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Sample records for epithelium-derived factor binds

  1. Recombinant pigment epithelium-derived factor PEDF binds vascular endothelial growth factor receptors 1 and 2.

    PubMed

    Johnston, Erin K; Francis, Mary K; Knepper, Janice E

    2015-08-01

    Angiogenesis, or the formation of new blood vessels, is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis. To explore the mechanism by which PEDF acts, recombinant PEDF was expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The PEDF fusion protein was confirmed to be active in inhibition of endothelial cell proliferation and migration. Direct binding of PEDF to both vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 was demonstrated in an in vitro assay similar to an enzyme-linked immunosorbent assay (ELISA). PEDF was shown by immune-confocal microscopy to be localized within treated endothelial cells. When VEGF-stimulated endothelial cells were incubated with PEDF the VEGF receptors showed intracellular localization. These data suggest that the interaction between PEDF and VEGFR-1 or VEGFR-2 may be a possible mechanism for inhibiting angiogenesis. PEDF may be binding to the VEGF receptors to promote their internalization and/or degradation to limit VEGF responses in treated cells.

  2. Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor*

    PubMed Central

    Kenealey, Jason; Subramanian, Preeti; Comitato, Antonella; Bullock, Jeanee; Keehan, Laura; Polato, Federica; Hoover, David; Marigo, Valeria; Becerra, S. Patricia

    2015-01-01

    The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98–114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98–114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors. PMID:26304116

  3. Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans.

    PubMed

    Sekiya, Atsushi; Okano-Kosugi, Hitomi; Yamazaki, Chisato M; Koide, Takaki

    2011-07-29

    Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.

  4. Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF Receptor (PEDF-R): identification of a functional ligand binding site.

    PubMed

    Subramanian, Preeti; Locatelli-Hoops, Silvia; Kenealey, Jason; DesJardin, Jacqueline; Notari, Luigi; Becerra, S Patricia

    2013-08-16

    The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

  5. Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells.

    PubMed

    Matsui, Takanori; Higashimoto, Yuichiro; Taira, Junichi; Yamagishi, Sho-ichi

    2013-11-15

    Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10(-7) M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.

  6. Airway epithelium-derived relaxing factor: myth, reality, or naivety?

    PubMed

    Vanhoutte, Paul M

    2013-05-01

    The presence of a healthy epithelium can moderate the contraction of the underlying airway smooth muscle. This is, in part, because epithelial cells generate inhibitory messages, whether diffusible substances, electrophysiological signals, or both. The epithelium-dependent inhibitory effect can be tonic (basal), synergistic, or evoked. Rather than a unique epithelium-derived relaxing factor (EpDRF), several known endogenous bronchoactive mediators, including nitric oxide and prostaglandin E2, contribute. The early concept that EpDRF diffuses all the way through the subepithelial layers to directly relax the airway smooth muscle appears unlikely. It is more plausible that the epithelial cells release true messenger molecules, which alter the production of endogenous substances (nitric oxide and/or metabolites of arachidonic acid) by the subepithelial layers. These substances then diffuse to the airway smooth muscle cells, conveying epithelium dependency.

  7. Comparison of plasma pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP-4), chitinase-3-like protein 1 (YKL-40) and brain-derived neurotrophic factor (BDNF) for the identification of insulin resistance.

    PubMed

    Toloza, F J K; Pérez-Matos, M C; Ricardo-Silgado, M L; Morales-Álvarez, M C; Mantilla-Rivas, J O; Pinzón-Cortés, J A; Pérez-Mayorga, M; Arévalo-García, M L; Tolosa-González, G; Mendivil, C O

    2017-09-01

    To evaluate and compare the association of four potential insulin resistance (IR) biomarkers (pigment-epithelium-derived factor [PEDF], retinol-binding-protein-4 [RBP-4], chitinase-3-like protein 1 [YKL-40] and brain-derived neurotrophic factor [BDNF]) with objective measures of IR. We studied 81 subjects with different metabolic profiles. All participants underwent a 5-point OGTT with calculation of multiple IR indexes. A subgroup of 21 participants additionally underwent a hyperinsulinemic-euglycemic clamp. IR was defined as belonging to the highest quartile of incremental area under the insulin curve (iAUCins), or to the lowest quartile of the insulin sensitivity index (ISI). PEDF was associated with adiposity variables. PEDF and RBP4 increased linearly across quartiles of iAUCins (for PEDF p-trend=0.029; for RBP-4 p-trend=0.053). YKL-40 and BDNF were not associated with any adiposity or IR variable. PEDF and RBP-4 levels identified individuals with IR by the iAUCins definition: A PEDF cutoff of 11.9ng/mL had 60% sensitivity and 68% specificity, while a RBP-4 cutoff of 71.6ng/mL had 70% sensitivity and 57% specificity. In multiple regression analyses simultaneously including clinical variables and the studied biomarkers, only BMI, PEDF and RBP-4 remained significant predictors of IR. Plasma PEDF and RBP4 identified IR in subjects with no prior diagnosis of diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Structural Properties of HIV Integrase. Lens Epithelium-derived Growth Factor Oligomers

    SciTech Connect

    Gupta, K.; Diamond, T; Hwang, Y; Bushman, F; Van Duyne, G

    2010-01-01

    Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN {center_dot} LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN {center_dot} LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.

  9. [Effect of cryotherapy over the expression of vascular endothelial growth factor and pigment epithelium-derived factor].

    PubMed

    Toscano-Garibay, Julia Dolores; Quiroz-Mercado, Hugo; Espitia-Pinzón, Clara; Gil-Carrasco, Félix; Flores-Estrada, José Javier

    2014-01-01

    Cryotherapy is a no invasive technique that uses intense cold to freeze and destroy cancer tissues. There are no descriptions of its effects over the expression of vascular endothelial growth factor and pigment epithelium-derived factor. Experimental study in cryogenic spot were applied in the right sclera of twelve pigs for ten minutes. Other 3 pigs were used as normal controls. Animals were sacrificed at 7, 14 and 21 and the tissues of choriodes and retina were dissected in areas of approximately 1 cm2 surrounding cryogenic spots. Expression levels of vascular endothelial growth factor and pigment epithelium-derived factor were determined analyzed using polymerase chain reaction coupled to reverse-transcription. Vascular endothelial growth factor was significantly downregulated (24%, p< 0.05) seven days post-treatment meanwhile pigment epithelium-derived factor levels increased 44.8% (p< 0.05) as compared to normal controls (untreated). Both vascular endothelial growth factor and pigment epithelium-derived factor levels remain the same until day 14 but returned to basal expression at day 21. This work expose the relation of cryotherapy with the expression of two factors related to angiogenesis. RESULTS showed significant changes on the expression of vascular endothelial growth factor and pigment epithelium-derived factor illustrating that both proteins are regulated in response to cryogenic treatment in relatively short periods (21 days).

  10. Pigment epithelium-derived factor as a multifunctional regulator of wound healing

    PubMed Central

    Wietecha, Mateusz S.; Król, Mateusz J.; Michalczyk, Elizabeth R.; Chen, Lin; Gettins, Peter G.

    2015-01-01

    During dermal wound repair, hypoxia-driven proliferation results in dense but highly permeable, disorganized microvascular networks, similar to those in solid tumors. Concurrently, activated dermal fibroblasts generate an angiopermissive, provisional extracellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for reestablishing tissue homeostasis. Mechanisms guiding wound resolution are poorly understood; one candidate regulator is pigment epithelium-derived factor (PEDF), a secreted glycoprotein. PEDF is a potent antiangiogenic in models of pathological angiogenesis and a promising cancer and cardiovascular disease therapeutic, but little is known about its physiological function. To examine the roles of PEDF in physiological wound repair, we used a reproducible model of excisional skin wound healing in BALB/c mice. We show that PEDF is abundant in unwounded and healing skin, is produced primarily by dermal fibroblasts, binds to resident microvascular endothelial cells, and accumulates in dermal ECM and epidermis. PEDF transcript and protein levels were low during the inflammatory and proliferative phases of healing but increased in quantity and colocalization with microvasculature during wound resolution. Local antibody inhibition of endogenous PEDF delayed vessel regression and collagen maturation during the remodeling phase. Treatment of wounds with intradermal injections of exogenous, recombinant PEDF inhibited nascent angiogenesis by repressing endothelial proliferation, promoted vascular integrity and function, and increased collagen maturity. These results demonstrate that PEDF contributes to the resolution of healing wounds by causing regression of immature blood vessels and stimulating maturation of the vascular microenvironment, thus promoting a return to tissue homeostasis after injury. PMID:26163443

  11. Pigment epithelium-derived factor as a multifunctional regulator of wound healing.

    PubMed

    Wietecha, Mateusz S; Król, Mateusz J; Michalczyk, Elizabeth R; Chen, Lin; Gettins, Peter G; DiPietro, Luisa A

    2015-09-01

    During dermal wound repair, hypoxia-driven proliferation results in dense but highly permeable, disorganized microvascular networks, similar to those in solid tumors. Concurrently, activated dermal fibroblasts generate an angiopermissive, provisional extracellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for reestablishing tissue homeostasis. Mechanisms guiding wound resolution are poorly understood; one candidate regulator is pigment epithelium-derived factor (PEDF), a secreted glycoprotein. PEDF is a potent antiangiogenic in models of pathological angiogenesis and a promising cancer and cardiovascular disease therapeutic, but little is known about its physiological function. To examine the roles of PEDF in physiological wound repair, we used a reproducible model of excisional skin wound healing in BALB/c mice. We show that PEDF is abundant in unwounded and healing skin, is produced primarily by dermal fibroblasts, binds to resident microvascular endothelial cells, and accumulates in dermal ECM and epidermis. PEDF transcript and protein levels were low during the inflammatory and proliferative phases of healing but increased in quantity and colocalization with microvasculature during wound resolution. Local antibody inhibition of endogenous PEDF delayed vessel regression and collagen maturation during the remodeling phase. Treatment of wounds with intradermal injections of exogenous, recombinant PEDF inhibited nascent angiogenesis by repressing endothelial proliferation, promoted vascular integrity and function, and increased collagen maturity. These results demonstrate that PEDF contributes to the resolution of healing wounds by causing regression of immature blood vessels and stimulating maturation of the vascular microenvironment, thus promoting a return to tissue homeostasis after injury. Copyright © 2015 the American Physiological Society.

  12. Pigment epithelium-derived factor protects cone photoreceptor-derived 661W cells from light damage through Akt activation.

    PubMed

    Rapp, Matthew; Woo, Grace; Al-Ubaidi, Muayyad R; Becerra, S Patricia; Subramanian, Preeti

    2014-01-01

    Pigment epithelium-derived factor (PEDF) can delay and prevent the death of photoreceptors in vivo. We investigated the survival activity of PEDF on cone photoreceptor-derived 661W cells in culture, the presence of PEDF receptor (PEDF-R) in these cells and the activation of prosurvival Akt. Cell death was induced by light exposure in the presence of 9-cis retinal. Cell viability assays showed that PEDF increased the number of 661W cells exposed to these conditions. Western blots showed that PEDF-treated 661W cells had a higher ratio of phosphorylated Akt to total Akt than untreated cells. The PEDF receptor PEDF-R was immunodetected in the plasma membrane fractions of 661W cells. The results demonstrated that PEDF can protect 661W cells against light-induced cell death and suggest that the binding of PEDF to cell surface PEDF-R triggers a prosurvival signaling pathway.

  13. Role of pigment epithelium-derived factor in the reproductive system.

    PubMed

    Chuderland, Dana; Ben-Ami, Ido; Bar-Joseph, Hadas; Shalgi, Ruth

    2014-10-01

    The physiological function of the female reproductive organs is hormonally controlled. In each cycle, the reproductive organs undergo tissue modifications that are accompanied by formation and destruction of blood vessels. Proper angiogenesis requires an accurate balance between stimulatory and inhibitory signals, provided by pro- and anti-angiogenic factors. As with many other tissues, vascular endothelial growth factor (VEGF) appears to be one of the major pro-angiogenic factors in the female reproductive organs. Pigment epithelium-derived factor (PEDF) is a non-inhibitory member of the serine protease inhibitors (serpin) superfamily, possessing potent physiologic anti-angiogenic activity that negates VEGF activity. The role of PEDF in decreasing abnormal neovascularization by exerting its anti-angiogenic effect that inhibits pro-angiogenic factors, including VEGF, has been investigated mainly in the eye and in cancer. This review summarizes the function of PEDF in the reproductive system, showing its hormonal regulation and its anti-angiogenic activity. Furthermore, some pathologies of the female reproductive organs, including endometriosis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, and others, are associated with a faulty angiogenic process. This review illuminates the role of PEDF in their pathogenesis and treatment. Collectively, we can conclude that although PEDF seems to play an essential role in the physiology and pathophysiology of the reproductive system, its full role and mechanism of action still need to be elucidated. © 2014 Society for Reproduction and Fertility.

  14. The pigment epithelium-derived factor (PEDF): an important potential therapeutic agent for infantile hemangioma.

    PubMed

    Li, Ming; Chen, Yanru; Guo, Zhihui; Xie, Yide; Zhou, Yakuan; Jiang, Chenghong; Chen, Xiaosong

    2017-04-01

    In previous studies, the expression and the role of proangiogenic factors in infantile hemangiomas have been well studied. However, the role of angiogenic inhibitors has been revealed rarely. The expression of PEDF, as the strongest and safe endogenous inhibitor, is still unrecognized until the current study. In order to investigate the expression and significance of the pigment epithelium-derived factor (PEDF) in the proliferating and regressing phases of infantile hemangiomas, the expression of PEDF, VEGF, Ki-67, and CD34 protein in hemangioma tissues was examined with immunohistochemical polymer HRP method in 42 cases during the proliferative phase, 40 cases during the regressing phase, and 11 cases of non-involuting congenital hemangiomas (NICHs). Meanwhile, the mRNA expression of these factors was detected with quantitative realtime RT-PCR. We found the protein and mRNA expression of PEDF in regressing phase was significantly higher than those in proliferative phase and NICHs (P < 0.001), while the protein and mRNA expression of VEGF were much lower (P < 0.001). The microvessel density (MVD), Ki-67 changes, and the expression of PEDF and VEGF were found significantly correlated. These results indicated that the reduction of VEGF and increase in PEDF are causative to the evolution of infantile hemangioma. PEDF may play a key role in the spontaneous regression of infantile hemangioma and may become an important potential therapeutic agent for infantile hemangioma.

  15. Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells.

    PubMed

    Chuderland, Dana; Ben-Ami, Ido; Kaplan-Kraicer, Ruth; Grossman, Hadas; Komsky, Alisa; Satchi-Fainaro, Ronit; Eldar-Boock, Anat; Ron-El, Raphael; Shalgi, Ruth

    2013-02-01

    Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

  16. Hormonal regulation of pigment epithelium-derived factor (PEDF) expression in the endometrium.

    PubMed

    Chuderland, Dana; Ben-Ami, Ido; Friedler, Shevach; Hasky, Noa; Ninio-Many, Lihi; Goldberg, Keren; Bar-Joseph, Hadas; Grossman, Hadas; Shalgi, Ruth

    2014-06-05

    Pigment epithelium-derived factor (PEDF) is highly expressed in the female reproductive system and is subjected to regulation by steroid hormones in the ovary. As the uterine endometrium exhibits morphological and functional changes in response to estrogen (E2) and progesterone (P4), we aimed at characterizing the expression of PEDF in this component of the female reproductive tract and further at exploring the hormonal regulation of its expression. We found that PEDF is expressed in human and mouse endometrium. We further showed that this expression is subjected to regulation by steroid hormones, both in vivo and in vitro, as follows: E2 decreased PEDF expression and P4 increased its levels. In human endometrial samples, PEDF levels were dynamically altered along the menstrual cycle; they were low at the proliferative and early secretory phases and significantly higher at the late secretory phase. The expression levels of PEDF were inversely correlated to that of vascular endothelial growth factor (VEGF). We also showed that PEDF receptor was expressed in the endometrium and that its stimulation reduced VEGF expression. Illustrating the pattern of PEDF expression during the menstrual cycle may contribute to our understanding of the endometrial complexity.

  17. Role of Pigment Epithelium-Derived Factor in Stem/Progenitor Cell-Associated Neovascularization

    PubMed Central

    Liu, Jung-Tung; Chen, Yuh-Lien; Chen, Wen-Chi; Chen, Huey-Yi; Lin, Yi-Wen; Wang, Shu-Huei; Man, Kee-Ming; Wan, Hui-Min; Yin, Wei-Hsian; Liu, Po-Len; Chen, Yung-Hsiang

    2012-01-01

    Pigment epithelium-derived factor (PEDF) was first identified in retinal pigment epithelium cells. It is an endogenously produced protein that is widely expressed throughout the human body such as in the eyes, liver, heart, and adipose tissue; it exhibits multiple and varied biological activities. PEDF is a multifunctional protein with antiangiogenic, antitumorigenic, antioxidant, anti-inflammatory, antithrombotic, neurotrophic, and neuroprotective properties. More recently, PEDF has been shown to be the most potent inhibitor of stem/progenitor cell-associated neovascularization. Neovascularization is a complex process regulated by a large, interacting network of molecules from stem/progenitor cells. PEDF is also involved in the pathogenesis of angiogenic eye disease, tumor growth, and cardiovascular disease. Novel antiangiogenic agents with tolerable side effects are desired for the treatment of patients with various diseases. Here, we review the value of PEDF as an important endogenous antiangiogenic molecule; we focus on the recently identified role of PEDF as a possible new target molecule to influence stem/progenitor cell-related neovascularization. PMID:22685380

  18. Protective role of small pigment epithelium-derived factor (PEDF) peptide in diabetic renal injury.

    PubMed

    Awad, Alaa S; Gao, Ting; Gvritishvili, Anzor; You, Hanning; Liu, Yanling; Cooper, Timothy K; Reeves, W Brian; Tombran-Tink, Joyce

    2013-09-15

    Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.

  19. Effects of pigment epithelium derived factor (PEDF) on malignant peripheral nerve sheath tumours (MPNSTs).

    PubMed

    Demestre, Maria; Terzi, Menderes Yusuf; Mautner, Victor; Vajkoczy, Peter; Kurtz, Andreas; Piña, Ana Luisa

    2013-12-01

    Neurofibromatosis type 1 (NF1) is an inherited genetic disease affecting 1 in 3,500 individuals. A prominent feature of NF1 is the formation of benign tumours of the peripheral nerve sheath (neurofibromas). However, these can become malignant and form highly metastatic malignant peripheral nerve sheath tumours (MPNST), which are usually fatal despite aggressive surgery, chemotherapy, and radiotherapy. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis in several kinds of tumours. The present study was designed to determine the in vitro and in vivo effects of PEDF on MPNST angiogenesis and tumour growth. PEDF inhibited proliferation and augmented apoptosis in S462 MPNST cells after 48 h of treatment in culture. In xenografts of S462 MPNST cells in athymic nude mice, PEDF suppressed MPNST tumour burden, due mainly to inhibition of angiogenesis. These results demonstrate for the first time inhibitory effects of PEDF on the growth of human MPNST via induction of anti-angiogenesis and apoptosis. Our results suggest that PEDF could be a novel approach for future therapeutic purposes against MPNST.

  20. Pigment epithelium-derived factor mediates impaired lung vascular development in neonatal hyperoxia.

    PubMed

    Chetty, Anne; Bennett, Michelle; Dang, Linh; Nakamura, Daisy; Cao, Gong-Jie; Mujahid, Sana; Volpe, MaryAnn; Herman, Ira; Becerra, S Patricia; Nielsen, Heber C

    2015-03-01

    Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

  1. Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

    PubMed

    Goldberg, Keren; Bar-Joseph, Hadas; Grossman, Hadas; Hasky, Noa; Uri-Belapolsky, Shiri; Stemmer, Salomon M; Chuderland, Dana; Shalgi, Ruth; Ben-Aharon, Irit

    2015-12-01

    Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies.

