to be frank, the ending was so oddly tragic that I ended up blotting considerable chunks from my mind. For the purpose of not jack-knifing this story to a confusing end however, I'll dredge up the blotted bits for a bit of re-assembling... could be closure!
We met at Scaletto, the bar that Liam was destined to drain of coffee throughout his stay and I have to say that my initial thought was, oh dear.
Belen had ...
Stonehaven to Grantown-on-Spey
After a good nights sleep and a fantastic breakfast we headed off to Dunnottar Castle.
Our first experience of a B&B certainly was everything we could have wanted, and even climbing the steep staircase to our room paled into insignificance due to all the other pluses.
Dunnottar Castle ruins sit on the top of a cliff top and is very impressive. We spent time there catching up on its magnificent history and picturing what it would have been like to live ...
Day Twenty-One - In Search of Nessie
... I have to admit I wasn't overly keen on the idea. Before coming to Inverness I had sworn to myself I would do something else besides a Loch Ness tour. It all seems like a big tourist trap to me. But I followed her advice and took a part-day trip out to Loch Ness on a guided tour. During the tour I got to take a 30 minute ride on Loch Ness, spend an hour at Urquhart Castle and Visitor Centre, and visit the Nessieland ...


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Grade 7 math
Here is a list of all of the math skills students learn in grade 7! These skills are organized into categories, and you can move your mouse over any skill name to view a sample question. To start practising, just click on any link. IXL will track your score, and the questions will automatically increase in difficulty as you improve!
Number theory
Operations with integers
- C.1Integer addition and subtraction rules
- C.2Add and subtract integers using counters
- C.3Add and subtract integers
- C.4Complete addition and subtraction sentences with integers
- C.5Add and subtract integers: word problems
- C.6Integer multiplication and division rules
- C.7Multiply and divide integers
- C.8Complete multiplication and division sentences with integers
- C.9Evaluate numerical expressions involving integers
Operations with decimals
- E.1Add and subtract decimals
- E.2Add and subtract decimals: word problems
- E.3Multiply decimals
- E.4Multiply decimals and whole numbers: word problems
- E.5Divide decimals
- E.6Divide decimals by whole numbers: word problems
- E.7Estimate sums, differences and products of decimals
- E.8Add, subtract, multiply and divide decimals: word problems
- E.9Multi-step inequalities with decimals
- E.10Maps with decimal distances
- E.11Evaluate numerical expressions involving decimals
Fractions and mixed numbers
- F.1Equivalent fractions
- F.2Simplify fractions
- F.3Understanding fractions: word problems
- F.4Least common denominator
- F.5Compare and order fractions
- F.6Compare fractions: word problems
- F.7Convert between mixed numbers and improper fractions
- F.8Compare mixed numbers and improper fractions
- F.9Round mixed numbers
Operations with fractions
- G.1Add and subtract fractions with like denominators
- G.2Add and subtract fractions with like denominators: word problems
- G.3Add fractions with unlike denominators
- G.4Subtract fractions with unlike denominators
- G.5Add and subtract fractions with unlike denominators: word problems
- G.6Add and subtract mixed numbers
- G.7Add and subtract mixed numbers: word problems
- G.8Inequalities with addition and subtraction of fractions and mixed numbers
- G.9Estimate sums and differences of mixed numbers
- G.10Multiply fractions and whole numbers
- G.11Multiply fractions and whole numbers: word problems
- G.12Multiply two fractions using models
- G.13Multiply fractions
- G.14Multiply fractions: word problems
- G.15Multiply three or more fractions and whole numbers
- G.16Divide whole numbers by unit fractions using models
- G.17Reciprocals
- G.18Divide whole numbers and unit fractions
- G.19Divide fractions
- G.20Divide fractions: word problems
- G.21Estimate products and quotients of fractions and mixed numbers
- G.22Add, subtract, multiply and divide fractions and mixed numbers: word problems
- G.23Evaluate numerical expressions involving fractions
Rational numbers
- H.1Identify rational numbers
- H.2Convert between decimals and fractions or mixed numbers
- H.3Absolute value of rational numbers
- H.4Compare rational numbers
- H.5Put rational numbers in order
- H.6Add and subtract rational numbers
- H.7Apply addition and subtraction rules
- H.8Multiply and divide rational numbers
- H.9Apply multiplication and division rules
Exponents and square roots
Ratios and proportions
- J.1Understanding ratios