  2. Pigment Epithelium Derived Factor Peptide Protects Murine Hepatocytes from Carbon Tetrachloride-Induced Injury

    PubMed Central

    Shih, Shou-Chuan; Ho, Tsung-Chuan; Chen, Show-Li; Tsao, Yeou-Ping

    2016-01-01

    Fibrogenesis is induced by repeated injury to the liver and reactive regeneration and leads eventually to liver cirrhosis. Pigment epithelium derived factor (PEDF) has been shown to prevent liver fibrosis induced by carbon tetrachloride (CCl4). A 44 amino acid domain of PEDF (44-mer) was found to have a protective effect against various insults to several cell types. In this study, we investigated the capability of synthetic 44-mer to protect against liver injury in mice and in primary cultured hepatocytes. Acute liver injury, induced by CCl4, was evident from histological changes, such as cell necrosis, inflammation and apoptosis, and a concomitant reduction of glutathione (GSH) and GSH redox enzyme activities in the liver. Intraperitoneal injection of the 44-mer into CCl4-treated mice abolished the induction of AST and ALT and markedly reduced histological signs of liver injury. The 44-mer treatment can reduce hepatic oxidative stress as evident from lower levels of lipid hydroperoxide, and higher levels of GSH. CCl4 caused a reduction of Bcl-xL, PEDF and PPARγ, which was markedly restored by the 44-mer treatment. Consequently, the 44-mer suppressed liver fibrosis induced by repeated CCl4 injury. Furthermore, our observations in primary culture of rat hepatocytes showed that PEDF and the 44-mer protected primary rat hepatocytes against apoptosis induced by serum deprivation and TGF-β1. PEDF/44-mer induced cell protective STAT3 phosphorylation. Pharmacological STAT3 inhibition prevented the antiapoptotic action of PEDF/44-mer. Among several PEDF receptor candidates that may be responsible for hepatocyte protection, we demonstrated that PNPLA2 was essential for PEDF/44-mer-mediated STAT3 phosphorylation and antiapoptotic activity by using siRNA to selectively knockdown PNPLA2. In conclusion, the PEDF 44-mer protects hepatocytes from single and repeated CCl4 injury. This protective effect may stem from strengthening the counter oxidative stress capacity and

  3. Leukostasis and pigment epithelium-derived factor in rat models of diabetic retinopathy

    PubMed Central

    Matsuoka, Masato; Minamino, Keizo; Matsumura, Miyo

    2007-01-01

    Purpose Spontaneously diabetic Torii (SDT) rats, an animal model of type 2 diabetes, have a low incidence of neovascular formation and an absence of non-perfused areas in their retinas at the proliferative stage that presents tractional retinal detachment with fibrous proliferation. The aim of this study was to determine whether leukostasis is present in the retina, to evaluate the levels of pigment epithelium-derived factor (PEDF) and intracellular adhesion molecule-1 (ICAM-1) levels in the blood of SDT rats, and to examine the effects of PEDF on leukostasis. Methods SDT rats, streptozotocin-induced diabetic (STZ) rats, and control Sprague-Dawley (SD) rats were studied. The index of leukostasis in the retina was determined immunohistochemically by counting the number of labeled adherent leukocytes. The levels of PEDF and the soluble intracellular adhesion molecule (sICAM)-1 in the plasma were measured. To investigate the effect of PEDF and vascular endothelial growth factor (VEGF) on leukostasis, the adhesion of monocytes to human umbilical vein endothelial cells (HUVECs) was assayed in vitro. Results SDT and STZ diabetic rats showed a significant increase of retinal leukostasis compared to that of control SD rats, but SDT rats had noteworthy lower levels of leukostasis than STZ rats in long term experiments. The sICAM-1 levels and PEDF expression were up-regulated in both STZ and SDT rats, but the SDT rats showed significantly higher levels of PEDF than STZ rats. In vitro studies showed that exposure of HUVECs to VEGF increased the number of adhering monocytes, and PEDF inhibited the VEGF-induced leukostasis in a dose-dependent manner. Conclusions The inhibition of the VEGF-induced leukostasis by PEDF is most likely responsible for the low incidence of capillary occlusion and retinal neovascularization in SDT rats. PMID:17653050

  4. Pigment epithelium-derived factor and matrix metalloproteinase-9 in liver cirrhosis.

    PubMed

    Kozlowska, Joanna; Mikula, Tomasz; Suchacz, Magdalena; Jabnonska, Joanna; Stanczak, Wojciech; Cianciara, Janusz; Wiercinska-Drapalo, Alicja

    2016-01-01

    The aim of this study was to assess the role of serum pigment epithelium-derived factor (PEDF) and matrix metalloproteinase-9 (MMP-9) in progression of liver cirrhosis and development of hepatocellular carcinoma (HCC). Serum levels of PEDF and MMP-9 were tested in 212 patients with liver cirrhosis and in a control group of 30 healthy volunteers. HCC was diagnosed in 45 of the 212 patients studied (21%). Serum PEDF and MMP-9 were higher in the study group than that in the control group (P < 0.001). In patients with alcoholic or mixed (alcoholic and viral hepatitis-related) cirrhosis, serum PEDF was higher than that in other patients (13970.2 ± 13406.9 ng/ml vs. 8563.5 ± 9602.7 ng/ml, P = 0.008). In patients with viral hepatitis-related cirrhosis, significantly higher PEDF levels were recorded in those with HCC (13429.1 ± 12045.8) than that in patients without HCC (6660.1 ± 7927.1; P = 0.04). There was a trend for higher serum MMP-9 in patients with HCC (5778.7 ± 12426.6 vs. 1389.8 ± 1944.7 in those without HCC; P = 0.07). Significant negative correlation between serum MMP-9 and serum alpha-fetoprotein in patients with HCC was observed (r = -0.54; P = 0.04). Serum PEDF and MMP-9 could be auxiliary markers in diagnosis of HCC, especially in patients with low alpha-fetoprotein level. Alcohol consumption can affect serum PEDF.

  5. Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin.

    PubMed

    Hong, Honghai; Zhou, Ti; Fang, Shuhuan; Jia, Minghan; Xu, Zumin; Dai, Zhiyu; Li, Cen; Li, Shuai; Li, Lei; Zhang, Ting; Qi, Weiwei; Bardeesi, Adham Sameer A; Yang, Zhonghan; Cai, Weibin; Yang, Xia; Gao, Guoquan

    2014-11-01

    Pigment epithelium-derived factor (PEDF) plays an important role in the tumor growth and metastasis inhibition. It has been reported that PEDF expression is significantly reduced in breast cancer, and associated with disease progression and poor patient outcome. However, the exact mechanism of PEDF on breast cancer metastasis including liver and lung metastasis remains unclear. The present study aims to reveal the impact of PEDF on breast cancer. The orthotopic tumor mice model inoculated by MDA-MB-231 cells stably expressing PEDF or control cells was used to assess liver and lung metastasis of breast cancer. In vitro, migration and invasion experiments were used to detect the metastatic abilities of MDA-MB-231 and SKBR3 breast cancer cells with or without overexpression of PEDF. The metastatic-related molecules including EMT makers, fibronectin, and p-AKT and p-ERK were detected by qRT-PCR, Western blot, and Fluorescent immunocytochemistry. PEDF significantly inhibited breast cancer growth and metastasis in vivo and in vitro. Mechanically, PEDF inhibited breast cancer cell migration and invasion by down-regulating fibronectin and subsequent MMP2/MMP9 reduction via p-ERK and p-AKT signaling pathways. However, PEDF had no effect on EMT conversion in the breast cancer cells which was usually involved in cancer metastasis. Furthermore, the study showed that laminin receptor mediated the down-regulation of fibronectin by PEDF. These results reported for the first time that PEDF inhibited breast cancer metastasis by down-regulating fibronectin via laminin receptor/AKT/ERK pathway. Our findings demonstrated PEDF as a dual effector in limiting breast cancer growth and metastasis and highlighted a new avenue to block breast cancer progression.

  6. Relationship between pigment epithelium-derived factor (PEDF) and renal function in patients with diabetic retinopathy

    PubMed Central

    Matsuyama, Kayako; Matsuoka, Masato; Shima, Chieko; Wada, Mitsumasa; Jo, Nobuo; Matsumura, Miyo

    2008-01-01

    Purpose Diabetic retinopathy and nephropathy are microvascular complications in patients with diabetes that are considered to be related. Pigment epithelium-derived factor (PEDF), a strong inhibitor of angiogenesis, is significantly elevated in the blood of diabetic patients, especially those with proliferative diabetic retinopathy (PDR). The level of PEDF in the blood, on the other hand, is reported to be low in a diabetic nephropathy. The aim of this study was to determine the relationship between PEDF and renal function in patients with diabetic retinopathy. Methods A total of 243 type 2 diabetic patients were studied. The relationship between the diabetic retinopathy and levels of PEDF, HbA1c, blood urea nitrogen (BUN), and creatinine were evaluated. Results The mean plasma PEDF level in patients with PDR (7.69±6.14 µg/ml; mean±standard error) was significantly higher than that of mild-to-moderate nonproliferative diabetic retinopathy (5.07±4.37 µg/ml, p=0.02). The level of BUN and creatinine increased significantly as the stage of diabetic retinopathy advanced. The plasma PEDF levels were significantly correlated with the levels of BUN and creatinine (r=0.54, p<0.0001; r=0.57, p<0.0001, respectively). Conclusions The levels of plasma PEDF increases with advances in both diabetic retinopathy and nephropathy. Thus, increased levels of PEDF in the blood may indicate microvascular damages in diabetic patients and may be predictor of the progression of retinopathy and nephropathy. PMID:18523656

  7. Molecular phylogeny of the antiangiogenic and neurotrophic serpin, pigment epithelium derived factor in vertebrates

    PubMed Central

    Xu, Xuming; Zhang, Samuel Shao-Min; Barnstable, Colin J; Tombran-Tink, Joyce

    2006-01-01

    Background Pigment epithelium derived factor (PEDF), a member of the serpin family, regulates cell proliferation, promotes survival of neurons, and blocks growth of new blood vessels in mammals. Defining the molecular phylogeny of PEDF by bioinformatic analysis is one approach to understanding the link between its gene structure and its function in these biological processes. Results From a comprehensive search of available DNA databases we identified a single PEDF gene in all vertebrate species examined. These included four mammalian and six non-mammalian vertebrate species in which PEDF had not previously been described. A five gene cluster around PEDF was found in an approximate 100 kb region in mammals, birds, and amphibians. In ray-finned fish these genes are scattered over three chromosomes although only one PEDF gene was consistently found. The PEDF gene is absent in invertebrates including Drosophila melanogaster (D. melanogaster), Caenorhabditis elegans (C. elegans), and sea squirt (C. intestinalis). The PEDF gene is transcribed in all vertebrate phyla, suggesting it is biologically active throughout vertebrate evolution. The multiple actions of PEDF are likely conserved in evolution since it has the same gene structure across phyla, although the size of the gene ranges from 48.3 kb in X. tropicalis to 2.9 kb in fugu, with human PEDF at a size of 15.6 kb. A strong similarity in the proximal 200 bp of the PEDF promoter in mammals suggests the existence of a possible regulatory region across phyla. Using a non-synonymous/synonymous substitution rate ratio we show that mammalian and fish PEDFs have similar ratios of <0.13, reflecting a strong purifying selection of PEDF gene. A large number of repetitive transposable elements of the SINE and LINE class were found with random distribution in both the promoter and introns of mammalian PEDF. Conclusion The PEDF gene first appears in vertebrates and our studies suggest that the regulation and biological actions

  8. Pigment epithelium-derived factor increases in type 2 diabetes after treatment with metformin.

    PubMed

    Akın, Safak; Aksoy, Duygu Yazgan; Cınar, Neşe; Aydın, Kadriye; Karaağaoğlu, Ergun; Arıyürek, Macit; Gülçelik, Neşe Ersöz; Usman, Aydan; Gürlek, Alper

    2012-12-01

    Pigment epithelium-derived factor (PEDF) has anti-angiogenic, immunomodulatory and anti-inflammatory properties. In addition to the significant role it plays in reducing diabetic complications, PEDF is now used in the treatment of certain cancers. It possibly plays a role in insulin resistance cases, too. However, whether metformin treatment has any significant effects on PEDF levels is not known. In this study, we investigated the regulation of PEDF in type 2 diabetes in relation to fat mass and insulin resistance before and after the use of metformin for treatment. Prospective cohort study. Thirty-six patients with newly diagnosed type 2 diabetes and 33 healthy individuals. Baseline weight, waist circumference (WC), fasting (FPG) and postprandial (PPPG) glucose, insulin, HbA1c, HOMA, PEDF and total/truncal fat mass were determined both in the diabetic and control subjects. Procedures were repeated in the diabetic group after a 6-month metformin treatment. Baseline FPG, PPPG, HbA1c, HOMA, weight, WC and truncal fat mass were higher in patients with diabetes whereas PEDF levels were found to be comparable with the controls. We completed the study with 31 of the 36 patients with diabetes we had selected for the study. We observed a decrease in the weight, WC, FPG, PPPG, HOMA, total and truncal fat mass of the patients while there was a significant rise in the PEDF levels (P = 0·002) after the metformin treatment. On the other hand, no significant correlation was observed between the change in PEDF levels and the clinical and laboratory findings. Our study is the first to identify a metformin-related increase in PEDF levels in diabetes. The increase observed in PEDF levels after the metformin treatment does not seem to be related to the changes in insulin resistance, fat mass or glycemic control. Hence, our results suggest that further investigation is necessary to determine the direct effects of metformin on PEDF gene and protein expression in vitro. © 2012

  9. Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

    PubMed

    Castro-Garcia, Paola; Díaz-Moreno, María; Gil-Gas, Carmen; Fernández-Gómez, Francisco J; Honrubia-Gómez, Paloma; Álvarez-Simón, Carmen Belén; Sánchez-Sánchez, Francisco; Cano, Juan Carlos Castillo; Almeida, Francisco; Blanco, Vicente; Jordán, Joaquín; Mira, Helena; Ramírez-Castillejo, Carmen

    2015-04-01

    We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model. © FASEB.

  10. Pigment epithelium-derived factor and its phosphomimetic mutant induce JNK-dependent apoptosis and p38-mediated migration arrest.

    PubMed

    Konson, Alexander; Pradeep, Sunila; D'Acunto, Cosimo Walter; Seger, Rony

    2011-02-04

    Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (ECs), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing a stronger effect. The stronger activity of EEE-PEDF was correlated with a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found regulated by differential activation of two distinct MAPK pathways, namely JNK and p38, respectively. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, whereas p38 leads to anti-migratory effect in both EC and cancer cells. These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.

  11. Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

    PubMed

    Ho, Tsung-Chuan; Chen, Show-Li; Shih, Shou-Chuan; Chang, Shing-Jyh; Yang, Su-Lin; Hsieh, Jui-Wen; Cheng, Huey-Chuan; Chen, Lee-Jen; Tsao, Yeou-Ping

    2011-10-14

    Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

  12. Pigment epithelium-derived factor (PEDF): a novel trophoblast-derived factor limiting feto-placental angiogenesis in late pregnancy.

    PubMed

    Loegl, Jelena; Nussbaumer, Erika; Hiden, Ursula; Majali-Martinez, Alejandro; Ghaffari-Tabrizi-Wizy, Nassim; Cvitic, Silvija; Lang, Ingrid; Desoye, Gernot; Huppertz, Berthold

    2016-07-01

    The rapidly expanding feto-placental vasculature needs tight control by paracrine and endocrine mechanisms. Here, we focused on paracrine influence by trophoblast, the placental epithelium. We aimed to identify differences in regulation of feto-placental angiogenesis in early versus late pregnancy. To this end, the effect of conditioned media (CM) from early and late pregnancy human trophoblast was tested on network formation, migration and proliferation of human feto-placental endothelial cells. Only CM of late pregnancy trophoblast reduced network formation and migration. Screening of trophoblast transcriptome for anti-angiogenic candidates identified pigment epithelium-derived factor (PEDF) with higher expression and protein secretion in late pregnancy trophoblast. Addition of a PEDF-neutralizing antibody restored the anti-angiogenic effect of CM from late pregnancy trophoblast. Notably, human recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two key players in VEGF-induced proliferation and migration, revealed that PEDF altered VEGF signaling, while PEDF alone did not affect phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and targets to modulate the intracellular effect of VEGF.

  13. Regulatory and Functional Connection of Microphthalmia-Associated Transcription Factor and Anti-Metastatic Pigment Epithelium Derived Factor in Melanoma1

    PubMed Central

    Fernández-Barral, Asunción; Orgaz, Jose Luis; Baquero, Pablo; Ali, Zaheer; Moreno, Alberto; Tiana, María; Gómez, Valentí; Riveiro-Falkenbach, Erica; Cañadas, Carmen; Zazo, Sandra; Bertolotto, Corine; Davidson, Irwin; Rodríguez-Peralto, Jose Luis; Palmero, Ignacio; Rojo, Federico; Jensen, Lasse Dahl; del Peso, Luis; Jiménez, Benilde

    2014-01-01

    Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma. PMID:25030625

  14. Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization.

    PubMed Central

    Petersen, Steen V; Valnickova, Zuzana; Enghild, Jan J

    2003-01-01

    Pigment epithelium-derived factor (PEDF) inhibits the formation of blood vessels in the eye by inducing apotosis in actively dividing endothelial cells. The activity of PEDF equals or supersedes that of other anti-angiogenic factors, including angiostatin, endostatin and thrombospondin-1. In addition, PEDF has the potential to promote the survival of neurons and affect their differentiation. Here we show that PEDF is present in plasma at a concentration of approx. 100 nM (5 microg/ml) or twice the level required to inhibit aberrant blood-vessel growth in the eye. Thus the systemic delivery of PEDF has the potential to affect angiogenesis or neurotrophic processes throughout the body, significantly expanding the putative physiological role of the protein. A complete map of all post-translational modifications revealed that authentic plasma PEDF carries an N-terminal pyroglutamate blocking group and an N-linked glycan at position Asn266. The pyroglutamate residue may regulate the activity of PEDF analogously to the manner in which it regulates thyrotropin-releasing hormone. PMID:12737624

  15. Alzheimer's disease: Elevated pigment epithelium-derived factor in the cerebrospinal fluid is mostly of systemic origin.

    PubMed

    Lang, Veronika; Zille, Marietta; Infante-Duarte, Carmen; Jarius, Sven; Jahn, Holger; Paul, Friedemann; Ruprecht, Klemens; Pina, Ana Luisa

    2017-04-15

    Pigment-epithelium derived factor (PEDF) is a neurotrophic factor with neuroprotective, anti-tumorigenic, and anti-angiogenic effects. Elevated levels of PEDF have previously been proposed as a cerebrospinal fluid (CSF) biomarker for Alzheimer's disease. However, the origin of PEDF in CSF, i.e. whether it is derived from the brain or from the systemic circulation, and the specificity of this finding hitherto remained unclear. Here, we analyzed levels of PEDF in paired CSF and serum samples by ELISA in patients with Alzheimer's disease (AD, n=12), frontotemporal dementia (FTD, n=6), vascular dementia (n=4), bacterial meningitis (n=8), multiple sclerosis (n=32), pseudotumor cerebri (n=36), and diverse non-inflammatory neurological diseases (n=19). We established CSF/serum quotient diagrams to determine the fraction of intrathecally synthesized PEDF in CSF. We found that PEDF is significantly increased in CSF of patients with AD, FTD, and bacterial meningitis. Remarkably, PEDF concentrations were also significantly elevated in serum of patients with AD. CSF/serum quotient diagrams demonstrated that elevated PEDF concentrations in CSF of patients with AD are mostly due to elevated PEDF concentrations in serum. These findings underscore the importance of relating concentrations of proteins in CSF to their respective concentrations in serum to avoid erroneous interpretations of increased protein concentrations in lumbar CSF.