- J.2Equivalent ratios
- J.3Equivalent ratios: word problems
- J.4Compare ratios: word problems
- J.5Unit rates
- J.6Do the ratios form a proportion?
- J.7Do the ratios form a proportion: word problems
- J.8Solve proportions
- J.9Solve proportions: word problems
- J.10Estimate population size using proportions
- J.11Rate of change
- J.12Constant rate of change
- J.13Scale drawings and scale factors
Proportional relationships
- K.1Find the constant of proportionality from a table
- K.2Identify graphs of proportional relationships
- K.3Find the constant of proportionality from a graph
- K.4Write equations for proportional relationships
- K.5Graph proportional relationships
- K.6Identify proportional relationships
- K.7Find the constant of proportionality: word problems
- K.8Solve problems involving proportional relationships
- L.1What percentage is illustrated?
- L.2Convert between percents, fractions and decimals
- L.3Compare percents to fractions and decimals
- L.4Estimate percents of numbers
- L.5Percents of numbers and money amounts
- L.6Percents of numbers: word problems
- L.7Solve percent equations
- L.8Solve percent equations: word problems
Consumer math
- M.1Add, subtract, multiply and divide money amounts: word problems
- M.2Price lists
- M.3Unit prices
- M.4Unit prices: find the total price
- M.5Percent of a number: tax, discount and more
- M.6Find the percent: tax, discount and more
- M.7Sale prices: find the original price
- M.8Multi-step problems with percents
- M.9Estimate tips
- M.10Simple interest
- M.11Compound interest
Problem solving and estimation
Coordinate plane
Number sequences
Variable expressions
- R.1Write variable expressions
- R.2Write variable expressions: word problems
- R.3Evaluate linear expressions
- R.4Evaluate multi-variable expressions
- R.5Evaluate absolute value expressions
- R.6Evaluate nonlinear expressions
- R.7Identify terms and coefficients
- R.8Add and subtract like terms
- R.9Add, subtract, and multiply linear expressions
- R.10Factor linear expressions
- R.11Identify equivalent linear expressions
One-variable equations
Two-variable equations
- U.1Does (x, y) satisfy the equation?
- U.2Identify independent and dependent variables
- U.3Solve word problems involving two-variable equations
- U.4Complete a table for a two-variable relationship
- U.5Write a two-variable equation
- U.6Identify the graph of an equation
- U.7Graph a two-variable equation
- U.8Interpret a graph: word problems
- U.9Write an equation from a graph using a table
- V.1Lines, line segments and rays
- V.2Parallel, perpendicular, intersecting
- V.3Construct the midpoint or perpendicular bisector of a segment
- V.4Construct a perpendicular line
- V.5Name, measure and classify angles
- V.6Identify complementary, supplementary, vertical, adjacent and congruent angles
- V.7Find measures of complementary, supplementary, vertical and adjacent angles
- V.8Construct an angle bisector
- V.9Construct a congruent angle
- V.10Transversal of parallel lines
- V.11Construct parallel lines
- V.12Classify triangles
- V.13Classify quadrilaterals
- V.14Find missing angles in triangles and quadrilaterals
- V.15Identify and classify polygons
- V.16Interior angles of polygons
- V.17Construct an equilateral triangle or regular hexagon
- V.18Similar and congruent figures
- V.19Similar figures: side lengths and angle measures
- V.20Similar figures and indirect measurement
- V.21Congruent figures: side lengths and angle measures
- V.22Congruence statements and corresponding parts
- V.23Perimeter
- V.24Area of rectangles and parallelograms
- V.25Area of triangles and trapezoids
- V.26Area with mixed numbers
- V.27Area and perimeter: word problems
- V.28Parts of a circle
- V.29Circles: calculate area, circumference, radius and diameter
- V.30Circles: word problems
- V.31Find lengths and measures of bisected lines and angles
- V.32Front, side and top view
- V.33Names and bases of 3-dimensional figures
- V.34Nets of 3-dimensional figures
- V.35Surface area
- V.36Volume
- V.37Perimeter, area and volume: changes in scale
- V.38Semicircles: calculate area, perimeter, radius and diameter
- V.39Quarter circles: calculate area, perimeter and radius
- V.40Area of compound figures with triangles, semicircles and quarter circles
- V.41Area between two shapes
Data and graphs
- X.1Interpret tables
- X.2Interpret line plots
- X.3Create line plots
- X.4Interpret stem-and-leaf plots
- X.5Interpret bar graphs
- X.6Create bar graphs
- X.7Interpret histograms
- X.8Create histograms
- X.9Create frequency charts
- X.10Interpret circle graphs
- X.11Circle graphs and central angles
- X.12Interpret line graphs
- X.13Create line graphs
- X.14Interpret box-and-whisker plots
- X.15Scatter plots
- X.16Choose the best type of graph


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just wondering if anyone has flown Virgin Premium Economy from Australia to Europe.
flight centre have a deal which is Melbourne to Sydney (economy), then Sydney to Abu Dhabi (premium economy) and then Etihad economy from Abu Dhabi to Paris.
If anyone has been on this type of flight can you tell me if the flight was worth paying the extra for. I am a bit concerned about the Virgin Premium economy product not being much better than economy.
If this is the case would Singapore Airlines economy to Paris be a better option.
thanks in advance


text:
E-mail: firstname.lastname@example.com
Hangzhou Hangshen Energy-Saving Furnace Co.,Ltd. Is the production of energy-saving electric resistance furnace and of the professional manufacturers of industrial furnaces, the first fastener industry association member units, zhejiang province, the company was founded in 1990, organized by super resistance to industrial company, hangzhou enterprise, in 1996 changed its name to hangzhou HangShen energy-saving furnaces co., LTD. Since its inception the company has always adhere to science and technology and the deepening reform. Over the years through the company all staff´s continuous efforts and explore, proud achievements obtained. Is district environmental protection bureau rated industrial furnace drainage standard. Hangzhou Hangshen Energy-Saving Funace Co.,Ltd is one of the manufacturers and suppliers of annealing furnace, gas generating furnace, galvanized iron wire annealing furnace, aluminum alloy wire tempering furnace products from China. As a very professional annealing furnace company, we have our own factory, we welcome you to buy our annealing furnace products in wholesale, cheap annealing furnace. Welcome to contact us.
Hangzhou Hangshen Energy-Saving Furnace Co.,Ltd
Sales manager: Mr.Qian
Tel: 86-0571-86172088
Fax: 86-0571-86173266
Mobile phone: 15990123123
Email: firstname.lastname@example.com
Add: Yanshan Industrial Zone, Chongxian Street, Yuhang, Hangzhou, Zhengjiang