  16. Identification of BPIFA1/SPLUNC1 as an epithelium-derived smooth muscle relaxing factor

    PubMed Central

    Wu, Tongde; Huang, Julianne; Moore, Patrick J.; Little, Michael S.; Walton, William G.; Fellner, Robert C.; Alexis, Neil E.; Peter Di, Y.; Redinbo, Matthew R.; Tilley, Stephen L.; Tarran, Robert

    2017-01-01

    Asthma is a chronic airway disease characterized by inflammation, mucus hypersecretion and abnormal airway smooth muscle (ASM) contraction. Bacterial permeability family member A1, BPIFA1, is a secreted innate defence protein. Here we show that BPIFA1 levels are reduced in sputum samples from asthmatic patients and that BPIFA1 is secreted basolaterally from healthy, but not asthmatic human bronchial epithelial cultures (HBECs), where it suppresses ASM contractility by binding to and inhibiting the Ca2+ influx channel Orai1. We have localized this effect to a specific, C-terminal α-helical region of BPIFA1. Furthermore, tracheas from Bpifa1−/− mice are hypercontractile, and this phenotype is reversed by the addition of recombinant BPIFA1. Our data suggest that BPIFA1 deficiency in asthmatic airways promotes Orai1 hyperactivity, increased ASM contraction and airway hyperresponsiveness. Strategies that target Orai1 or the BPIFA1 deficiency in asthma may lead to novel therapies to treat this disease. PMID:28165446

  17. Pigment Epithelium-derived Factor (PEDF) Blocks Wnt3a Protein-induced Autophagy in Pancreatic Intraepithelial Neoplasms.

    PubMed

    Gong, Jingjing; Belinsky, Glenn; Sagheer, Usman; Zhang, Xuchen; Grippo, Paul J; Chung, Chuhan

    2016-10-14

    An increase in autophagy characterizes pancreatic carcinogenesis, but the signals that regulate this process are incompletely understood. Because canonical Wnt/β-catenin signaling is necessary for the transition from early to advanced pancreatic intraepithelial neoplasia (PanIN) lesions, we assessed whether Wnt ligands and endogenous inhibitors of Wnt signaling modulate autophagy. In this study, canonical Wnt3a ligand induced autophagy markers and vacuoles in murine PanIN cells. Furthermore, pigment epithelium-derived factor (PEDF), a secreted glycoprotein known for its anti-tumor properties, blocked Wnt3a-directed induction of autophagy proteins. Autophagy inhibition was complemented by reciprocal regulation of the oxidative stress enzymes, superoxide dismutase 2 (SOD2) and catalase. Transcriptional control of Sod2 expression was mediated by PEDF-induced NFκB nuclear translocation. PEDF-dependent SOD2 expression in PanIN lesions was recapitulated in a murine model of PanIN formation where PEDF was deleted. In human PanIN lesions, co-expression of PEDF and SOD2 was observed in the majority of early PanIN lesions (47/50, 94%), whereas PEDF and SOD2 immunolocalization in high-grade human PanIN-2/3 was uncommon (7/50, 14%). These results indicate that PEDF regulates autophagy through coordinate Wnt signaling blockade and NFκB activation.

  18. Anti-tumor effects of pigment epithelium-derived factor (PEDF): implication for cancer therapy. A mini-review.

    PubMed

    Belkacemi, Louiza; Zhang, Shaun Xiaoliu

    2016-01-08

    Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein and a non-inhibitory member of the serine protease inhibitor (serpin) family. It is widely expressed in human fetal and adult tissues but its expression decreases with age and in malignant tissues. The main anti-cancer activities of PEDF derive from its dual effects, either indirectly on the tumor microenvironment (indirect antitumor action) or directly on the tumor itself (direct antitumor influence). The indirect antitumor activities of PEDF were uncovered from the early findings that it stimulates retinoblastoma cell differentiation and that additionally it possesses anti-angiogenic, anti-tumorigenic and anti-metastatic properties. The mechanisms of its direct antitumor effect, however, have not been fully elucidated. This review highlights recent progress in our understanding of the multifunctional activities of PEDF and, in particular, its anti-cancer signaling mechanisms. Additionally, we discuss the possibility of using novel phosphaplatin compounds that can upregulate PEDF expression as a chemotherapy for cancer treatment.

  19. Host pigment epithelium-derived factor (PEDF) prevents progression of liver metastasis in a mouse model of uveal melanoma.

    PubMed

    Lattier, John M; Yang, Hua; Crawford, Susan; Grossniklaus, Hans E

    2013-12-01

    Uveal melanoma (UM) has a 30 % 5-year mortality rate, primarily due to liver metastasis. Both angiogenesis and stromagenesis are important mechanisms for the progression of liver metastasis. Pigment epithelium-derived factor (PEDF), an anti-angiogenic and anti-stromagenic protein, is produced by hepatocytes. Exogenous PEDF suppresses metastasis progression; however, the effects of host-produced PEDF on metastasis progression are unknown. We hypothesize that host PEDF inhibits liver metastasis progression through a mechanism involving angiogenesis and stromagenesis. Mouse melanoma cells were injected into the posterior ocular compartment of PEDF-null mice and control mice. After 1 month, the number, size, and mean vascular density (MVD) of liver metastases were determined. The stromal component of hepatic stellate cells (HSCs) and the type III collagen they produce was evaluated by immunohistochemistry. Host PEDF inhibited the total area of liver metastasis and the frequency of macrometastases (diameter >200 μm) but did not affect the total number of metastases. Mice expressing PEDF exhibited significantly lower MVD and less type III collagen production in metastases. An increase in activated HSCs was seen in the absence of PEDF, but this result was not statistically significant. In conclusion, host PEDF inhibits the progression of hepatic metastases in a mouse model of UM, and loss of PEDF is accompanied by an increase in tumor blood vessel density and type III collagen.

  20. Pigment epithelium-derived factor: clinical significance in estrogen-dependent tissues and its potential in cancer therapy

    PubMed Central

    Franco-Chuaire, María Liliana; Ramírez-Clavijo, Sandra; Chuaire-Noack, Lilian

    2015-01-01

    Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the family of non-inhibitory serpins. The broad spectrum of PEDF biological activity is evident when considering its effects in promoting cell survival and proliferation, as well as its antiangiogenic, antitumor, and anti-metastatic properties. Although the structural domains of the PEDF gene that mediate such diverse effects and their mechanisms of action have not been completely elucidated, there is a large body of evidence describing their diverse range of activities; this evidence combined with the regulation of PEDF expression by sex steroids and their receptors have led to the idea that PEDF is not only a diagnostic and prognostic marker for certain diseases such as cancer, but is also a potential therapeutic target. In this manner, this paper aims to generally review the regulation of PEDF expression and PEDF interactions, as well as the findings that relate PEDF to the role of estrogens and estrogen receptors. In addition, this manuscript will review major advances toward potential therapeutic applications of PEDF. PMID:26523216

  1. Pigment epithelium-derived factor enhances tumor response to radiation through vasculature normalization in allografted lung cancer in mice.

    PubMed

    Xu, Z; Dong, Y; Peng, F; Yu, Z; Zuo, Y; Dai, Z; Chen, Y; Wang, J; Hu, X; Zhou, Q; Ma, H; Bao, Y; Gao, G; Chen, M

    2015-03-01

    This study aimed to explore the potential therapeutic effects of the combination of pigment epithelium-derived factor (PEDF) and radiation on lung cancer. The Lewis lung cancer (LLC) allografts in nude mice were treated with radiation, PEDF and PEDF combined with radiation. The morphologic changes of tumor vasculature and the hypoxic fraction of tumor tissues were evaluated. Significant inhibition of tumor growth was observed when radiation was applied between the 3rd and 7th day (the vasculature normalization window) after the initiation of PEDF treatment. During the vasculature normalization window, the tumor blood vessels in PEDF-treated mice were less tortuous and more uniform than those in the LLC allograft tumor treated with phosphate-buffered saline. Meanwhile, the thickness of the basement membrane was remarkably reduced and pericyte coverage was significantly increased with the PEDF treatment. We also found that tumor hypoxic fraction decreased during the 3rd to the 7th day after PEDF treatment, suggesting improved intratumoral oxygenation. Taken together, our results show that PEDF improved the effects of radiation therapy on LLC allografts by inducing a vascular normalization window from the 3rd to the 7th day after PEDF treatment. Our findings provide a basis for treating lung cancer with the combination of PEDF and radiation.

  2. Pigment epithelium-derived factor delays cellular senescence of human mesenchymal stem cells in vitro by reducing oxidative stress.

    PubMed

    Cao, Yukun; Yang, Ting; Gu, Chunhu; Yi, Dinghua

    2013-04-01

    Mesenchymal stem cells (MSCs) are multipotent progenitor cells that represent a promising approach in the field of regenerative medicine; however, this potential diminishes with senescence. Pigment epithelium-derived factor (PEDF) gives some protection by reducing oxidative stress, which is known to accelerate cellular senescence. Thus we hypothesized that PEDF could delay senescence during MSC expansion by reducing oxidative stress. Proliferation and differentiation potentials, oxidative stress, senescence and p53/p16 expressions have been examined. In MSCs cultured under normoxic conditions treated with PEDF, proliferative lifespan in vitro was significantly increased compared with control group not given PEDF, with ∼10 additional population doublings (PD) occurring before terminal growth arrest. Most of the MSCs cultured under normoxic conditions ceased to proliferate after 20-28 PD, while few senescent cells were found in the hypoxic, PEDF-hypoxic and PEDF-normoxic cultures; this was associated with downregulation of p53 and p16 expression and decreased oxidative stress. PEDF also preserved differentiation potentials of MSCs compared with the control group. Thus PEDF suppression of oxidative stress delays cellular senescence and allows greater expansion of MSCs.

  3. Pigment epithelium-derived factor, insulin sensitivity, and adiposity in polycystic ovary syndrome: impact of exercise training.

    PubMed

    Joham, Anju E; Teede, Helena J; Hutchison, Samantha K; Stepto, Nigel K; Harrison, Cheryce L; Strauss, Boyd J; Paul, Eldho; Watt, Matthew J

    2012-12-01

    Pigment epithelium-derived factor (PEDF) is upregulated in obese rodents and is involved in the development of insulin resistance (IR). We aim to explore the relationships between PEDF, adiposity, insulin sensitivity, and cardiovascular risk factors in obese women with polycystic ovary syndrome (PCOS) and weight-matched controls and to examine the impact of endurance exercise training on PEDF. This prospective cohort intervention study was based at a tertiary medical center. Twenty obese PCOS women and 14 non-PCOS weight-matched women were studied at baseline. PEDF, cardiometabolic markers, detailed body composition, and euglycemic-hyperinsulinemic clamps were performed and measures were repeated in 10 PCOS and 8 non-PCOS women following 12 weeks of intensified aerobic exercise. Mean glucose infusion rate (GIR) was 31.7% lower (P = 0.02) in PCOS compared to controls (175.6 ± 96.3 and 257.2 ± 64.3 mg.m(-2).min(-1)) at baseline, yet both PEDF and BMI were similar between groups. PEDF negatively correlated to GIR (r = -0.41, P = 0.03) and high-density lipoprotein (HDL) (r = -0.46, P = 0.01), and positively to cardiovascular risk factors, systolic (r = 0.41, P = 0.02) and diastolic blood pressure (r = 0.47, P = 0.01) and triglycerides (r = 0.49, P = 0.004). The correlation with GIR was not significant after adjusting for fat mass (P = 0.07). Exercise training maintained BMI and increased GIR in both groups; however, plasma PEDF was unchanged. In summary, PEDF is not elevated in PCOS, is not associated with IR when adjusted for fat mass, and is not reduced by endurance exercise training despite improved insulin sensitivity. PEDF was associated with cardiovascular risk factors, suggesting PEDF may be a marker of cardiovascular risk status.

  4. Insulin resistance is associated with elevated serum pigment epithelium-derived factor (PEDF) levels in morbidly obese patients.

    PubMed

    Gattu, Arijeet K; Birkenfeld, Andreas L; Jornayvaz, Francois; Dziura, James; Li, Fangyong; Crawford, Susan E; Chu, Xin; Still, Christopher D; Gerhard, Glenn S; Chung, Chuhan; Samuel, Varman

    2012-12-01

    Obesity is a significant risk factor for developing diabetes. Pigment epithelium-derived factor (PEDF) has been identified by experimental and clinical studies as both a causative and counter-regulatory factor in the metabolic syndrome. We set out to determine whether serum PEDF levels correlated with the degree of insulin resistance in morbidly obese patients. Sera from 53 patients who were evaluated prior to gastric bypass surgery were analyzed for PEDF levels using a commercial ELISA. None of the patients were on diabetes medications prior to enrollment. Baseline data included BMI, serum glucose and insulin, and homeostasis model assessment (HOMA) scores. Patients were stratified based on HOMA score and glucose levels into three groups: insulin sensitive (IS): HOMA <2 and glucose <126; insulin resistant (IR): HOMA >2 and glucose ≤126; and diabetes mellitus (DM): HOMA >2 and glucose >126. Pre- and post-gastric bypass sera from 12 patients were obtained for serial assessment of metabolic parameters and PEDF levels. PEDF secretion was assessed in primary human hepatocytes, HCC cells, and cultured adipocytes in the absence and presence of high glucose media. No significant differences in age, gender, and BMI were found among the three groups. PEDF levels were similar between IR patients and the other groups, but were significantly higher in DM compared to IS patients (p = 0.01). Serum PEDF in individual patients declined significantly after gastric bypass (p = 0.006). High glucose media led to significantly higher PEDF release by human hepatocytes in vitro (p = 0.016). These data demonstrate that serum PEDF concentrations better relate to insulin resistance than to adiposity and suggest that PEDF expression is closely linked to the development of insulin resistance.

  5. Eye drop delivery of pigment epithelium-derived factor-34 promotes retinal ganglion cell neuroprotection and axon regeneration.

    PubMed

    Vigneswara, Vasanthy; Esmaeili, Maryam; Deer, Louise; Berry, Martin; Logan, Ann; Ahmed, Zubair

    2015-09-01

    Axotomised retinal ganglion cells (RGCs) die rapidly by apoptosis and fail to regenerate because of the limited availability of neurotrophic factors and a lack of axogenic stimuli. However, we have recently showed that pigment epithelium-derived factor (PEDF) promotes RGC survival and axon regeneration after optic nerve crush injury. PEDF has multiple fragments of the native peptide that are neuroprotective, anti-angiogenic and anti-inflammatory. Here we investigated the neuroprotective and axogenic properties of a fragment of PEDF, PEDF-34, in retinal neurons in vitro and when delivered by intravitreal injection and eye drops in vivo. We found that PEDF-34 was 43% more neuroprotective and 52% more neuritogenic than PEDF-44 in vitro. Moreover, in vivo, intravitreal delivery of 1.88nM PEDF-34 was 71% RGC neuroprotective at 21days after optic nerve crush compared to intact controls, whilst daily eye drops containing 1.88nM PEDF-34 promoted 87% RGC survival. After topical eye drop delivery, PEDF-34 was detected in the vitreous body within 30min and attained physiologically relevant concentrations in the retina by 4h peaking at 1.4±0.05nM by 14days. In eye drop- compared to intravitreal-treated PEDF-34 animals, 55% more RGC axons regenerated 250μm beyond the optic nerve lesion. We conclude that daily topical eye drop application of PEDF-34 is superior to weekly intravitreal injections in promoting RGC survival and axon regeneration through both direct effects on retinal neurons and indirect effects on other retinal cells. Copyright © 2015. Published by Elsevier Inc.

  6. Pigment Epithelium-Derived Factor Inhibits Retinal Microvascular Dysfunction Induced By 12/15-Lipoxygenase-Derived Eicosanoids

    PubMed Central

    Ibrahim, Ahmed S.; Tawfik, Amany M.; Hussein, Khaled A; Elshafey, Sally; Markand, Shanu; Rizk, Nasser; Duh, Elia J.; Smith, Sylvia B.; Al-Shabrawey, Mohamed

    2015-01-01

    We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and Pigment Epithelium-Derived Factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12/15HETE with/without PEDF. Thereafter, Fluorescein Angiography (FA) was used to evaluate the vascular leakage, followed by Optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETE and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR

  7. Delayed Treatment with a Small Pigment Epithelium Derived Factor (PEDF) Peptide Prevents the Progression of Diabetic Renal Injury.

    PubMed

    Awad, Alaa S; You, Hanning; Gao, Ting; Gvritishvili, Anzor; Cooper, Timothy K; Tombran-Tink, Joyce

    2015-01-01

    Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.

  8. Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

    PubMed

    Yoshida, Takafumi; Akiba, Jun; Matsui, Takanori; Nakamura, Kazuo; Hisamoto, Takao; Abe, Mitsuhiko; Ikezono, Yu; Wada, Fumitaka; Iwamoto, Hideki; Nakamura, Toru; Koga, Hironori; Yamagishi, Sho-Ichi; Torimura, Takuji

    2017-06-01

    Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1β, IL-6, TGF-β, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

  9. Nucleotide structure and expression of equine pigment epithelium-derived factor during repair of experimentally induced wounds in horses.

    PubMed

    Ipiña, Zoë; Lussier, Jacques G; Theoret, Christine L

    2009-01-01

    To clone full-length equine pigment epithelium-derived factor (PEDF) complementary DNA (cDNA) and to evaluate its temporal expression during repair of wounds in horses. 4 clinically normal 2-to 3-year-old Standardbred mares. Full-length equine PEDF cDNA was cloned by screening size-selected cDNA libraries derived from biopsy specimens obtained from the wound edge 7 days after experimental creation of a 6.25-cm(2) full-thickness wound in the skin of the lateral thoracic wall. Expression was evaluated in normal skin and in biopsy specimens obtained weekly from experimentally induced wounds on the trunk and limbs of horses. Temporal gene expression was determined by use of reverse transcriptase PCR assay. Equine PEDF shared 87% sequence and 88% peptide homology with human PEDF. Wounding caused upregulation of PEDF mRNA, which did not return to baseline by the end of the study in either anatomic location. Relative overexpression was evident in wounds on the trunk, compared with expression for wounds on the limbs. This study characterized full-length equine cDNA for PEDF and determined that the gene for PEDF appeared to be upregulated in response to dermal wounding. Although the cause of exuberant granulation tissue is probably multifactorial, these data suggested that PEDF, via its potent antiangiogenic capabilities, may contribute to superior healing in wounds on the trunks of horses by protecting such wounds from excessive formation of vascular granulation tissue that characterizes wounds on the limbs of this species.

  10. Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ*S⃞

    PubMed Central

    Bartholomeeusen, Koen; Christ, Frauke; Hendrix, Jelle; Rain, Jean-Christophe; Emiliani, Stéphane; Benarous, Richard; Debyser, Zeger; Gijsbers, Rik; De Rijck, Jan

    2009-01-01

    Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function. PMID:19244240

  11. Genetic variants in pigment epithelium-derived factor influence response of polypoidal choroidal vasculopathy to photodynamic therapy.

    PubMed

    Nakata, Isao; Yamashiro, Kenji; Yamada, Ryo; Gotoh, Norimoto; Nakanishi, Hideo; Hayashi, Hisako; Tsujikawa, Akitaka; Otani, Atsushi; Ooto, Sotaro; Tamura, Hiroshi; Saito, Masaaki; Saito, Kuniharu; Iida, Tomohiro; Oishi, Akio; Kurimoto, Yasuo; Matsuda, Fumihiko; Yoshimura, Nagahisa

    2011-07-01

    with the AA genotype showed significantly worse visual prognosis after PDT (P = 0.013). Pigment epithelium-derived factor (SERPINF1 or PEDF) polymorphisms may influence the initial response to and visual prognosis after PDT for PCV. Our findings may lead to understanding the pathogenesis of PCV and modification of the effects of PDT. Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  12. [Effects of insulin on pigment epithelium-derived factor expression in adipocytes of type 2 diabetic mellitus in rats].