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3850 State St, Santa Barbara, CA, 93105, United States of America, 866-925-8648
Best Western Plus Pepper Tree Inn, Santa Barbara
Lowest price
Best Price Guarantee
3-star hotel in Santa Barbara with outdoor pool, restaurant
- Free WiFi and free parking
Main amenities
- 150 guestrooms
- Restaurant and bar/lounge
- Outdoor pool
- Breakfast available
- Free airport shuttle
- Fitness center
- Massage/treatment rooms
- 24-hour business center
- 24-hour front desk
- Coffee/tea in a common area
- Daily housekeeping
- Lobby fireplace
What's around
- In Upper State Street
- Mission Santa Barbara (2.4 mi / 3.9 km)
- Santa Barbara Natural History Museum (2.2 mi / 3.6 km)
- Santa Barbara Museum of Art (3.1 mi / 4.9 km)
- La Cumbre Plaza (0.3 mi / 0.4 km)
- Santa Barbara Golf Club (0.8 mi / 1.3 km)
For families
- Refrigerator
- Private bathroom
- Premium TV channels
- Free toiletries
- Daily housekeeping
- Laundry facilities
See all 404 Hotels.com reviews
Fantastic service
Great pool
Great location
Bad breakfast
TripAdvisor Traveller Rating: 4
From 1,000 reviews
Best Western Plus Pepper Tree Inn, Santa Barbara
Hotel Info: 866-925-8648from $175
- Standard Room, 1 King Bed
- Double Room, 2 Double Beds
- Deluxe Room, 1 King Bed with Sofabed
- Standard Room, 2 Double Beds, Refrigerator, Poolside
- Standard Room, 1 King Bed
- Deluxe Room, 1 King Bed with Sofabed
- Double Room, 2 Double Beds
- Standard Room, 2 Queen Beds, Refrigerator, Poolside
- Deluxe Room, 1 King Bed, Non Smoking, Refrigerator & Microwave
- Standard Room
- Standard Room, 1 King Bed, Refrigerator, Poolside
- Standard Room, 1 Queen Bed, Accessible, Non Smoking
- Standard Room, 1 Queen Bed, Non Smoking, Refrigerator & Microwave
- Deluxe Room, 1 King Bed, Refrigerator & Microwave, Courtyard View
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 150 rooms
- This hotel is arranged over 2 floors
- Check-in time starts at 3 PM
- Check-out time is 11 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Children (12 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- No cribs (infant beds)
- Pets not allowed (service dogs only)
- Free WiFi in public areas
- Free WiFi in rooms
- Free airport shuttle
- Free self parking
Nearby Three Star Santa Barbara hotels
In the hotel
Food and drink
- Breakfast (surcharge)
- Restaurant
- Bar/lounge
- Coffee/tea in a common area
Things to do
- Outdoor pool
- Fitness facilities
- Spa treatment room(s)
- Use of nearby fitness center (discount)
Working away
- 24-hour business center
- Conference space
- Conference space size (feet) - 3000
- Conference space size (meters) - 279
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Hair salon
- Free newspapers in lobby
- Multilingual staff
- Porter/bellhop
- Safe-deposit box at front desk
- Fireplace in lobby
- Accessible bathroom
- In-room accessibility
In the room
Home comforts
- In-room climate control (air conditioning)
- Air conditioning
- Coffee/tea maker
- Bathrobes
- Iron/ironing board
Sleep well
- Hypo-allergenic bedding available
- Blackout drapes/curtains
Things to enjoy
- In-room massage available
- Balcony
Freshen up
- Private bathroom
- Shower/tub combination
- Designer toiletries
- Hair dryer
Be entertained
- Premium TV channels
- DVD player
Stay connected
- Desk
- Free weekday newspaper
- Free WiFi
- Free local calls
Food and drink
- Refrigerator
- Microwave (on request)
- Free bottled water
- Daily housekeeping
- In-room safe
- Access via exterior corridors
Top Santa Barbara hotels
Recently reviewed hotels in Santa Barbara
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"Noisy AC/heater, small tub, average bed, bath, & furnishing quality, and parking lot view definitely does not justify the $450+ per night! Never saw pool or hottub. Just because…"Jun 16, 2016
Nearby hotels
Special features
DiningTreehouse Restaurant - This family restaurant serves breakfast, lunch, and dinner. Guests can enjoy drinks at the bar.
On Site
- Fitness facilities
- Personal motorized watercraft on site
- Bicycle rentals nearby
- Golfing nearby
- Hiking/biking trails nearby
- Mountain biking nearby
- Parasailing nearby
- Sailing nearby
- Scuba diving nearby
- Skydiving nearby
- Surfing/boogie boarding nearby
- Windsurfing nearby
Best Western Plus Pepper Tree Inn, Santa Barbara's small print
Also known as
- Best Western Pepper
- Best Western Plus Pepper Tree Hotel Santa Barbara
- Best Western Pepper Inn
- Best Western Pepper Tree
- Best Western Pepper Tree Inn
- Best Western Plus Pepper
- Best Western Plus Pepper Tree
- Best Western Plus Pepper Tree Inn
- Best Western Plus Pepper Tree Inn Santa Barbara
- Best Western Plus Pepper Tree Santa Barbara
Optional extras
Breakfast costs between USD 3 and USD 20 per person (approximately)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