    PubMed

    Xu, Fen; Lin, Beisi; Zhou, Yinli; Deng, Hongrong; Bi, Yan; Liang, Hua

    2014-10-21

    To explore the effects of insulin therapy on the expression of pigment epithelium-derived factor (PEDF) in adipocytes of type 2 diabetic mellitus (T2DM) in rats. A total of 22 newly diagnosed type 2 diabetics received a 2-week intensive insulin therapy. The levels of fasting plasma glucose (FPG), serum triglyceride and PEDF were measured before and after therapy. T2DM was induced by a high-fat diet and a low-dose streptozotocin (STZ). The Spraque-Dawley rats were divided randomly into diabetic, insulin treatment and gliclazide treatment groups. Another group with a chow diet was designated as normal controls. Differentiated 3T3-L1 adipocytes were then incubated with tumor necrosis factor-alpha (TNF-α) and (or) insulin for 24 h. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of PEDF in adipose tissue or adipocytes. The PEDF levels in both sera and cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Glucose uptake was detected after treatment of PEDF or anti-PEDF antibody simultaneously together with insulin in mature 3T3-L1 adipocytes. Insulin therapy decreased the serum levels of FPG and triglyceride of T2DM patients ((12.9 ± 2.8) vs (5.9 ± 1.4) mmol/L, (3.1 ± 1.8) vs (1.7 ± 0.8) mmol/L, P < 0.05) while the serum level of PEDF decreased significantly after therapy ((22.85 ± 5.73) vs (18.38 ± 5.28) µg/L, P < 0.05). Consistently the serum level of PEDF of diabetic rats was remarkably higher than that of normal controls and insulin-treated group ((28.6 ± 0.5) vs (25.4 ± 0.6) and (25.3 ± 0.6) µg/L, P < 0.05). And the elevated levels of PEDF, TNF-α mRNA and protein in adipose tissue (P < 0.05) could be reduced by insulin treatment (P < 0.05). However, no obvious change was detected in gliclazide treatment group. Further evidences suggested that TNF-α could induce more secretion and expression of PEDF in 3T3-L1 adipocyte while this effect became ameliorated

  13. Influence of lactic acid on differential expression of vascular endothelial growth factor and pigment epithelium-derived factor in explants of rat retina.

    PubMed

    Zhu, Dongqing; Zhou, Jibo; Xu, Xun

    2012-11-01

    To investigate the influence of lactate on expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in rat retina. Retinal explants from neonatal Sprague Dawley rats were incubated with media containing 10, 20, or 30 mM of lactic acid. The 10 mM group was used as a control. At 24 h after incubation, retinas were sectioned for light microscopy, and expressions of VEGF and PEDF measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. The architecture of cultured retinas appeared to be intact. Compared with control, both RT-PCR and Western blot analysis showed that 30 mM of lactic acid significantly increased the levels of VEGF, but not PEDF. Stimulation of production of retinal VEGF by lactate is dependent on the concentration of lactate. Lactate has no effect on the expression of PEDF in rat retinal explants.

  14. Lens epithelium-derived growth factor/p75 qualifies as a target for HIV gene therapy in the NSG mouse model.

    PubMed

    Vets, Sofie; Kimpel, Janine; Volk, Andreas; De Rijck, Jan; Schrijvers, Rik; Verbinnen, Bert; Maes, Wim; Von Laer, Dorothee; Debyser, Zeger; Gijsbers, Rik

    2012-05-01

    Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.

  15. Lens Epithelium-derived Growth Factor/p75 Qualifies as a Target for HIV Gene Therapy in the NSG Mouse Model

    PubMed Central

    Vets, Sofie; Kimpel, Janine; Volk, Andreas; De Rijck, Jan; Schrijvers, Rik; Verbinnen, Bert; Maes, Wim; Von Laer, Dorothee; Debyser, Zeger; Gijsbers, Rik

    2012-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF325–530) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD4+ T-cells were transduced with lentiviral vectors encoding LEDGF325–530, the interaction-deficient mutant LEDGF325–530D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF325–530 overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD4+ T-cells expressing LEDGF325–530 in comparison with engraftment with LEDGF325–530D366N cells. Four weeks after transplantation with LEDGF325–530D366N cells, 70% of the CD4+ cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF325–530 cells only a 20% decrease in CD4+ cells was measured. Liver and spleen sections of LEDGF325–530 mice contained less HIV than LEDGF325–530D366N mice as measured by p24 antigen detection. LEDGF325–530 overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD4+ T-cells. PMID:22334021

  16. Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization.

    PubMed

    Li, Feng; Armstrong, Gillian B; Tombran-Tink, Joyce; Niyibizi, Christopher

    2016-09-23

    Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.

  17. Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells

    PubMed Central

    Kim, Tae Hoon; Hong, Jun Hee; Kim, Youn-Jae; Jin, Xiong; Kang, Sangjo; Jung, Ji-Eun; Kim, Jeong-Yub; Yun, Hyeongsun; Lee, Jeong Eun; Kim, Minkyung; Chung, Junho; Kim, Hyunggee; Nakano, Ichiro; Gwak, Ho-Shin; Yoo, Heon; Yoo, Byong Chul; Kim, Jong Heon; Hur, Eun-Mi; Lee, Jeongwu; Lee, Seung-Hoon; Park, Myung-Jin; Park, Jong Bae

    2015-01-01

    Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications. PMID:25992628

  18. Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

    PubMed

    Yin, Jinlong; Park, Gunwoo; Kim, Tae Hoon; Hong, Jun Hee; Kim, Youn-Jae; Jin, Xiong; Kang, Sangjo; Jung, Ji-Eun; Kim, Jeong-Yub; Yun, Hyeongsun; Lee, Jeong Eun; Kim, Minkyung; Chung, Junho; Kim, Hyunggee; Nakano, Ichiro; Gwak, Ho-Shin; Yoo, Heon; Yoo, Byong Chul; Kim, Jong Heon; Hur, Eun-Mi; Lee, Jeongwu; Lee, Seung-Hoon; Park, Myung-Jin; Park, Jong Bae

    2015-05-01

    Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications.

  19. Involvement of pigment epithelium-derived factor, docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery.

    PubMed

    Kenchegowda, S; He, J; Bazan, H E P

    2013-01-01

    Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier.

    PubMed

    Deeg, Cornelia A; Altmann, Frank; Hauck, Stefanie M; Schoeffmann, Stephanie; Amann, Barbara; Stangassinger, Manfred; Ueffing, Marius

    2007-05-01

    Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.

  1. Localization of the gene for pigment epithelium-derived factor (PEDF) to chromosome 17p13. 1 and expression in cultured human retinoblastoma cells

    SciTech Connect

    Tombran-Tink, J.; Rodriguez, I.; Chader, G.J. ); Pawar, H.; Swaroop, A. )

    1994-01-15

    The gene for pigment epithelium-derived factor (PEDF) was localized to chromosome 17 by the analysis of three independent somatic cell hybrid panels. Fluorescence in situ hybridization shows a specific hybridization signal at the terminal portion of the short arm of chromosome 17. PCR analysis of somatic cell hybrids containing specific regions of 17 was subsequently used to sublocalize PEDF to 17p13.1-pter. PEDF thus maps to a region containing a number of cancer-related loci and thus must be considered a candidate gene for these cancers. Preliminary studies with cultured human Y79 retinoblastoma cells indicate that expression of PEDF is associated with relatively undifferentiated, proliferating cells rather than their differentiated, slow-growing counterparts. This and the fact that the PEDF protein can act as a potent neurotrophic differentiating agent suggest that PEDF is linked to proliferative events that terminate in final phenotypic determination within specific cell lineages. 19 refs., 5 figs.

  2. Pigment epithelium-derived factor (PEDF) regulates metabolism and insulin secretion from a clonal rat pancreatic beta cell line BRIN-BD11 and mouse islets.

    PubMed

    Chen, Younan; Carlessi, Rodrigo; Walz, Nikita; Cruzat, Vinicius Fernandes; Keane, Kevin; John, Abraham N; Jiang, Fang-Xu; Carnagarin, Revathy; Dass, Crispin R; Newsholme, Philip

    2016-05-05

    Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat β-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced β-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation.

  3. Pigment Epithelium-Derived Factor (PEDF) Protects Osteoblastic Cell Line from Glucocorticoid-Induced Apoptosis via PEDF-R.

    PubMed

    Yao, Shengcheng; Zhang, Yingnan; Wang, Xiaoyu; Zhao, Fengchao; Sun, Maji; Zheng, Xin; Dong, Hongyan; Guo, Kaijin

    2016-05-13

    Pigment epithelial-derived factor (PEDF) is known as a widely expressed multifunctional secreted glycoprotein whose biological actions are cell-type dependent. Recent studies demonstrated that PEDF displays cytoprotective activity in several cell types. However, it remains unknown whether PEDF is involved in glucocorticoid-induced osteoblast death. The aim of this study was to examine the role of PEDF in osteoblast survival in response to dexamethasone, an active glucocorticoid analogue, and explore the underlying mechanism. In the present study, dexamethasone (DEX) was used to induce MC3T3-E1 pre-osteoblast apoptosis. PEDF mRNA and protein levels and cell apoptosis were determined respectively. Then PEDF receptor (PEDF-R)- and lysophosphatidic acid (LPA)-related signal transductions were assessed. Here we show that DEX down-regulates PEDF expression, which contributes to osteoblast apoptosis. As a result, exogenous recombinant PEDF (rPEDF) inhibited DEX-induced cell apoptosis. We confirmed that PEDF-R was expressed on MC3T3-E1 pre-osteoblast membrane and could bind to PEDF which increased the level of LPA and activated the phosphorylation of Akt. Our results suggest that PEDF attenuated DEX-induced apoptosis in MC3T3-E1 pre-osteoblasts through LPA-dependent Akt activation via PEDF-R.

  4. Pigment Epithelium-Derived Factor (PEDF) Protects Osteoblastic Cell Line from Glucocorticoid-Induced Apoptosis via PEDF-R

    PubMed Central

    Yao, Shengcheng; Zhang, Yingnan; Wang, Xiaoyu; Zhao, Fengchao; Sun, Maji; Zheng, Xin; Dong, Hongyan; Guo, Kaijin

    2016-01-01

    Pigment epithelial-derived factor (PEDF) is known as a widely expressed multifunctional secreted glycoprotein whose biological actions are cell-type dependent. Recent studies demonstrated that PEDF displays cytoprotective activity in several cell types. However, it remains unknown whether PEDF is involved in glucocorticoid-induced osteoblast death. The aim of this study was to examine the role of PEDF in osteoblast survival in response to dexamethasone, an active glucocorticoid analogue, and explore the underlying mechanism. In the present study, dexamethasone (DEX) was used to induce MC3T3-E1 pre-osteoblast apoptosis. PEDF mRNA and protein levels and cell apoptosis were determined respectively. Then PEDF receptor (PEDF-R)- and lysophosphatidic acid (LPA)-related signal transductions were assessed. Here we show that DEX down-regulates PEDF expression, which contributes to osteoblast apoptosis. As a result, exogenous recombinant PEDF (rPEDF) inhibited DEX-induced cell apoptosis. We confirmed that PEDF-R was expressed on MC3T3-E1 pre-osteoblast membrane and could bind to PEDF which increased the level of LPA and activated the phosphorylation of Akt. Our results suggest that PEDF attenuated DEX-induced apoptosis in MC3T3-E1 pre-osteoblasts through LPA-dependent Akt activation via PEDF-R. PMID:27187377

  5. C1q/TNF-related protein-3 (CTRP-3) and pigment epithelium-derived factor (PEDF) concentrations in patients with type 2 diabetes and metabolic syndrome.

    PubMed

    Choi, Kyung Mook; Hwang, Soon Young; Hong, Ho Cheol; Yang, Sae Jeong; Choi, Hae Yoon; Yoo, Hye Jin; Lee, Kwan Woo; Nam, Moon Suk; Park, Yong Soo; Woo, Jeong Taek; Kim, Young Seol; Choi, Dong Seop; Youn, Byung-Soo; Baik, Sei Hyun

    2012-11-01

    Recent studies have suggested that a novel adipokine, C1q/tumor necrosis factor-related protein-3 (CTRP-3), a paralog of adiponectin, may play an important role in the regulation of glucose metabolism and innate immunity. Pigment epithelium-derived factor (PEDF), a multifunctional protein with antioxidant and anti-inflammatory properties, is associated with insulin resistance and metabolic syndrome. We examined circulating CTRP-3 and PEDF concentrations in 345 subjects with diverse glucose tolerance statuses. Furthermore, we evaluated the involvement of CTRP-3 and PEDF with cardiometabolic risk factors including insulin resistance, high-sensitivity C-reactive protein (hsCRP), estimated glomerular filtration rate (eGFR), and brachial-ankle pulse wave velocity (baPWV). CTRP-3 concentrations were significantly higher in patients with type 2 diabetes or prediabetes than the normal glucose tolerance group, whereas PEDF levels were not different. Subjects with metabolic syndrome showed significantly higher levels of both CTRP-3 and PEDF compared with subjects without metabolic syndrome. Both CTRP-3 and PEDF were significantly associated with cardiometabolic parameters, including waist-to-hip ratio, triglycerides, HDL-cholesterol, alanine aminotransferase, eGFR, hsCRP, and baPWV. In conclusion, circulating CTRP-3 concentrations were elevated in patients with glucose metabolism dysregulation. Both CTRP-3 and PEDF concentrations were increased in subjects with metabolic syndrome and associated with various cardiometabolic risk factors.

  6. Adenoviral E4 Gene Stimulates Secretion of Pigmental Epithelium Derived Factor (PEDF) that Maintains Long-term Survival of Human Glomerulus-derived Endothelial Cells*

    PubMed Central

    Jerebtsova, Marina; Kumari, Namita; Obuhkov, Yuri; Nekhai, Sergei

    2012-01-01

    Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region also supported survival of human glomerulus-derived endothelial cells in serum-free media. Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT-PCR, Western blot and ELISA analysis. PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells. PMID:22915824

  7. Mesenchymal Stem Cells Engineered to Secrete Pigment Epithelium-Derived Factor Inhibit Tumor Metastasis and the Formation of Malignant Ascites in a Murine Colorectal Peritoneal Carcinomatosis Model.

    PubMed

    Yang, Liping; Zhang, Yuwei; Cheng, Liuliu; Yue, Dan; Ma, Jinhu; Zhao, Da; Hou, Xiaoming; Xiang, Rong; Cheng, Ping

    2016-03-01

    The therapeutic effects of conventional treatments for advanced colorectal cancer with colorectal peritoneal carcinomatosis (CRPC) and malignant ascites are not very encouraging. Vascular endothelial growth factor-A/vascular permeability factors (VEGF-A/VPF) play key roles in the formation of malignant ascites. In previous work, we demonstrated that pigment epithelium-derived factor (PEDF) antagonized VEGF-A and could repress tumor growth and suppress metastasis in several cancer types. Thus, PEDF may be a therapeutic candidate for treating malignant ascites. Mesenchymal stem cells (MSCs) are promising tools for delivering therapeutic agents in cancer treatment. In the study, MSCs derived from bone marrow were efficiently engineered to secrete human PEDF by adenoviral transduction. Then, intraperitoneal Ad-PEDF-transduced MSCs were analyzed with respect to CRPC and malignant ascites in a CT26 CRPC model. MSCs engineered to secrete PEDF through adenoviral transduction significantly inhibited tumor metastasis and malignant ascites formation in CT26 CRPC mice. Antitumor mechanisms of MSCs-PEDF (MSCs transduced with Ad-PEDF: MOI 500) were associated with inhibiting tumor angiogenesis, inducing apoptosis, and restoring the VEGF-A/sFLT-1 ratio in ascites. Moreover, MSC-mediated Ad-PEDF delivery reduced production of adenovirus-neutralizing antibodies, prolonged PEDF expression, and induced MSCs-PEDF migration toward tumor cells. As a conclusion, MSCs engineered to secrete PEDF by adenoviral transduction may be a therapeutic approach for suppressing tumor metastasis and inhibiting malignant ascites production in CRPC.

  8. Role of pigment epithelium-derived factor in the involution of hemangioma: Autocrine growth inhibition of hemangioma-derived endothelial cells

    SciTech Connect

    Kim, Kyung-Jin; Yun, Jang-Hyuk; Heo, Jong-Ik; Lee, Eun Hui; Min, Hye Sook; Choi, Tae Hyun; Cho, Chung-Hyun

    2014-11-14

    Highlights: • PEDF was expressed and induced during the involuting phase of IH. • PEDF inhibited the cell growth of the involuting HemECs in an autocrine manner. • PEDF suppression restored the impaired cell growth of the involuting HemECs. - Abstract: Hemangioma is a benign tumor derived from abnormal blood vessel growth. Unlike other vascular tumor counterparts, a hemangioma is known to proliferate during its early stage but it is followed by a stage of involution where regression of the tumor occurs. The critical onset leading to the involution of hemangioma is currently not well understood. This study focused on the molecular identities of the involution of hemangioma. We demonstrated that a soluble factor released from the involuting phase of hemangioma-derived endothelial cells (HemECs) and identified pigment epithelium-derived factor (PEDF) as an anti-angiogenic factor that was associated with the growth inhibition of the involuting HemECs. The growth inhibition of the involuting HemECs was reversed by suppression of PEDF in the involuting HemECs. Furthermore, we found that PEDF was more up-regulated in the involuting phase of hemangioma tissues than in the proliferating or the involuted. Taken together, we propose that PEDF accelerates the involution of hemangioma by growth inhibition of HemECs in an autocrine manner. The regulatory mechanism of PEDF expression could be a potential therapeutic target to treat hemangiomas.

  9. Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules.

    PubMed

    Windschüttl, S; Kampfer, C; Mayer, C; Flenkenthaler, F; Fröhlich, T; Schwarzer, J U; Köhn, F M; Urbanski, H; Arnold, G J; Mayerhofer, A

    2015-09-03

    Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well.

  10. Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content.

    PubMed

    Gattu, Arijeet K; Swenson, E Scott; Iwakiri, Yasuko; Samuel, Varman T; Troiano, Nancy; Berry, Ryan; Church, Christopher D; Rodeheffer, Matthew S; Carpenter, Thomas O; Chung, Chuhan

    2013-11-01

    Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt-β-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-β-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.

  11. Core-shell fibrous scaffold as a vehicle for sustained release of retinal pigmented epithelium-derived factor (PEDF) for photoreceptor differentiation of conjunctiva mesenchymal stem cells.

    PubMed

    Nasehi, Fatemeh; Karshenas, Mohsen; Nadri, Samad; Barati, Ghasem; Salim, Ahdiye

    2017-08-10

    Coaxial electrospinning technique was introduced as a flexible and promising technique for the fabrication of core-shell fibrous scaffold from poly ethylene glycol/poly caprolactone (PEG/PCL), where retinal pigmented epithelium-derived factor (PEDF) was encapsulated in the core, for photoreceptor differentiation of conjunctiva mesenchymal stem cells (CJMSCs) seed on scaffolds. The morphology and structure of fibers were characterized using SEM and TEM and photoreceptor differentiation was examined by quantitative real time PCR (qPCR). Release study showed that, a sustained release of PEDF from PEG/PCL scaffold was observed over 14 days. qPCR analysis demonstrated that rhodopsin (as a main photoreceptor gene) was significantly expressed in CJMSCs cultured on scaffold loaded with PEDF. According to the result, the core-shell scaffold loaded with PEDF (PEG+PEDF)/PCL) has superior control over factor release profile and has a potential for guiding photoreceptor differentiation of mesenchymal stem cells and promoting retinal regeneration. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.