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Author has written 2 stories for Naruto.
Well, as the name suggests, I'm a science major in college who also happens to love karate, music (especially metal), art, and of course Japanese anime!
Not much to say. I enjoy seinen anime and tasteful romance. None of the unnecessarily mushy stuff, please.
I will probably add more to my profile later. That's all you need to know about me right now.


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The College of Liberal Arts is the cultural center of Northeastern State University. We offer a variety of programs in the social sciences, performing arts, fine arts, humanities, communication and military science fields.
Phone: 918-444-3600
Fax: 918-458-2348
Email: email@example.com
Seminary Hall - Room 141
609 N. Grand Ave.
Tahlequah, OK 74464-2302
Liberal Arts building G
3100 E New Orleans St.
Broken Arrow, OK 74014
Dean Phil Bridgmon, Ph.D.
Dean's Welcome Letter
Contact Us


text:
8 of 8 people found the following review helpful
Right up there with Drunken Master 2,
By A Customer
This review is from: Fist of Legend [VHS] (VHS Tape)
To all the viewers who say the action is great but the storyline isn't, they need to get a grip. The reason you watch any action film, hollywood made or HK made isn't because of the plot but the action in the movie. Jet Li is the best Kung-Fu star out there now but please don't compare him to Jackie or Bruce. Jackie Chan has a style all his own and has dominated the HK scene for a long time. Bruce Lee made more realistic films in terms of choreography. People need to realize that in a real fight, you would probably fight more like bruce than Jet or Jackie. So stop saying that Jet Li would beat Bruce because it just wouldn't happen. That being said, this is by far the most superior film made in the last 5 years. The story is good(based on real life events)and the choreography is amazing...although for everyone who thinks that Jet Li really moves that fast, sorry...they speed the film up slightly during certain fight scenes. I like how they incorporated so many different styles of fighting into one movie, you had boxing, karate, jujitsu, aidkido, hapkido, chi-na joint locks, even a little bit of capoiera(sp?) I would have to say that this movie showed that jet can act as well as he can fight...more emotional range than bruce lee ever showed. Only Drunken Master 2 can challenge it for overall best martial arts film ever.
Review Details
A Customer


text:
Transfer Zone
Guardiola reveals Sane & Stones talks
The Manchester City manager reemphasised the club's interest in the Schalke forward and revealed that they are also trying to sign the Everton man
The Manchester City manager reemphasised the club's interest in the Schalke forward and revealed that they are also trying to sign the Everton man
Goal's Sam Lee was pitchside in Shenzhen to get a closer look at two of the top coaches in world football as Man City beat Borussia Dortmund
The Swedish striker has started life with his new club, joining his team-mates for his first practice session on Thursday at Carrington
On Thursday, the Manchester United manager is set to tell nine players if they have a future at the club - get all the latest rumours with Transfer Talk!
Christian Pusilic levelled after Sergio Aguero's opener, sending the match to penalties, with Jose Tasende scoring the winning spot kick for the Citizens
Franck Ribery netted a brace but it wasn't enough as Bayern fell to Milan on penalties after a 3-3 draw in the International Champions Cup.
Goals from Paulo Dybala and Mehdi Benatia gave the Italian champions the win over Spurs, who pulled one back through Erick Lamela
The striker has claimed the Italian called him recently to ask him to move to Stamford Bridge this summer but the Blues boss has refused to discuss the matter further
The Uruguayan was on the receiving end of a nutmeg by his Barca team-mate as the Spanish champions trained in front of Roy Keane at St George's Park
The Gunners midfielder, who joined from Borussia Monchengladbach earlier in the summer, has been filmed singing the song in Albanian in front of his team-mates
The former Manchester United midfielder gave everyone a glimpse of his talent in a pre-season friendly as he continues to be linked with a move back to England