  12. Pigment epithelium-derived factor (PEDF) suppresses IL-1β-mediated c-Jun N-terminal kinase (JNK) activation to improve hepatocyte insulin signaling.

    PubMed

    Gattu, Arijeet K; Birkenfeld, Andreas L; Iwakiri, Yasuko; Jay, Steven; Saltzman, Mark; Doll, Jennifer; Protiva, Petr; Samuel, Varman T; Crawford, Susan E; Chung, Chuhan

    2014-04-01

    Pigment epithelium-derived factor (PEDF) is an antiinflammatory protein that circulates at high levels in the metabolic syndrome. Metabolic studies of PEDF knockout (KO) mice were conducted to investigate the relationship between PEDF, inflammatory markers, and metabolic homeostasis. Male PEDF KO mice demonstrated a phenotype consisting of increased adiposity, glucose intolerance, and elevated serum levels of metabolites associated with the metabolic syndrome. Genome expression analysis revealed an increase in IL-1β signaling in the livers of PEDF KO mice that was accompanied by impaired IRS and Akt signaling. In human hepatocytes, PEDF blocked the effects of an IL-1β challenge by suppressing activation of the inflammatory mediator c-Jun N-terminal kinase while restoring Akt signaling. RNA interference of PEDF in human hepatocytes was permissive for c-Jun N-terminal kinase activation and decreased Akt signaling. A metabolomics profile identified elevated circulating levels of tricarboxyclic acid cycle intermediates including succinate, an inducer of IL-1β, in PEDF KO mice. Succinate-dependent IL-1β expression was blocked by PEDF in PEDF KO, but not wild-type hepatocytes. In vivo, PEDF restoration reduced hyperglycemia and improved hepatic insulin signaling in PEDF KO mice. These findings identify elevated PEDF as a homeostatic mechanism in the human metabolic syndrome.

  13. Pigment epithelium-derived factor (PEDF) peptide promotes the expansion of hepatic stem/progenitor cells via ERK and STAT3-dependent signaling.

    PubMed

    Shih, Shou-Chuan; Ho, Tsung-Chuan; Chen, Show-Li; Tsao, Yeou-Ping

    2017-01-01

    Hepatic stem/progenitor cells (HPC) have been considered as a potential cell source of an alternative to liver transplantation. Production of large numbers of autologous HPC from small pieces of live tissue is crucial for the application of HPC-based liver therapy. In this study, we demonstrated that a synthetic 44-amino acid peptide (44-mer) derived from pigment epithelium-derived factor (PEDF) can facilitate the production of a large number of actively dividing HPC from normal adult rat livers in a 35-day culture period. The phenotypic properties of HPC were characterized by morphological observation, colony formation and high expression of classical HPC markers including epithelial cell adhesion molecule (EpCAM), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and tumor-associated calcium signal transducer (TROP2). The 44-mer stimulated HPC proliferation in vitro and in mouse livers injured by a single intraperitoneal injection of carbon tetrachloride. In addition, the 44-mer induced the phosphorylation of ERK1/2 and STAT3 in HPC. Blocking the activity of ERK or STAT3 with pharmacological inhibitors attenuated the effects of the 44-mer on the induction of HPC proliferation. The long-term expanded HPC still possessed a bipotent ability to differentiate towards bile duct cells and mature hepatocytes. These results imply that the PEDF peptide may be a simple and effective agent to improve HPC-based liver therapy.

  14. Pigment epithelium-derived factor (PEDF) peptide promotes the expansion of hepatic stem/progenitor cells via ERK and STAT3-dependent signaling

    PubMed Central

    Shih, Shou-Chuan; Ho, Tsung-Chuan; Chen, Show-Li; Tsao, Yeou-Ping

    2017-01-01

    Hepatic stem/progenitor cells (HPC) have been considered as a potential cell source of an alternative to liver transplantation. Production of large numbers of autologous HPC from small pieces of live tissue is crucial for the application of HPC-based liver therapy. In this study, we demonstrated that a synthetic 44-amino acid peptide (44-mer) derived from pigment epithelium-derived factor (PEDF) can facilitate the production of a large number of actively dividing HPC from normal adult rat livers in a 35-day culture period. The phenotypic properties of HPC were characterized by morphological observation, colony formation and high expression of classical HPC markers including epithelial cell adhesion molecule (EpCAM), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and tumor-associated calcium signal transducer (TROP2). The 44-mer stimulated HPC proliferation in vitro and in mouse livers injured by a single intraperitoneal injection of carbon tetrachloride. In addition, the 44-mer induced the phosphorylation of ERK1/2 and STAT3 in HPC. Blocking the activity of ERK or STAT3 with pharmacological inhibitors attenuated the effects of the 44-mer on the induction of HPC proliferation. The long-term expanded HPC still possessed a bipotent ability to differentiate towards bile duct cells and mature hepatocytes. These results imply that the PEDF peptide may be a simple and effective agent to improve HPC-based liver therapy. PMID:28386338

  15. Pigment epithelium-derived factor attenuates myocardial fibrosis via inhibiting Endothelial-to-Mesenchymal Transition in rats with acute myocardial infarction

    PubMed Central

    Zhang, Hao; Hui, Hongliang; Li, Zhimin; Pan, Jiajun; Jiang, Xia; Wei, Tengteng; Cui, Huazhu; Li, Lei; Yuan, Xulong; Sun, Teng; Liu, Zhiwei; Zhang, Zhongming; Dong, Hongyan

    2017-01-01

    Endothelial mesenchymal transition (EndMT) plays a critical role in the pathogenesis and progression of interstitial and perivascular fibrosis after acute myocardial infarction (AMI). Pigment epithelium-derived factor (PEDF) is shown to be a new therapeutic target owing to its protective role in cardiovascular disease. In this study, we tested the hypothesis that PEDF is an endogenous inhibitor of EndMT and represented a novel mechanism for its protective effects against overactive cardiac fibrosis after AMI. Masson’s trichrome (MTC) staining and picrosirius red staining revealed decreased interstitial and perivascular fibrosis in rats overexpressing PEDF. The protective effect of PEDF against EndMT was confirmed by co-labeling of cells with the myofibroblast and endothelial cell markers. In the endothelial cells of microvessels in the ischemic myocardium, the inhibitory effect of PEDF against nuclear translocation of β-catenin was observed through confocal microscopic imaging. The correlation between antifibrotic effect of PEDF and inactivation of β-catenin was confirmed by co-transfecting cells with lentivirus carrying PEDF or PEDF RNAi and plasmids harboring β-catenin siRNA(r) or constitutive activation of mutant β-catenin. Taken together, these results establish a novel finding that PEDF could inhibit EndMT related cardiac fibrosis after AMI by a mechanism dependent on disruption of β-catenin activation and translocation. PMID:28167820

  16. Positive association of circulating levels of advanced glycation end products (AGEs) with pigment epithelium-derived factor (PEDF) in a general population.

    PubMed

    Yamagishi, Sho-Ichi; Matsui, Takanori; Adachi, Hisashi; Takeuchi, Masayoshi

    2010-02-01

    We have recently found that serum levels of pigment epithelium-derived factor (PEDF), a glycoprotein with anti-oxidative and anti-inflammatory properties, are elevated in proportion to the accumulation of the number of the components of the metabolic syndrome. Since formation and accumulation of advanced glycation end products (AGEs) progress under the metabolic syndrome and that PEDF could inhibit the AGE-elicited tissue damage, it is conceivable that PEDF levels may be increased as a counter-system against AGEs in patients with the metabolic syndrome. However, correlation between circulating levels of AGEs and PEDF in humans remains to be elucidated. In this study, we investigated the relationship between serum AGE and PEDF levels in a general population and examined the effects of AGEs on PEDF gene expression in vitro. One hundred ninety-six Japanese subjects in a general population underwent a complete history and physical examination, determination of blood chemistries, including serum levels of AGEs and PEDF. In multiple regression analyses, creatinine, body mass index, triglycerides, AGEs and insulin were independently correlated with serum PEDF levels. AGEs dose-dependently increased PEDF gene expression in cultured adipocytes and liver cells. Our present study demonstrated first that circulating AGEs were one of the independent correlates of serum levels of PEDF. Adipose tissue and liver may be target organs for the AGE-induced PEDF overexpression in humans. Serum PEDF levels may be elevated in response to circulating AGEs as a counter-system against the AGE-elicited tissue damage.

  17. Virtual-screening targeting Human Immunodeficiency Virus type 1 integrase-lens epithelium-derived growth factor/p75 interaction for drug development.

    PubMed

    Gu, Wan-Gang; Liu, Bai-Nan; Yuan, Jun-Fa

    2015-02-01

    Three integrase (IN) inhibitors have been approved by FDA for clinical treatment of Human Immunodeficiency Virus (HIV) infection. This stimulates more researchers to focus their studies on this target for anti-HIV drug development. Three steps regarding of IN activity have been validated for inhibitor discovery: strand transfer, 3'-terminal processing, and IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction. Among them, IN-LEDGF/p75 interaction is a new target validated in recent years. Emergence of drug-resistant virus strains makes this target appealing to pharmacologists. Compared with the traditional screening methods such as AlphaScreen and cell-based screening developed for IN inhibitor discovery, virtual screening is a powerful technique in modern drug discovery. Here we summarized the recent advances of virtual-screening targeting IN-LEDFG/p75 interaction. The combined application of virtual screening and experiments in drug discovery against IN-LEDFG/p75 interaction sheds light on anti-HIV research and drug discovery.

  18. 1,4-Bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene, a small molecule, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular Lens epithelium-derived growth factor.

    PubMed

    Gu, Wan-gang; Ip, Denis Tsz-Ming; Liu, Si-jie; Chan, Joseph H; Wang, Yan; Zhang, Xuan; Zheng, Yong-tang; Wan, David Chi-Cheong

    2014-04-25

    Translocation of viral integrase (IN) into the nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). As the first discovered cellular factor to interact with IN, Lens epithelium-derived growth factor (LEDGF/p75) plays an important role in the process of integration. Disruption of the LEDGF/p75-IN interaction has provided a great interest for anti-HIV agent discovery. In this work, we reported that one small molecular compound, 1,4-bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene(Compound 15), potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution at 1 μM. The putative binding mode of Compound 15 was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Compound 15 suppressed viral replication by measuring p24 antigen production in HIV-1IIIB acute infected C8166 cells with EC50 value of 11.19 μM. Compound 15 might supply useful structural information for further anti-HIV agent discovery. Copyright © 2014. Published by Elsevier Ireland Ltd.

  19. Pigment epithelium-derived factor (PEDF) inhibits survival and proliferation of VEGF-exposed multiple myeloma cells through its anti-oxidative properties.

    PubMed

    Seki, Ritsuko; Yamagishi, Sho-ichi; Matsui, Takanori; Yoshida, Takafumi; Torimura, Takuji; Ueno, Takato; Sata, Michio; Okamura, Takashi

    2013-02-22

    Vascular endothelial growth factor (VEGF) has been reported not only to induce angiogenesis within the bone marrow, but also directly stimulate the proliferation and survival of multiple myeloma cells, thus being involved in the development and progression of this second most common hematological malignancy. We, along with others, have found that pigment epithelium-derived factor (PEDF) has anti-angiogenic and anti-vasopermeability properties both in cell culture and animal models by counteracting the biological actions of VEGF. However, effects of PEDF on VEGF-exposed myeloma cells remain unknown. In this study, we examined whether and how PEDF could inhibit the VEGF-induced proliferation and survival of myeloma cells. PEDF, a glutathione peroxidase mimetic, ebselen, or an inhibitor of NADPH oxidase, diphenylene iodonium significantly inhibited the VEGF-induced reactive oxygen species (ROS) generation, increase in anti-apoptotic and growth-promoting factor, myeloid cell leukemia 1 (Mcl-1) expression, and proliferation in U266 myeloma cells. VEGF blocked apoptosis of multiple myeloma cells isolated from patients, which was prevented by PEDF. PEDF also reduced p22phox levels in VEGF-exposed U266 cells. Furthermore, overexpression of dominant-negative human Rac-1 mutant mimicked the effects of PEDF on ROS generation and Mcl-1 expression in U266 cells. Our present study suggests that PEDF could block the VEGF-induced proliferation and survival of multiple myeloma U266 cells through its anti-oxidative properties via suppression of p22phox, one of the membrane components of NADPH oxidase. Suppression of VEGF signaling by PEDF may be a novel therapeutic target for multiple myeloma.

  20. Pigment epithelium-derived factor has a role in the progression of papillary thyroid carcinoma by affecting the HIF1α-VEGF signaling pathway

    PubMed Central

    Lv, Yichen; Sun, Yu; Shi, Tiefeng; Shi, Chenlei; Qin, Huadong; Li, Zhaozhu

    2016-01-01

    The progression mechanism of papillary thyroid carcinoma (PTC) remains largely unknown. Accumulating evidence has suggested that various targets of pigment epithelium-derived factor (PEDF) are able to inhibit cancer progression. The aim of the present study was to examine PEDF expression in PTC patients and to investigate its relationship with aggressive clinicopathological features, as well as to explore whether PEDF affects the progression of PTC via the hypoxia-inducible factor 1α (HIF1α)-vascular endothelial growth factor (VEGF) pathway. A total of 271 patients with PTC, including 24 men and 247 women, were enrolled in the present study. Relevant patient data, including demographic features, preoperative clinical features and pathological features, were collected for analysis. The protein expression levels of PEDF in PTC tissues were detected using immunohistochemical staining, and the mRNA expression levels of PEDF, VEGF and HIF1α in 15 PTC tissues with lymph node metastasis (LNM) and 10 tissues without LNM were detected using reverse transcription-quantitative polymerase chain reaction. Immunohistochemical staining with an anti-PEDF antibody detected PEDF expression in 74.5% of the PTC tissues. PEDF expression levels were significantly correlated with LNM, extrathyroid invasion, a high TNM stage, the presence of the BRAFV600E mutation and tumor size. PEDF mRNA expression levels were significantly decreased in PTC tissues with LNM, as compared with PTC tissues without LNM, while the mRNA expression levels of HIF1α and VEGF were markedly increased in PTC tissues with LNM. Taken together, the results of the present study suggested that PEDF plays a role in the progression of PTC, and that PEDF may exert an anti-angiogenesis role by affecting the HIF1α-VEGF pathway, eventually inhibiting the metastasis of PTC. PMID:28105231

  1. Pigment epithelium-derived factor 34-mer peptide prevents liver fibrosis and hepatic stellate cell activation through down-regulation of the PDGF receptor.

    PubMed

    Tsai, Tung-Han; Shih, Shou-Chuan; Ho, Tsung-Chuan; Ma, Hsin-I; Liu, Ming-Ying; Chen, Show-Li; Tsao, Yeou-Ping

    2014-01-01

    Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl4)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.

  2. Augmented expression and secretion of adipose-derived pigment epithelium-derived factor does not alter local angiogenesis or contribute to the development of systemic metabolic derangements.

    PubMed

    Lakeland, Thomas V; Borg, Melissa L; Matzaris, Maria; Abdelkader, Amany; Evans, Roger G; Watt, Matthew J

    2014-06-15

    Impaired coupling of adipose tissue expansion and vascularization is proposed to lead to adipocyte hypoxia and inflammation, which in turn contributes to systemic metabolic derangements. Pigment epithelium-derived factor (PEDF) is a powerful antiangiogenic factor that is secreted by adipocytes, elevated in obesity, and implicated in the development of insulin resistance. We explored the angiogenic and metabolic role of adipose-derived PEDF through in vivo studies of mice with overexpression of PEDF in adipocytes (PEDF-aP2). PEDF expression in white adipocytes and PEDF secretion from adipose tissue was increased in transgenic mice, but circulating levels of PEDF were not increased. Overexpression of PEDF did not alter vascularization, the partial pressure of O2, cellular hypoxia, or gene expression of inflammatory markers in adipose tissue. Energy expenditure and metabolic substrate utilization, body mass, and adiposity were not altered in PEDF-aP2 mice. Whole body glycemic control was normal as assessed by glucose and insulin tolerance tests, and adipocyte-specific glucose uptake was unaffected by PEDF overexpression. Adipocyte lipolysis was increased in PEDF-aP2 mice and associated with increased adipose triglyceride lipase and decreased perilipin 1 expression. Experiments conducted in mice rendered obese by high-fat feeding showed no differences between PEDF-aP2 and wild-type mice for body mass, adiposity, whole body energy expenditure, glucose tolerance, or adipose tissue oxygenation. Together, these data indicate that adipocyte-generated PEDF enhances lipolysis but question the role of PEDF as a major antiangiogenic or proinflammatory mediator in adipose tissue in vivo. Copyright © 2014 the American Physiological Society.

  3. Chronic intermittent hypoxia increases encoding pigment epithelium-derived factor gene expression, although not that of the protein itself, in the temporal cortex of rats*,**

    PubMed Central

    Julian, Guilherme Silva; de Oliveira, Renato Watanabe; Favaro, Vanessa Manchim; de Oliveira, Maria Gabriela Menezes; Perry, Juliana Cini; Tufik, Sergio; Chagas, Jair Ribeiro

    2015-01-01

    Objective: Obstructive sleep apnea syndrome is mainly characterized by intermittent hypoxia (IH) during sleep, being associated with several complications. Exposure to IH is the most widely used animal model of sleep apnea, short-term IH exposure resulting in cognitive and neuronal impairment. Pigment epithelium-derived factor (PEDF) is a hypoxia-sensitive factor acting as a neurotrophic, neuroprotective, and antiangiogenic agent. Our study analyzed performance on learning and cognitive tasks, as well as PEDF gene expression and PEDF protein expression in specific brain structures, in rats exposed to long-term IH. Methods: Male Wistar rats were exposed to IH (oxygen concentrations of 21-5%) for 6 weeks-the chronic IH (CIH) group-or normoxia for 6 weeks-the control group. After CIH exposure, a group of rats were allowed to recover under normoxic conditions for 2 weeks (the CIH+N group). All rats underwent the Morris water maze test for learning and memory, PEDF gene expression and PEDF protein expression in the hippocampus, frontal cortex, and temporal cortex being subsequently assessed. Results: The CIH and CIH+N groups showed increased PEDF gene expression in the temporal cortex, PEDF protein expression remaining unaltered. PEDF gene expression and PEDF protein expression remained unaltered in the frontal cortex and hippocampus. Long-term exposure to IH did not affect cognitive function. Conclusions: Long-term exposure to IH selectively increases PEDF gene expression at the transcriptional level, although only in the temporal cortex. This increase is probably a protective mechanism against IH-induced injury. PMID:25750673

  4. The effects of calcium channel blockers on nephropathy and pigment epithelium-derived factor in the treatment of hypertensive patients with type 2 diabetes mellitus.

    PubMed

    Tabur, Suzan; Oğuz, Elif; Sabuncu, Tevfik; Korkmaz, Hakan; Çelik, Hakim

    2015-01-01

    The aim of this study was to compare the effects of a dihidropiridin calcium channel blocker amlodipin and a non-dihidropiridin calcium channel blocker verapamil on nephropathy and serum pigment epithelium-derived factor (PEDF) levels of type 2 diabetic patients with hypertension. Forty-one type 2 diabetic patients with uncontrolled hypertension in spite of using angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) were enrolled in the study. The patients were randomized in two groups. First group received amlodipin (5-10 mg/d) and second group verapamil (120-240 mg/d) for 6 weeks. The difference between two calcium channel blocker treatments was investigated by analyzing urinary albumin excretion and plasma PEDF levels of patients at the end of 6 weeks. Urinary microalbumin/creatinine values were decreased in both amlodipin and verapamil groups but it was not statistically significant. Plasma PEDF levels also decreased significantly in both groups at the end of the treatment (p < 0.001 and p < 0.001, respectively). At the end of the treatment there was no significant difference between changes in values of systolic BP, diastolic BP, microalbumin/creatinine and PEDF percentage in both groups (p = 0.788, p = 0.926, p = 0.908, p = 0.140, respectively). PEDF values showed a positive correlation with microalbumin/creatinine, hb A1c, FBS, systolic and diastolic BP levels. It was observed that both of the drugs have similar effects on nefhropathy and PEDF at the end of the treatment. In this study, we suggest that calcium channel blockers may have renoprotective effects by different mechanisms except their antihypertensive effects and this may be important to determine the selection of antihypertensive drug combinations in diabetic nephropathy.

  5. Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects

    PubMed Central

    Fedan, Jeffrey S.; Thompson, Janet A.; Ismailoglu, U. Burcin; Jing, Yi

    2013-01-01

    In asthmatic patients, inhalation of hyperosmolar saline or D-mannitol (D-M) elicits bronchoconstriction, but in healthy subjects exercise causes bronchodilation. Hyperventilation causes drying of airway surface liquid (ASL) and increases its osmolarity. Hyperosmolar challenge of airway epithelium releases epithelium-derived relaxing factor (EpDRF), which relaxes the airway smooth muscle. This pathway could be involved in exercise-induced bronchodilation. Little is known of ASL hyperosmolarity effects on epithelial function. We investigated the effects of osmolar challenge maneuvers on dispersed and adherent guinea-pig tracheal epithelial cells to examine the hypothesis that EpDRF-mediated relaxation is associated with epithelial cell shrinkage. Enzymatically-dispersed cells shrank when challenged with ≥10 mOsM added D-M, urea or NaCl with a concentration-dependence that mimics relaxation of the of isolated perfused tracheas (IPT). Cells shrank when incubated in isosmolar N-methyl-D-glucamine (NMDG) chloride, Na gluconate (Glu), NMDG-Glu, K-Glu and K2SO4, and swelled in isosmolar KBr and KCl. However, isosmolar challenge is not a strong stimulus of relaxation in IPTs. In previous studies amiloride and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells. Confocal microscopy in tracheal segments showed that adherent epithelium is refractory to low hyperosmolar concentrations that induce dispersed cell shrinkage and relaxation of IPT. Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells. Our studies dissociate relaxation of IPT from cell shrinkage after hyperosmolar challenge of airway epithelium. PMID:24130533

  6. Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.

    PubMed

    Wu, Qinjie; He, Shasha; Wei, Xiawei; Shao, Bin; Luo, Shuntao; Guo, Fuchun; Zhang, Hailong; Wang, Yongsheng; Gong, Changyang; Yang, Li

    2014-09-01

    Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia

  7. A Novel IFITM5 Mutation in Severe Atypical Osteogenesis Imperfecta Type VI Impairs Osteoblast Production of Pigment Epithelium-Derived Factor

    PubMed Central

    Farber, Charles R; Reich, Adi; Barnes, Aileen M; Becerra, Patricia; Rauch, Frank; Cabral, Wayne A; Bae, Alison; Quinlan, Aaron; Glorieux, Francis H; Clemens, Thomas L; Marini, Joan C

    2015-01-01

    Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5′-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and

  8. Association of single nucleotide polymorphisms in the lens epithelium-derived growth factor (LEDGF/p75) with HIV-1 infection outcomes in Brazilian HIV-1+ individuals.

    PubMed

    Passaes, Caroline Pereira Bittencourt; Cardoso, Cynthia Chester; Caetano, Diogo Gama; Teixeira, Sylvia Lopes Maia; Guimarães, Monick Lindenmeyer; Campos, Dayse Pereira; Veloso, Valdilea Gonçalves; Babic, Dunja Z; Stevenson, Mario; Moraes, Milton Ozório; Morgado, Mariza Gonçalves

    2014-01-01

    The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

  9. Structure based Virtual Screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75).

    PubMed

    Panwar, Umesh; Singh, Sanjeev Kumar

    2017-09-26

    HIV -1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to because of its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine- Z742267384, Maybridge- HTS02400, and Specs- AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behaviour and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.

  10. Lens epithelium-derived growth factor relieves transforming growth factor-beta1-induced transcription repression of heat shock proteins in human lens epithelial cells.

    PubMed

    Sharma, Preeti; Fatma, Nigar; Kubo, Eri; Shinohara, Toshimichi; Chylack, Leo T; Singh, Dhirendra P

    2003-05-30

    Lens epithelium-cell derived growth factor (LEDGF) is a transcriptional activator. It protects the cells by binding to cis-stress response ((A/T)GGGG(T/A)), and heat shock (HSE; nGAAn) elements in the stress genes and activating their transcription. Transforming growth factor-beta (TGF-beta) has been implicated in the control of tissue homeostasis, terminal differentiation, and apoptosis. Here we provide evidence that TGF-beta1 down-regulates LEDGF expression and diminishes its affinity for DNA during TGF-beta1-induced phenotypic changes and apoptosis in human lens epithelial cells. Surprisingly, TGF-beta1 treatment for 48 h markedly decreased the LEDGF, Hsp27, and alphaB-crystallin promoter activities with the decrease of abundance of LEDGF mRNA and protein. Deletion mutants of the LEDGF promoter showed that one TGF-beta1 inhibitory element (TIE) like sequence nnnTTGGnnn (-444 to -433) contributed to this negative regulation. Mutation of TIE (TTGG to TATT) abolished the down-regulation of the LEDGF promoter. Gel mobility and supershift assays showed that LEDGF in the nuclear extracts of TGF-beta1-treated human lens epithelial cells did not bind to stress-response elements and HSE. The TGF-beta1-induced down-regulation of LEDGF, Hsp27, and alphaB-crystallin promoters activity was reversed by cotransfection with a plasmid expressing LEDGF. Because overexpression of LEDGF was able to relieve TGF-beta1 and/or stress-induced changes, it would be a candidate molecule to postpone age-related degenerating disorders.

  11. Dynamic Modulation of HIV-1 Integrase Structure and Function by Cellular Lens Epithelium-derived Growth Factor (LEDGF) Protein*S⃞

    PubMed Central

    McKee, Christopher J.; Kessl, Jacques J.; Shkriabai, Nikolozi; Dar, Mohd Jamal; Engelman, Alan; Kvaratskhelia, Mamuka

    2008-01-01

    The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target. PMID:18801737

  12. Proliferative diabetic retinopathy is associated with a low level of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor. a pilot study.

    PubMed

    Boehm, B O; Lang, G; Feldmann, B; Kurkhaus, A; Rosinger, S; Volpert, O; Lang, G K; Bouck, N

    2003-06-01

    Retinopathy is the most common microvascular diabetes complication and represents a major threat to the eyesight. The aim of this study was to address the role of pro- and anti-angiogenic molecules in diabetic retinopathy in the aqueous humor of the eye. Aqueous humor was collected at cataract surgery from 19 diabetic patients and from 13 age- and sex-matched normoglycemic controls. Levels of pro-angiogenic vascular endothelial growth factor (VEGF) and angiogenic inhibitor pigment epithelium-derived factor (PEDF) were determined. Angiogenic activity of the aqueous humor was quantified by measuring its effect on the migration of capillary endothelial cells. In the aqueous fluid, VEGF levels were increased in diabetics (mean values: 501 vs. 367 pg/ml; p = 0.05), compared to controls. PEDF was found to be decreased in diabetics (mean values: 2080 vs. 5780 ng/ml; p = 0.04) compared to controls. In seven diabetic patients with proliferative retinopathy, the most profound finding was a significant decrease of the PEDF level (mean value: 237 ng/ml), whereas VEGF levels were comparable to diabetic patients without proliferation (mean value: 3153; p = 0.003). Angiogenic activity in samples of patients from the control group was generally inhibitory due to PEDF, and inhibition was blocked by neutralizing antibodies to PEDF. Likewise, in diabetics without proliferation, angiogenic activity was also blocked by antibodies to PEDF. We will demonstrate here that the level of the natural ocular anti-angiogenic agent PEDF is inversely associated with proliferative retinopathy. PEDF is an important negative regulator of angiogenic activity of aqueous humor. Our data may have implications for the development of novel regimens for diabetic retinopathy.

  13. Inhibition of corneal neovascularization with the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18) vector in a rat corneal experimental angiogenesis model.

    PubMed

    Kuo, Chien-Neng; Chen, Chung-Yi; Chen, San-Ni; Yang, Lin-Cheng; Lai, Li-Ju; Lai, Chien-Hsiung; Chen, Miao-Fen; Hung, Chia-Hui; Chen, Ching-Hsein

    2013-04-16

    Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18(p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups(Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and D: 10 μg + 10 μg) of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μgof p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV.The bFGF and PEDF genes were successfully expressed as shown by western blot analysis,and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival bevacizumab administration combined with a relatively safe profile and low toxicity.

  14. Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2.

    PubMed

    Sanchez, A; Tripathy, D; Yin, X; Luo, J; Martinez, J; Grammas, P

    2012-01-01

    Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.

  15. Neural Stem Cell-based Intraocular Administration of Pigment Epithelium-derived Factor Promotes Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Crush Injury in Rat: An Experimental Study

    PubMed Central

    Zhang, Wei-Min; Zhang, Zhi-Ren; Zhang, Yong-Gang; Gao, Yan-Sheng

    2016-01-01

    Background: Pigment epithelium-derived factor (PEDF) is regarded as a multifunctional protein possessing neurotrophic and neuroprotective properties. PEDF has a very short half-life, and it would require multiple injections to maintain a therapeutically relevant level without a delivery system. However, multiple injections are prone to cause local damage or infection. To overcome this, we chose a cell-based system that provided sustained delivery of PEDF and compared the effect of weekly injections of PEDF and neural stem cell (NSC)-based intraocular administration of PEDF on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve injury. Methods: Seventy-two rats were randomly assigned to 3 groups: group with injections of phosphate buffered saline (PBS) (n=24), group with weekly injections of PEDF (n=24), and group with NSC-based administration of PEDF (n=24). Western blot was used to analyze the PEDF protein level 2 weeks after injection. Retinal flat mounts and immunohistochemistry were employed to analyze RGC survival and axon regeneration 2 weeks and 4 weeks after injection. The data were analyzed with one-way ANOVA in SPSS (version 19.0). A P<0.05 was considered significant. Results: The PEDF protein level in the group with NSC-based administration of PEDF increased compared with that in the groups with injections of PEDF and PBS (P<0.05). The PEDF-modified NSCs differentiated into GFAP-positive astrocytes andβ-tubulin-III-positive neurons. NSC-based administration of PEDF effectively increased RGC survival and improved the axon regeneration of the optic nerve compared with weekly injections of PEDF. Conclusion: Subretinal space transplantation of PEDF-secreting NSCs sustained high concentrations of PEDF, differentiated into neurons and astrocytes, and significantly promoted RGC survival and axon regeneration after optic nerve injury. PMID:27582587

  16. Nuclear receptor co-repressor is required to maintain proliferation of normal intestinal epithelial cells in culture and down-modulates the expression of pigment epithelium-derived factor.

    PubMed

    Doyon, Geneviève; St-Jean, Stéphanie; Darsigny, Mathieu; Asselin, Claude; Boudreau, Francois

    2009-09-11

    Stem cells of the gut epithelium constantly produce precursors that progressively undergo a succession of molecular changes resulting in growth arrest and commitment to a specific differentiation program. Few transcriptional repressors have been identified that maintain the normal intestinal epithelial cell (IEC) proliferation state. Herein, we show that the nuclear receptor co-repressor (NCoR1) is differentially expressed during the proliferation-to-differentiation IEC transition. Silencing of NCoR1 expression in proliferating cells of crypt origin resulted in a rapid growth arrest without associated cell death. A genechip profiling analysis identified several candidate genes to be up-regulated in NCoR1-deficient IEC. Pigment epithelium-derived factor (PEDF, also known as serpinf1), a suspected tumor suppressor gene that plays a key role in the inhibition of epithelial tissue growth, was significantly up-regulated in these cells. Chromatin immunoprecipitation experiments showed that the PEDF gene promoter was occupied by NCoR1 in proliferating epithelial cells. Multiple retinoid X receptor (RXR) heterodimers interacting sites of the PEDF promoter were confirmed to interact with RXR and retinoid acid receptor (RAR). Cotransfection assays showed that RXR and RAR were able to transactivate the PEDF promoter and that NCoR1 was repressing this effect. Finally, forced expression of PEDF in IEC resulted in a slower rate of proliferation. These observations suggest that NCoR1 expression is required to maintain IEC in a proliferative state and identify PEDF as a novel transcriptional target for NCoR1 repressive action.

  17. Coelomic epithelium-derived cells in visceral morphogenesis.

    PubMed

    Ariza, Laura; Carmona, Rita; Cañete, Ana; Cano, Elena; Muñoz-Chápuli, Ramón

    2016-03-01

    Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans.

  18. Synthetic heparin-binding growth factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  19. Synthetic heparin-binding factor analogs

    DOEpatents

    Pena, Louis A [Poquott, NY; Zamora, Paul O [Gaithersburg, MD; Lin, Xinhua [Plainview, NY; Glass, John D [Shoreham, NY

    2010-04-20

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  20. A Potential Profibrogenic Role of Biliary Epithelium-Derived Cardiotrophin-1 in Pediatric Cholestatic Liver Disease.

    PubMed

    Hua, Xiangwei; Shan, Yuhua; Li, Dawei; Xu, Dongwei; Zhang, Jiang; Yang, Taihua; Han, Longzhi; Shen, Conghuan; Xia, Yun; Chen, Qimin; Ma, Xiong; Zhang, Jianjun; Xia, Qiang

    2015-08-01

    As a cytokine of the interleukin-6 family, cardiotrophin-1 (CT-1) has been shown to be an important endogenous protector in liver injury. Our study aimed to investigate the role of CT-1 in liver fibrosis in pediatric cholestatic liver disease (PCLD). CT-1 mRNA and protein expression levels were upregulated in PCLD liver biopsy tissues compared with controls. Immunohistochemistry and confocal microscopy of liver sections showed that CT-1 was predominantly expressed by biliary epithelium cells. Serum CT-1 was elevated significantly in the children with PCLD compared with controls. Serum CT-1 levels exhibited a moderate positive correlation with the Scheuer stage of hepatic fibrosis and serum TB levels and a weak correlation with serum ALP levels. In vitro analysis indicated that LX-2 cells preconditioned with CT-1 exhibited significant increments in proliferation and accumulation of extracellular matrix components, while also positively regulating the STAT3 and p38MAPK pathways. In conclusion, biliary epithelium-derived CT-1 may exert a profibrogenic potential in PCLD.

  1. Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors

    PubMed Central

    Bulyk, Martha L.; Johnson, Philip L. F.; Church, George M.

    2002-01-01

    We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement. PMID:11861919

  2. Transcription factor binding energy vs. biological function

    NASA Astrophysics Data System (ADS)

    Djordjevic, M.; Grotewold, E.

    2007-03-01

    Transcription factors (TFs) are proteins that bind to DNA and regulate expression of genes. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of gene regulatory networks. Recent theoretical advances that we developed [1,2], allow us to infer TF-DNA interaction parameters from in-vitro selection experiments [3]. We use more than 6000 binding sequences [3], assembled under controlled conditions, to obtain protein-DNA interaction parameters for a mammalian TF with up to now unprecedented accuracy. Can one accurately identify biologically functional TF binding sites (i.e. the binding sites that regulate gene expression), even with the best possible protein-DNA interaction parameters? To address this issue we i) compare our prediction of protein binding with gene expression data, ii) use evolutionary comparison between related mammalian genomes. Our results strongly suggest that in a genome there exists a large number of randomly occurring high energy binding sites that are not biologically functional. [1] M Djordjevic, submitted to Biomol. Eng. [2] M. Djordjevic and A. M. Sengupta, Phys. Biol. 3: 13, 2006. [3] E. Roulet et al., Nature Biotech. 20: 831, 2002.

  3. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  4. Genetics Home Reference: core binding factor acute myeloid leukemia

    MedlinePlus

    ... acute myeloid leukemia core binding factor acute myeloid leukemia Printable PDF Open All Close All Enable Javascript ... collapse boxes. Description Core binding factor acute myeloid leukemia (CBF-AML) is one form of a cancer ...

  5. Recent advances in epithelium-derived cytokines (IL-33, IL-25 and TSLP) and allergic inflammation

    PubMed Central

    Divekar, Rohit; Kita, Hirohito

    2015-01-01

    Purpose of review Allergic diseases are thought to be driven by aberrant immune responses. Epithelium responds to various environmental factors by releasing key cytokines, such as thymic stromal lymphopoietin (TSLP), IL-33 and IL-25. While there are important differences among these cytokines, there are also similarities which confound a clear understanding of exact roles of these cytokines. The purpose of this review is to analyze advances in biology and functions of these cytokines over recent years, elucidate their differences and similarities, and provide new conceptual understanding as to their roles in allergic diseases. Recent findings There are distinct differences in the timing, onset, and kinetics of the responses and perhaps in potency of action of TSLP, IL-33 and IL-25. Newer roles of these cytokines have been described, including airway remodeling and fibrosis-related functions (TSLP, IL-33 and IL-25), fetal-maternal interface (IL-33 and TSLP), T cell biology (TSLP), group 2 innate lymphoid cell (ILC2) biology (TSLP, IL-33 and IL-25), and mast cell-neutrophil axis (IL-33). Novel roles of these cytokines in in pathogenesis of atopic dermatitis and asthma have also been described. Summary TSLP, IL-25 and IL-33 are increasingly recognized to play important roles in pathophysiology of allergic diseases. More clear recognition of the differences and similarities of the immunological pathways mediated by these cytokines would help optimize treatment for allergic diseases. PMID:25479313

  6. Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line.

    PubMed

    Seki, Satoru; Itagaki, Shirou; Kobayashi, Masaki; Hirano, Takeshi; Iseki, Ken

    2008-07-01

    Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T(3)), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 microM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 microM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC(50) value of DEA for A549 cell viability: 25.4+/-2.4 microM) was more pronounced than that by DEA alone (IC(50) value: 11.5+/-3.0 microM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.

  7. Physical factors affecting chloroquine binding to melanin.

    PubMed

    Schroeder, R L; Pendleton, P; Gerber, J P

    2015-10-01

    Chloroquine is an antimalarial drug but is also prescribed for conditions such as rheumatoid arthritis. Long-term users risk toxic side effects, including retinopathy, thought to be caused by chloroquine accumulation on ocular melanin. Although the binding potential of chloroquine to melanin has been investigated previously, our study is the first to demonstrate clear links between chloroquine adsorption by melanin and system factors including temperature, pH, melanin type, and particle size. In the current work, two Sepia melanins were compared with bovine eye as a representative mammalian melanin. Increasing the surface anionic character due to a pH change from 4.7 to 7.4 increased each melanin's affinity for chloroquine. Although the chloroquine isotherms exhibited an apparently strong interaction with each melanin, isosteric heat analysis indicated a competitive interaction. Buffer solution cations competed effectively at low surface coverage; chloroquine adsorption occurs via buffer cation displacement and is promoted by temperature-influenced secondary structure swelling.

  8. Mouse models for core binding factor leukemia.

    PubMed

    Chin, D W L; Watanabe-Okochi, N; Wang, C Q; Tergaonkar, V; Osato, M

    2015-10-01

    RUNX1 and CBFB are among the most frequently mutated genes in human leukemias. Genetic alterations such as chromosomal translocations, copy number variations and point mutations have been widely reported to result in the malfunction of RUNX transcription factors. Leukemias arising from such alterations in RUNX family genes are collectively termed core binding factor (CBF) leukemias. Although adult CBF leukemias generally are considered a favorable risk group as compared with other forms of acute myeloid leukemia, the 5-year survival rate remains low. An improved understanding of the molecular mechanism for CBF leukemia is imperative to uncover novel treatment options. Over the years, retroviral transduction-transplantation assays and transgenic, knockin and knockout mouse models alone or in combination with mutagenesis have been used to study the roles of RUNX alterations in leukemogenesis. Although successful in inducing leukemia, the existing assays and models possess many inherent limitations. A CBF leukemia model which induces leukemia with complete penetrance and short latency would be ideal as a platform for drug discovery. Here, we summarize the currently available mouse models which have been utilized to study CBF leukemias, discuss the advantages and limitations of individual experimental systems, and propose suggestions for improvements of mouse models.