text: Editor's note: Now president of the South African Medical Research Council and a principal investigator for the Fred Hutch-based global HIV Vaccine Trials Network, Dr. Glenda Gray spent much of her career as a pediatrician at an enormous hospital in the township of Soweto. As a clinician, researcher and activist, she successfully fought to prevent mother-to-child HIV transmission. Here she recalls the days before prevention or treatment was available, and she remembers one child in particular who received international acclaim after addressing the 2000 AIDS conference in Durban, South Africa. Gray gave this talk to a gathering of colleagues from that time attending the 2016 AIDS conference, held again in Durban.


text:
Book your room at La Quinta Inn & Suites Dallas Arlington South
Get an overview of this hotelArlington hotel in Dalworthington Gardens with free breakfast and outdoor pool
Located in Dalworthington Gardens, this hotel is 1 mi (1.6 km) from Arlington Highlands and 2.7 mi (4.3 km) from MetroCenter. University of Texas at Arlington and Maverick Stadium are also within 6 mi (10 km).
Hotel Features
This hotel features an outdoor pool, a fitness center, and a spa tub. Free continental breakfast, free WiFi in public areas, and free self parking are also provided. Additionally, coffee/tea in a common area, a meeting room, and laundry facilities are onsite.
Room Amenities
All 128 rooms offer free WiFi, premium bedding, and flat-screen TVs with cable channels. Guests will also find coffee makers, free local calls, and video-game consoles.Would you like to give us feedback? Share it! Opens in a new window
La Quinta Inn & Suites Dallas Arlington South4001 Scot's Legacy Drive Arlington TX 76015
Hotel Amenities
Hotel Amenities
In addition to an outdoor pool, La Quinta Inn & Suites Dallas Arlington South provides a spa tub and a fitness center. High-speed wireless Internet access is complimentary. A complimentary breakfast is offered. Event facilities measuring 700 square feet (63 square meters) include a meeting room. This Arlington hotel also offers coffee/tea in a common area, laundry facilities, and a television in a common area. Onsite self parking is complimentary.
- 24-hour front desk
- Coffee/tea in common areas
- Conference space size (feet) - 700
- Conference space size (meters) - 63
- Elevator/lift
- Fitness facilities
- Free breakfast
- Free self parking
- Free WiFi
- Laundry facilities
- Number of floors - 5
- One meeting room
- Outdoor pool
- Spa tub
- Television in common areas
- Total number of rooms - 128
Family Friendly Amenities
- Free Wi-Fi
- Laundry facilities
- Outdoor pool
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Air conditioning
- Cable TV service
- Coffee/tea maker
- Free toiletries
- Cribs/infant beds available
- Daily housekeeping
- Desk
- Flat-panel TV
- Free local calls
- Free WiFi
- Hair dryer
- Iron/ironing board
- Premium bedding
- Premium TV channels
- Private bathroom
- Shower/tub combination
- Video-game console
Where to Eat
Guests are offered a complimentary continental breakfast.
Nearby Things to Do
An outdoor pool and a spa tub are on site. Other recreational amenities include a fitness center.
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
Hotel Policies
Check-in time ends at Midnight
Check-in time starts at 3 PM
Check-out time is Noon
Payment types
- Pets stay free
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
Pets allowed (no fees)
Hotel Name
- La Quinta Dallas Arlington South
- La Quinta Inn Dallas Arlington South
- Quinta Inn Dallas Arlington South
- Quinta Dallas Arlington South
- La Quinta Inn Suites Dallas Arlington South
We should mention
Children 18 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.