  9. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  10. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  11. Binding Site Turnover Produces Pervasive Quantitative Changes in Transcription Factor Binding between Closely Related Drosophila Species

    PubMed Central

    Trapnell, Cole; Davidson, Stuart; Pachter, Lior; Chu, Hou Cheng; Tonkin, Leath A.; Biggin, Mark D.; Eisen, Michael B.

    2010-01-01

    Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances. PMID:20351773

  12. High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints.

    PubMed

    Guo, Yuchun; Mahony, Shaun; Gifford, David K

    2012-01-01

    An essential component of genome function is the syntax of genomic regulatory elements that determine how diverse transcription factors interact to orchestrate a program of regulatory control. A precise characterization of in vivo spacing constraints between key transcription factors would reveal key aspects of this genomic regulatory language. To discover novel transcription factor spatial binding constraints in vivo, we developed a new integrative computational method, genome wide event finding and motif discovery (GEM). GEM resolves ChIP data into explanatory motifs and binding events at high spatial resolution by linking binding event discovery and motif discovery with positional priors in the context of a generative probabilistic model of ChIP data and genome sequence. GEM analysis of 63 transcription factors in 214 ENCODE human ChIP-Seq experiments recovers more known factor motifs than other contemporary methods, and discovers six new motifs for factors with unknown binding specificity. GEM's adaptive learning of binding-event read distributions allows it to further improve upon previous methods for processing ChIP-Seq and ChIP-exo data to yield unsurpassed spatial resolution and discovery of closely spaced binding events of the same factor. In a systematic analysis of in vivo sequence-specific transcription factor binding using GEM, we have found hundreds of spatial binding constraints between factors. GEM found 37 examples of factor binding constraints in mouse ES cells, including strong distance-specific constraints between Klf4 and other key regulatory factors. In human ENCODE data, GEM found 390 examples of spatially constrained pair-wise binding, including such novel pairs as c-Fos:c-Jun/USF1, CTCF/Egr1, and HNF4A/FOXA1. The discovery of new factor-factor spatial constraints in ChIP data is significant because it proposes testable models for regulatory factor interactions that will help elucidate genome function and the implementation of combinatorial

  13. Retroactivity effects dependency on the transcription factors binding mechanisms.

    PubMed

    Pantoja-Hernández, Libertad; Álvarez-Buylla, Elena; Aguilar-Ibáñez, Carlos F; Garay-Arroyo, Adriana; Soria-López, Alberto; Martínez-García, Juan Carlos

    2016-12-07

    Downstream connection effects on transcription are caused by retroactivity. When biomolecular dynamical systems interconnect retroactivity is a property that becomes important. The biological functional meaning of these effects is increasingly becoming an area of interest. Downstream targets, which are operator binding sites in transcriptional networks, may induce behaviors such as ultrasensitive responses or even represent an undesired issue in regulation. To the best of our knowledge, the role of the binding mechanisms of transcription factors in relation to minimizing - or enhancing - retroactivity effects has not been previously addressed. Our aim is to evaluate retroactivity effects considering how the binding mechanism impacts the number of free functional transcription factor (FFTF) molecules using a simple model via deterministic and stochastic simulations. We study four transcription factor binding mechanisms (BM): simple monomer binding (SMB), dimer binding (DB), cooperative sequential binding (CSB) and cooperative sequential binding with dimerization (CSB_D). We consider weak and strong binding regimes for each mechanism, where we contrast the cases when the FFTF is bound or unbound to the downstream loads. Upon interconnection, the number of FFTF molecules changed less for the SMB mechanism while for DB they changed the most. Our results show that for the chosen mechanisms (in terms of the corresponding described dynamics), retroactivity effects depend on transcription binding mechanisms. This contributes to the understanding of how the transcription factor regulatory function-such as decision making-and its dynamic needs for the response, may determine the nature of the selected binding mechanism.

  14. Pigment epithelium-derived factor (PEDF) normalizes matrix defects in iPSCs derived from Osteogenesis imperfecta Type VI.

    PubMed

    Belinsky, Glenn S; Ward, Leanne; Chung, Chuhan

    2016-01-01

    Osteogenesis imperfecta (OI) Type VI is characterized by a defect in bone mineralization, which results in multiple fractures early in life. Null mutations in the PEDF gene, Serpinf1, are the cause of OI VI. Whether PEDF restoration in a murine model of OI Type VI could improve bone mass and function was previously unknown. In Belinsky et al, we provided evidence that PEDF delivery enhanced bone mass and improved parameters of bone function in vivo. Further, we demonstrated that PEDF temporally inhibits Wnt signaling to enhance osteoblast differentiation. Here, we demonstrate that generation of induced pluripotent stem cells (iPSCs) from a PEDF null patient provides additional evidence for PEDF's role in regulating extracellular matrix proteins secreted from osteoblasts. PEDF null iPSCs have marked abnormalities in secreted matrix proteins, capturing a key feature of human OI Type VI, which were normalized by exogenous PEDF. Lastly, we place our recent findings within the broader context of PEDF biology and the developmental signaling pathways that are implicated in its actions.

  15. Pigment Epithelium-Derived Factor (PEDF) is a Determinant of Stem Cell Fate: Lessons from an Ultra-Rare Disease.

    PubMed

    Sagheer, Usman; Gong, Jingjing; Chung, Chuhan

    2015-12-01

    PEDF is a secreted glycoprotein that is widely expressed by multiple organs. Numerous functional contributions have been attributed to PEDF with antiangiogenic, antitumor, anti-inflammatory, and neurotrophic properties among the most prominent. The discovery that null mutations in the PEDF gene results in Osteogenesis Imperfecta Type VI, a rare autosomal recessive bone disease characterized by multiple fractures, highlights a critical developmental function for this protein. This ultra-rare orphan disease has provided biological insights into previous studies that noted PEDF's effects on various stem cell populations. In addition to bone development, PEDF modulates resident stem cell populations in the brain, muscle, and eye. Functional effects on human embryonic stem cells have also been demonstrated. An overview of recent advances in our understanding by which PEDF regulates stem cells and their potential clinical applications will be evaluated in this review.

  16. Generation of tooth-periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells.

    PubMed

    Zhang, Yancong; Li, Yongliang; Shi, Ruirui; Zhang, Siqi; Liu, Hao; Zheng, Yunfei; Li, Yan; Cai, Jinglei; Pei, Duanqing; Wei, Shicheng

    2017-06-08

    A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. An optimized induction method was established to promote the differentiation of mES cells into dental

  17. Effects of Quercetin Treatment on Epithelium-derived Cytokines and Epithelial Cell Apoptosis in Allergic Airway Inflammation Mice Model.

    PubMed

    Caglayan Sozmen, Sule; Karaman, Meral; Cilaker Micili, Serap; Isik, Sakine; Bagriyanik, Alper; Arikan Ayyildiz, Zeynep; Uzuner, Nevin; Anal, Ozden; Karaman, Ozkan

    2016-12-01

    .05). These findings suggest that quercetin improves chronic histopathological changes except basal membrane thickness in lung tissue and its beneficial effects on inflammation might be related to modulating epithelium derived cytokines and epithelial apoptosis.

  18. Sequential coagulation factor VIIa domain binding to tissue factor

    SciTech Connect

    Oesterlund, Maria; Persson, Egon; Freskgard, Per-Ola . E-mail: msv@ifm.liu.se

    2005-12-02

    Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the {gamma}-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.

  19. Why Transcription Factor Binding Sites Are Ten Nucleotides Long

    PubMed Central

    Stewart, Alexander J.; Hannenhalli, Sridhar; Plotkin, Joshua B.

    2012-01-01

    Gene expression is controlled primarily by transcription factors, whose DNA binding sites are typically 10 nt long. We develop a population-genetic model to understand how the length and information content of such binding sites evolve. Our analysis is based on an inherent trade-off between specificity, which is greater in long binding sites, and robustness to mutation, which is greater in short binding sites. The evolutionary stable distribution of binding site lengths predicted by the model agrees with the empirical distribution (5–31 nt, with mean 9.9 nt for eukaryotes), and it is remarkably robust to variation in the underlying parameters of population size, mutation rate, number of transcription factor targets, and strength of selection for proper binding and selection against improper binding. In a systematic data set of eukaryotic and prokaryotic transcription factors we also uncover strong relationships between the length of a binding site and its information content per nucleotide, as well as between the number of targets a transcription factor regulates and the information content in its binding sites. Our analysis explains these features as well as the remarkable conservation of binding site characteristics across diverse taxa. PMID:22887818

  20. DNA-binding small molecules as inhibitors of transcription factors.

    PubMed

    Leung, Chung-Hang; Chan, Daniel Shiu-Hin; Ma, Victor Pui-Yan; Ma, Dik-Lung

    2013-07-01

    Accumulating evidence implicating the role of aberrant transcription factor signaling in the pathogenesis of various human diseases such as cancer and inflammation has stimulated the development of small molecule ligands capable of targeting transcription factor activity and modulating gene expression. The use of DNA-binding small molecules to selectively inhibit transcription factor-DNA interactions represents one possible approach toward this goal. In this review, we summarize the development of DNA-binding small molecule inhibitors of transcription factors from 2004 to 2011, and their binding mode and therapeutic potential will be discussed. © 2012 Wiley Periodicals, Inc.

  1. Modulation of DNA binding by gene-specific transcription factors.

    PubMed

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  2. The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein.

    PubMed

    Kwon, H; Green, M R

    1994-12-02

    The accurate transcription of human rRNA genes by RNA polymerase I requires two transcription factors, upstream binding factor (UBF) and promoter selectivity factor (SL1). Human SL1 (hSL1) is a multisubunit complex, one of whose components is TATA box-binding protein (TBP). hSL1 binds to the core region of the rRNA promoter, but does so inefficiently in the absence of human UBF (hUBF). hUBF interacts with the upstream control element of the rRNA promoter and facilitates binding of hSL1. The molecular basis by which hUBF increases binding of hSL1 remains to be elucidated. In this report, we use an immobilized protein binding assay to identify and purify a 95-kDa TBP-binding polypeptide. Microsequence analysis reveals that the 95-kDa TBP-binding protein is hUBF. We show that hUBF is stably associated with TBP and is present in large TBP-containing complexes. Our results indicate that the cooperative binding of hUBF and hSL1 on the rRNA promoter is mediated by direct interaction between hUBF and TBP. We also provide evidence that hUBF associates with TFIID, a TBP-containing RNA polymerase II transcription factor.

  3. Transcription factor binding predicts histone modifications in human cell lines

    PubMed Central

    Benveniste, Dan; Sonntag, Hans-Joachim; Sanguinetti, Guido; Sproul, Duncan

    2014-01-01

    Gene expression in higher organisms is thought to be regulated by a complex network of transcription factor binding and chromatin modifications, yet the relative importance of these two factors remains a matter of debate. Here, we show that a computational approach allows surprisingly accurate prediction of histone modifications solely from knowledge of transcription factor binding both at promoters and at potential distal regulatory elements. This accuracy significantly and substantially exceeds what could be achieved by using DNA sequence as an input feature. Remarkably, we show that transcription factor binding enables strikingly accurate predictions across different cell lines. Analysis of the relative importance of specific transcription factors as predictors of specific histone marks recapitulated known interactions between transcription factors and histone modifiers. Our results demonstrate that reported associations between histone marks and gene expression may be indirect effects caused by interactions between transcription factors and histone-modifying complexes. PMID:25187560

  4. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  5. Transcription Factor Binding Sites Prediction Based on Modified Nucleosomes

    PubMed Central

    Talebzadeh, Mohammad; Zare-Mirakabad, Fatemeh

    2014-01-01

    In computational methods, position weight matrices (PWMs) are commonly applied for transcription factor binding site (TFBS) prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP) predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, “modified nucleosomes neighboring” and “modified nucleosomes occupancy”, to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method for TFBS

  6. Scatter factor binds to thrombospondin and other extracellular matrix components.

    PubMed Central

    Lamszus, K.; Joseph, A.; Jin, L.; Yao, Y.; Chowdhury, S.; Fuchs, A.; Polverini, P. J.; Goldberg, I. D.; Rosen, E. M.

    1996-01-01

    Scatter factor (SF) is an angiogenic growth factor that stimulates motility and invasion of carcinoma cells. SF is present in the extracellular matrix (ECM) of breast cancers, where it might act to promote tumor cell invasion and angiogenesis. To investigate how SF is incorporated into the ECM, we studied the binding of SF to various ECM components using a solid-phase binding assay based on the SF enzyme-linked immunosorbent assay. We found that SF binds to a variety of ECM molecules, with different binding capacities. The highest SF binding capacities were observed for thrombospondin-1 (TSP-1), fibronectin (Fn), and heparan sulfate proteoglycan, although SF did not bind to albumin. Mature two-chain SF and precursor single-chain SF bound approximately equally well to TSP-1 and Fn. Moreover, two SF alpha-chain peptides (NK1 and NK2) both bound to TSP-1 and Fn, suggesting that the whole SF molecule is not required for binding. Based on binding competition assays, TSP-1 exhibited higher affinity for SF than did nine other ECM molecules, including Fn and heparan sulfate proteoglycan. Although heparin in solution potently inhibited the binding of SF to TSP-1-coated surfaces, even very high concentrations of heparin could not elute SF already bound to TSP-1. SF binding was modulated by binding interactions among ECM molecules (TSP-1-Fn, TSP-1-collagen I, and Fn-collagen I), suggesting that the matrix capacity to bind SF depends upon its exact composition. SF bound in a dose-dependent fashion to ECMs secreted by three human breast carcinoma cell lines. Binding of SF to matrices from all three cell lines was significantly inhibited by preincubation of the matrices with antibodies against TSP-1, whereas antibodies against several other ECM components were less effective or ineffective in inhibiting SF binding. In addition, TSP-1 markedly inhibited chemotaxis of microvascular endothelial cells toward SF and SF-induced angiogenesis in the rat cornea neovascularization assay

  7. Transcription factor binding dynamics during human ESC differentiation

    PubMed Central

    Tsankov, Alexander M.; Gu, Hongcang; Akopian, Veronika; Ziller, Michael J.; Donaghey, Julie; Amit, Ido; Gnirke, Andreas; Meissner, Alexander

    2015-01-01

    Summary Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation. PMID:25693565

  8. Zinc rapidly induces a metal response element-binding factor.

    PubMed Central

    Czupryn, M; Brown, W E; Vallee, B L

    1992-01-01

    Metal activation of metallothionein gene transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex. Addition to the growth medium of zinc, cadmium, or copper--metals known to induce MT biosynthesis in vivo--resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied. This result was not seen when the metals were added directly to the extracts from control cells. DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2. MRE-BF1 has an apparent molecular mass of approximately 86 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of approximately 28 kDa and binds to the hMREa in metal-treated cells. EDTA and o-phenanthroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA. Images PMID:1332048

  9. Identifying differential transcription factor binding in ChIP-seq.

    PubMed

    Wu, Dai-Ying; Bittencourt, Danielle; Stallcup, Michael R; Siegmund, Kimberly D

    2015-01-01

    ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.

  10. Identifying differential transcription factor binding in ChIP-seq

    PubMed Central

    Wu, Dai-Ying; Bittencourt, Danielle; Stallcup, Michael R.; Siegmund, Kimberly D.

    2015-01-01

    ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement. PMID:25972895

  11. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  12. DNA-binding specificities of human transcription factors.

    PubMed

    Jolma, Arttu; Yan, Jian; Whitington, Thomas; Toivonen, Jarkko; Nitta, Kazuhiro R; Rastas, Pasi; Morgunova, Ekaterina; Enge, Martin; Taipale, Mikko; Wei, Gonghong; Palin, Kimmo; Vaquerizas, Juan M; Vincentelli, Renaud; Luscombe, Nicholas M; Hughes, Timothy R; Lemaire, Patrick; Ukkonen, Esko; Kivioja, Teemu; Taipale, Jussi

    2013-01-17

    Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Design of Meningococcal Factor H Binding Protein Mutant Vaccines That Do Not Bind Human Complement Factor H

    PubMed Central

    Pajon, Rolando; Beernink, Peter T.

    2012-01-01

    Meningococcal factor H binding protein (fHbp) is a human species-specific ligand for the complement regulator, factor H (fH). In recent studies, fHbp vaccines in which arginine at position 41 was replaced by serine (R41S) had impaired fH binding. The mutant vaccines elicited bactericidal responses in human fH transgenic mice superior to those elicited by control fHbp vaccines that bound human fH. Based on sequence similarity, fHbp has been classified into three variant groups. Here we report that although R41 is present in fHbp from variant groups 1 and 2, the R41S substitution eliminated fH binding only in variant group 1 proteins. To identify mutants in variant group 2 with impaired fH binding, we generated fHbp structural models and predicted 63 residues influencing fH binding. From these, we created 11 mutants with one or two amino acid substitutions in a variant group 2 protein and identified six that decreased fH binding. Three of these six mutants retained conformational epitopes recognized by all six anti-fHbp monoclonal antibodies (MAbs) tested and elicited serum complement-mediated bactericidal antibody titers in wild-type mice that were not significantly different from those obtained with the control vaccine. Thus, fHbp amino acid residues that affect human fH binding differ across variant groups. This result suggests that fHbp sequence variation induced by immune selection also affects fH binding motifs via coevolution. The three new fHbp mutants from variant group 2, which do not bind human fH, retained important epitopes for eliciting bactericidal antibodies and may be promising vaccine candidates. PMID:22615247

  14. Design of meningococcal factor H binding protein mutant vaccines that do not bind human complement factor H.

    PubMed

    Pajon, Rolando; Beernink, Peter T; Granoff, Dan M

    2012-08-01

    Meningococcal factor H binding protein (fHbp) is a human species-specific ligand for the complement regulator, factor H (fH). In recent studies, fHbp vaccines in which arginine at position 41 was replaced by serine (R41S) had impaired fH binding. The mutant vaccines elicited bactericidal responses in human fH transgenic mice superior to those elicited by control fHbp vaccines that bound human fH. Based on sequence similarity, fHbp has been classified into three variant groups. Here we report that although R41 is present in fHbp from variant groups 1 and 2, the R41S substitution eliminated fH binding only in variant group 1 proteins. To identify mutants in variant group 2 with impaired fH binding, we generated fHbp structural models and predicted 63 residues influencing fH binding. From these, we created 11 mutants with one or two amino acid substitutions in a variant group 2 protein and identified six that decreased fH binding. Three of these six mutants retained conformational epitopes recognized by all six anti-fHbp monoclonal antibodies (MAbs) tested and elicited serum complement-mediated bactericidal antibody titers in wild-type mice that were not significantly different from those obtained with the control vaccine. Thus, fHbp amino acid residues that affect human fH binding differ across variant groups. This result suggests that fHbp sequence variation induced by immune selection also affects fH binding motifs via coevolution. The three new fHbp mutants from variant group 2, which do not bind human fH, retained important epitopes for eliciting bactericidal antibodies and may be promising vaccine candidates.