text:
Texas Longhorns NCAA Football News
Texas Longhorns NCAA Football Sports news continually updated from thousands of sources around the net.
NFL linebacker Emmanuel Acho joins Austin PD for #BecomeOne
Thursday Jul 14 | KXAN-TV Austin | Comment?
Generals defeat Strykers an hour ago The Victoria Generals were not...
Tuesday Jul 12 | The Victoria Advocate | Comment?
Akinosun named alternate for Team USA during Olympic Track & Field Trials
Monday Jul 4 | Daily Texan | Comment?
Texas Longhorns NCAA Football news is powered by NewsRank™
Trending Now
Updated: Tue Jul 26, 2016 03:37 pm
Copyright © 2016 Topix LLC


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Sugar Beach Oceanfront Ocean View Penthouse 2/2 Great Rates!
From $325 / night
balance of due
Description from the manager
Description from the manager
Bedrooms: 2/
Bathrooms: 2/
Sleeps: 4/
Type: Apt. / Condo
Book this 2 bedroom, 2 bathroom apt. / condo in Kihei today! A total of 4 people can sleep here comfortably.
Guests can enjoy a pool, hot tub, and air conditioning among other amenities offered by this rental.
No travelers have shared a review of this property yet.
Costs vary, with the average weekly rate as low as $2,275.00 to as high as $3,493.00, depending on travel dates.
The owner last updated the property's calendar today.
About the manager
About the manager
Rentals Maui
Rentals Maui
- Response rate: n/a
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- Calendar updated: today
Overall rating:
4.5 / 5.0


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Top Definition
This is when a girl is getting fucked up the ass so hard that both her and the person fucking her knees buckle from the forces of fucking and getting fucked. Best observed in any porno where anal poundings are involved.
Dude me and my girl were fucking, it was Knee Buckling Butt Fucking night, she was so tired after that she fell asleep with my cock in her ass, what a night!
Knee Buckling Ass Fucking is the way to go
by Overloard October 08, 2010
Trending Now


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Of course this singer was strong
| July 29, 2008 - 4:10 pm
Share < br />this article:
Of course this singer was
strong enough
to beat breast cancer!
Sheryl Crow
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text:
and CBS will loose this suit. Law 101 -you must mitigate your damages. Stern should have been taken off the air or sensored by CBS. To not do so and to say "I knew I could alwas sue you" has lost him and CBS this case even before they hear one retort in a US Court.END OF STORYBUY ON THE DIP
i tink is name is moonfez. that's albanian for hat
Moon??? did you say moon??? yes, siri to the moon like a rocketship. to the moon i say. yes, to the moon real soon now!!!!
Failure to Mitigate Damages. CBS already lost.Plus from a PR standpoint this makes them look like a loser that has to strike back.It confirms Stern and Sirius hurting them.
"Failure to Mitigate Damages. CBS already lost."CBS did NOT lose. Stern settled for an UNDISCLOSED amount.
I'm not a legal expert but it makes perfect sense.


text:
Is there a solution for Towers of Hanoi whose running time is less than O(2n) where n is the number of disks to move? My solution takes O(2n) time.
Also, the below solution is with recursion. Can we use Dynamic Programming with the concept of memoization to solve this in a lesser time?
public void towersOfHanoi(
int num,
MyStack<Integer> from,
MyStack<Integer> to,
MyStack<Integer> spare
) {
if (num == 1) {
int i = from.pop();
System.out.println("Move "+i+" from "+from.getName()+" to " + to.getName());
towersOfHanoi(num - 1, from, spare, to);
towersOfHanoi(1, from, to, spare);
towersOfHanoi(num - 1, spare, to, from);
MyStack is an extended version of
Stack class in Java that adds a
name field and accessor.
Also, are there any variations of the same problem?


text:
Stay Matte Pressed Powder with Minerals maximum powder presentation. Lightweight formula, with high coverage for long lasting shine control. Up to 5 hours natural shine control. Helps minimize the appearance of pores.
Product Form: Pressed Powder
For Use On: Face
Beauty Purpose: Skin Tone Improvement
Color Palette: Translucent
Color Family: Beige
Travel Size: No
Product Warning: no warning applicable
other Info.
- Made in the USA or Imported