  15. Interaction of HRP-2 isoforms with HDGF: chromatin binding of a specific heteromer.

    PubMed

    Thakar, Ketan; Votteler, Ina; Kelkar, Dipti; Shidore, Teja; Gupta, Shivangi; Kelm, Sørge; Dietz, Frank

    2012-03-01

    Hepatoma-derived growth-factor-related protein 2 (HRP-2) belongs to a family with five additional members: hepatoma-derived growth factor (HDGF); lens epithelium-derived growth factor; and the HDGF-related proteins -1, -3 and -4. Very little is known regarding the function of HRP-2 in particular. This study shows for the first time heteromer formation of different members of the HRP family; HDGF and HRP-2. In addition, we discovered a previously unknown splice variant of HRP-2 mRNA encoding for a protein with a 53-amino acid deletion in its hath region. This HRP-2 isoform c interacts preferentially with a processed form of HDGF probably because of the loss of an α helix of HRP-2. Furthermore, in contrast to other isoforms of HRP-2, isoform c binds to chromatin similar to its most closely related family member lens epithelium-derived growth factor with potential consequences regarding its function in HIV integration. Interestingly, only the new HRP-2 isoform c and a processed form of HDGF are displaced from condensed mitotic metaphase chromatin. In conclusion, these observations provide a new perspective for understanding the biological functions of HDGF and related proteins.

  16. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  17. Architecture and RNA binding of the human negative elongation factor

    PubMed Central

    Vos, Seychelle M; Pöllmann, David; Caizzi, Livia; Hofmann, Katharina B; Rombaut, Pascaline; Zimniak, Tomasz; Herzog, Franz; Cramer, Patrick

    2016-01-01

    Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF. DOI: http://dx.doi.org/10.7554/eLife.14981.001 PMID:27282391

  18. Specific binding of atrial natriuretic factor in brain microvessels

    SciTech Connect

    Chabrier, P.E.; Roubert, P.; Braquet, P.

    1987-04-01

    Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using /sup 125/I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of /sup 125/I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.

  19. The Next Generation of Transcription Factor Binding Site Prediction

    PubMed Central

    Mathelier, Anthony; Wasserman, Wyeth W.

    2013-01-01

    Finding where transcription factors (TFs) bind to the DNA is of key importance to decipher gene regulation at a transcriptional level. Classically, computational prediction of TF binding sites (TFBSs) is based on basic position weight matrices (PWMs) which quantitatively score binding motifs based on the observed nucleotide patterns in a set of TFBSs for the corresponding TF. Such models make the strong assumption that each nucleotide participates independently in the corresponding DNA-protein interaction and do not account for flexible length motifs. We introduce transcription factor flexible models (TFFMs) to represent TF binding properties. Based on hidden Markov models, TFFMs are flexible, and can model both position interdependence within TFBSs and variable length motifs within a single dedicated framework. The availability of thousands of experimentally validated DNA-TF interaction sequences from ChIP-seq allows for the generation of models that perform as well as PWMs for stereotypical TFs and can improve performance for TFs with flexible binding characteristics. We present a new graphical representation of the motifs that convey properties of position interdependence. TFFMs have been assessed on ChIP-seq data sets coming from the ENCODE project, revealing that they can perform better than both PWMs and the dinucleotide weight matrix extension in discriminating ChIP-seq from background sequences. Under the assumption that ChIP-seq signal values are correlated with the affinity of the TF-DNA binding, we find that TFFM scores correlate with ChIP-seq peak signals. Moreover, using available TF-DNA affinity measurements for the Max TF, we demonstrate that TFFMs constructed from ChIP-seq data correlate with published experimentally measured DNA-binding affinities. Finally, TFFMs allow for the straightforward computation of an integrated TF occupancy score across a sequence. These results demonstrate the capacity of TFFMs to accurately model DNA

  20. Telomere-binding factors in the regulation of DNA replication.

    PubMed

    Masai, Hisao; Kanoh, Yutaka; Moriyama, Kenji; Yamazaki, Satoshi; Yoshizawa, Naoko; Matsumoto, Seiji

    2017-06-30

    Recent studies have indicated new roles for telomere-binding factors in the regulation of DNA replication, not only at the telomeres but also at the arm regions of the chromosome. Among these factors, Rif1, a conserved protein originally identified in yeasts as a telomere regulator, play a major role in the spatiotemporal regulation of DNA replication during S phase. Its ability to interact with phosphatases and to create specific higher-order chromatin structures is central to the mechanism by which Rif1 exerts this function. In this review, we discuss recent progress in elucidating the roles of Rif1 and other telomere-binding factors in the regulation of chromosome events occurring at locations other than telomeres.

  1. Cooperative binding of Ets-1 and core binding factor to DNA.

    PubMed Central

    Wotton, D; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1994-01-01

    Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription. Images PMID:8264651

  2. Soluble spiroperidol binding factors from bovine caudate nucleus.

    PubMed

    Winkler, M H; Berl, S

    1982-09-01

    Several properties of soluble spiroperidol binding factors separated from bovine caudate nucleus have been investigated by a previously unreported procedure. Data consistent with high particle weight and rapid binding equilibration are reported for high-affinity (+)butaclamol-sensitive components of a digitonin extract. A slower sedimenting component is found that also exhibits high affinity for spiroperidol but is not sensitive to (+)butaclamol. Centrifugation of a caudate nucleus homogenate yields a supernatant that appears to contain a component that exhibits spiroperidol binding that is more sensitive to displacement by (-) than by (+)butaclamol. The procedure used effects rapid separation of bound from unbound tritiated ligand on short columns of Sephadex G-15 followed by extrusion and sectioning of the Sephadex. The radioactivity remaining with each section is determined. The procedure is very rapid; the addition of active phases or the changing of the ionic environment, which may disturb the equilibrium, is avoided; and recovery of the protein free of bound ligand is easily affected.

  3. Insulin-Like Growth Factor Binding Proteins: A Structural Perspective

    PubMed Central

    Forbes, Briony E.; McCarthy, Peter; Norton, Raymond S.

    2012-01-01

    Insulin-like growth factor binding proteins (IGFBP-1 to -6) bind insulin-like growth factors-I and -II (IGF-I and IGF-II) with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation, and survival via the type 1 IGF receptor. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that regulate processes such as cell migration and apoptosis by modulating gene transcription. IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, linker, and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarizes structural studies reported so far and highlights features important for binding not only IGF but also other partners. We also highlight future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease. PMID:22654863

  4. Mechanisms of Unphosphorylated STAT3 Transcription Factor Binding to DNA*

    PubMed Central

    Timofeeva, Olga A.; Chasovskikh, Sergey; Lonskaya, Irina; Tarasova, Nadya I.; Khavrutskii, Lyuba; Tarasov, Sergey G.; Zhang, Xueping; Korostyshevskiy, Valeriy R.; Cheema, Amrita; Zhang, Lihua; Dakshanamurthy, Sivanesan; Brown, Milton L.; Dritschilo, Anatoly

    2012-01-01

    Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ (gamma)-activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to the GAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer. PMID:22378781

  5. Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII.

    PubMed

    Ganz, P R; Atkins, J S; Palmer, D S; Dudani, A K; Hashemi, S; Luison, F

    1991-10-15

    Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.

  6. Predicting DNA-Binding Specificities of Eukaryotic Transcription Factors

    PubMed Central

    Schröder, Adrian; Eichner, Johannes; Supper, Jochen; Eichner, Jonas; Wanke, Dierk; Henneges, Carsten; Zell, Andreas

    2010-01-01

    Today, annotated amino acid sequences of more and more transcription factors (TFs) are readily available. Quantitative information about their DNA-binding specificities, however, are hard to obtain. Position frequency matrices (PFMs), the most widely used models to represent binding specificities, are experimentally characterized only for a small fraction of all TFs. Even for some of the most intensively studied eukaryotic organisms (i.e., human, rat and mouse), roughly one-sixth of all proteins with annotated DNA-binding domain have been characterized experimentally. Here, we present a new method based on support vector regression for predicting quantitative DNA-binding specificities of TFs in different eukaryotic species. This approach estimates a quantitative measure for the PFM similarity of two proteins, based on various features derived from their protein sequences. The method is trained and tested on a dataset containing 1 239 TFs with known DNA-binding specificity, and used to predict specific DNA target motifs for 645 TFs with high accuracy. PMID:21152420

  7. Variable structure motifs for transcription factor binding sites.

    PubMed

    Reid, John E; Evans, Kenneth J; Dyer, Nigel; Wernisch, Lorenz; Ott, Sascha

    2010-01-14

    Classically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable

  8. Factor H-binding protein, a unique meningococcal vaccine antigen.

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

    GNA1870, also named factor H-binding protein (fHbp) or rLP-2086, is a genome-derived antigen and one of the components of a rationally designed vaccine against Neisseria meningitidis serogroup B, which has entered phase III clinical trials. It has been classified into three main non-cross-protective variant groups. GNA1870 has also been termed fHbp because of its ability to bind factor H, a key regulatory component of the alternative complement pathway. fHbp is important for survival in human blood, human sera, and in presence of antimicrobial peptides, independently of its expression level. All these properties make fHbp a unique vaccine antigen.

  9. OCTAMER-BINDING TRANSCRIPTION FACTORS: GENOMICS AND FUNCTIONS

    PubMed Central

    Zhao, Feng-Qi

    2015-01-01

    The Octamer-binding proteins (Oct) are a group of highly conserved transcription factors that specifically bind to the octamer motif (ATGCAAAT) and closely related sequences that are found in promoters and enhancers of a wide variety of both ubiquitously expressed and cell type-specific genes. Oct factors belong to the larger family of POU domain factors that are characterized by the presence of a highly conserved bipartite DNA binding domain, consisting of an amino-terminal specific subdomain (POUS) and a carboxyl-terminal homeo-subdomain (POUH). Eleven Oct proteins have been named (Oct1-11), and currently, eight genes encoding Oct proteins (Oct1, Oct2, Oct3/4, Oct6, Oct7, Oct8, Oct9, and Oct11) have been cloned and characterized. Oct1 and Oct2 are widely expressed in adult tissues, while other Oct proteins are much more restricted in their expression patterns. Oct proteins are implicated in crucial and versatile biological events, such as embryogenesis, neurogenesis, immunity, and body glucose and amino acid metabolism. The aberrant expression and null function of Oct proteins have also been linked to various diseases, including deafness, diabetes and cancer. In this review, I will report both the genomic structure and major functions of individual Oct proteins in physiological and pathological processes. PMID:23747866

  10. Saccharin and cyclamate inhibit binding of epidermal growth factor.

    PubMed Central

    Lee, L S

    1981-01-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit. PMID:6262753

  11. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    NASA Astrophysics Data System (ADS)

    Lee, L. S.

    1981-02-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

  12. Exposure to multi-walled carbon nanotubes results in aggravation of airway inflammation and remodeling and in increased production of epithelium-derived innate cytokines in a mouse model of asthma.

    PubMed

    Ronzani, Carole; Casset, Anne; Pons, Françoise

    2014-02-01

    With the development of nanotechnologies, the potential adverse effects of nanomaterials such as multi-walled carbon nanotubes (MWCNT) on the respiratory tract of asthmatics are questioned. Furthermore, investigations are necessary to understand how these effects might arise. In the present study, we hypothesized that epithelium-derived innate cytokines that are considered as important promoting factors in allergy may contribute to an aggravating effect of MWCNT on asthma. We investigated in the mouse the effect of MWCNT on systemic immune response and airway inflammation and remodeling induced by the most frequent allergen so far associated with asthma, house dust mite (HDM), and we examined the production of the innate cytokines thymic stromal lymphopoietin (TSLP), IL-25, IL-33, and GM-CSF. Mice exposed to HDM exhibited specific IgG1 in serum and inflammatory cell infiltration, and increased Th2 cytokine production, mucus hyperproduction, and collagen deposition in the airways when compared to naïve animals. Levels of total IgG1 and HDM-specific IgG1, influx of macrophages, eosinophils and neutrophils, production of collagen, TGF-β1, and mucus, as well as levels of IL-13, eotaxin, and TARC, were dose-dependently increased in mice exposed to HDM and MWCNT compared to HDM alone. These effects were associated with an increased production of TSLP, IL-25, IL-33, and GM-CSF in the airways. Our data demonstrate that MWCNT increase in a dose-dependent manner systemic immune response, as well as airway allergic inflammation and remodeling induced by HDM in the mouse. Our data suggest also a role for airway epithelium and innate cytokines in these effects.

  13. Imputation for transcription factor binding predictions based on deep learning

    PubMed Central

    Qin, Qian

    2017-01-01

    Understanding the cell-specific binding patterns of transcription factors (TFs) is fundamental to studying gene regulatory networks in biological systems, for which ChIP-seq not only provides valuable data but is also considered as the gold standard. Despite tremendous efforts from the scientific community to conduct TF ChIP-seq experiments, the available data represent only a limited percentage of ChIP-seq experiments, considering all possible combinations of TFs and cell lines. In this study, we demonstrate a method for accurately predicting cell-specific TF binding for TF-cell line combinations based on only a small fraction (4%) of the combinations using available ChIP-seq data. The proposed model, termed TFImpute, is based on a deep neural network with a multi-task learning setting to borrow information across transcription factors and cell lines. Compared with existing methods, TFImpute achieves comparable accuracy on TF-cell line combinations with ChIP-seq data; moreover, TFImpute achieves better accuracy on TF-cell line combinations without ChIP-seq data. This approach can predict cell line specific enhancer activities in K562 and HepG2 cell lines, as measured by massively parallel reporter assays, and predicts the impact of SNPs on TF binding. PMID:28234893

  14. Imputation for transcription factor binding predictions based on deep learning.

    PubMed

    Qin, Qian; Feng, Jianxing

    2017-02-01

    Understanding the cell-specific binding patterns of transcription factors (TFs) is fundamental to studying gene regulatory networks in biological systems, for which ChIP-seq not only provides valuable data but is also considered as the gold standard. Despite tremendous efforts from the scientific community to conduct TF ChIP-seq experiments, the available data represent only a limited percentage of ChIP-seq experiments, considering all possible combinations of TFs and cell lines. In this study, we demonstrate a method for accurately predicting cell-specific TF binding for TF-cell line combinations based on only a small fraction (4%) of the combinations using available ChIP-seq data. The proposed model, termed TFImpute, is based on a deep neural network with a multi-task learning setting to borrow information across transcription factors and cell lines. Compared with existing methods, TFImpute achieves comparable accuracy on TF-cell line combinations with ChIP-seq data; moreover, TFImpute achieves better accuracy on TF-cell line combinations without ChIP-seq data. This approach can predict cell line specific enhancer activities in K562 and HepG2 cell lines, as measured by massively parallel reporter assays, and predicts the impact of SNPs on TF binding.

  15. Binding.

    ERIC Educational Resources Information Center

    Rebsamen, Werner

    1981-01-01

    Categorizes contemporary methods of binding printed materials in terms of physical preservation--hand binding (archival restoration), edition binding (paperback, hardcover), publication binding (magazines), textbook binding (sidesewn), single-sheet binding (loose-leaf, mechanical), and library binding (oversewn, sidesewn). Seven references are…

  16. Understanding variation in transcription factor binding by modeling transcription factor genome-epigenome interactions.

    PubMed

    Chen, Chieh-Chun; Xiao, Shu; Xie, Dan; Cao, Xiaoyi; Song, Chun-Xiao; Wang, Ting; He, Chuan; Zhong, Sheng

    2013-01-01

    Despite explosive growth in genomic datasets, the methods for studying epigenomic mechanisms of gene regulation remain primitive. Here we present a model-based approach to systematically analyze the epigenomic functions in modulating transcription factor-DNA binding. Based on the first principles of statistical mechanics, this model considers the interactions between epigenomic modifications and a cis-regulatory module, which contains multiple binding sites arranged in any configurations. We compiled a comprehensive epigenomic dataset in mouse embryonic stem (mES) cells, including DNA methylation (MeDIP-seq and MRE-seq), DNA hydroxymethylation (5-hmC-seq), and histone modifications (ChIP-seq). We discovered correlations of transcription factors (TFs) for specific combinations of epigenomic modifications, which we term epigenomic motifs. Epigenomic motifs explained why some TFs appeared to have different DNA binding motifs derived from in vivo (ChIP-seq) and in vitro experiments. Theoretical analyses suggested that the epigenome can modulate transcriptional noise and boost the cooperativity of weak TF binding sites. ChIP-seq data suggested that epigenomic boost of binding affinities in weak TF binding sites can function in mES cells. We showed in theory that the epigenome should suppress the TF binding differences on SNP-containing binding sites in two people. Using personal data, we identified strong associations between H3K4me2/H3K9ac and the degree of personal differences in NFκB binding in SNP-containing binding sites, which may explain why some SNPs introduce much smaller personal variations on TF binding than other SNPs. In summary, this model presents a powerful approach to analyze the functions of epigenomic modifications. This model was implemented into an open source program APEG (Affinity Prediction by Epigenome and Genome, http://systemsbio.ucsd.edu/apeg).

  17. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6.

    PubMed

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H; Orian-Rousseau, Véronique

    2015-06-29

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.

  18. LASAGNA: A novel algorithm for transcription factor binding site alignment

    PubMed Central

    2013-01-01

    Background Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provides scoring matrices for TFs, nearly 78% of the TFs in the public release do not have matrices available. As work on TFBS alignment algorithms has been limited, it is highly desirable to have an alignment algorithm tailored to TFBSs. Results We designed a novel algorithm named LASAGNA, which is aware of the lengths of input TFBSs and utilizes position dependence. Results on 189 TFs of 5 species in the TRANSFAC database showed that our method significantly outperformed ClustalW2 and MEME. We further compared a PSSM method dependent on LASAGNA to an alignment-free TFBS search method. Results on 89 TFs whose binding sites can be located in genomes showed that our method is significantly more precise at fixed recall rates. Finally, we described LASAGNA-ChIP, a more sophisticated version for ChIP (Chromatin immunoprecipitation) experiments. Under the one-per-sequence model, it showed comparable performance with MEME in discovering motifs in ChIP-seq peak sequences. Conclusions We conclude that the LASAGNA algorithm is simple and effective in aligning variable-length binding sites. It has been integrated into a user-friendly webtool for TFBS search and visualization called LASAGNA-Search. The tool currently stores precomputed PSSM models for 189 TFs and 133 TFs built from TFBSs in the TRANSFAC Public database (release 7.0) and the ORegAnno database (08Nov10 dump), respectively. The webtool is available at http://biogrid.engr.uconn.edu/lasagna_search/. PMID:23522376

  19. Structural factors governing azide and cyanide binding to mammalian metmyoglobins.

    PubMed

    Brancaccio, A; Cutruzzolá, F; Allocatelli, C T; Brunori, M; Smerdon, S J; Wilkinson, A J; Dou, Y; Keenan, D; Ikeda-Saito, M; Brantley, R E

    1994-05-13

    The structural factors governing azide and cyanide binding have been examined by measuring the effects of 46 mutations at key topological positions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His64 (E7) with smaller amino acids results in dramatic increases in the association rate constant for azide binding primarily due to relief of steric hindrance imposed by the imidazole side chain. Gln64 and His64 (native) metmyoglobins have abnormally low rate constants for azide dissociation (0.1-0.3 s-1) due to direct hydrogen bonding between the N epsilon atoms of these residues and the bound ligand. Mutations at positions 67(E10) and 68(E11) produce large but complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, influence the rate of anion movement into the distal pocket, and affect the stability of the Fe-N3 bond. Replacement of Phe46 with Leu or Val and substitution of Arg(Lys)45 with Glu and Ser cause disorder in the position of the distal histidine side chain and result in 4-700-fold increases in both k'N3 and kN3 but produce little change in overall azide affinity. All of these results suggest strongly that azide enters the distal pocket of native myoglobin through a polar channel that is regulated by a His64 "gate." In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the distal histidine is replaced with apolar residues. His64, Gln64, and distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.

  20. Insulin-Like Growth Factor Binding Proteins--an Update.

    PubMed

    Bach, Leon A

    2015-12-01

    The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

  1. Insulin-like growth factor binding proteins 4-6.

    PubMed

    Bach, Leon A

    2015-10-01

    Insulin-like growth factor binding proteins (IGFBPs) 4-6 have important roles as modulators of IGF actions. IGFBP-4 and IGFBP-6 predominantly inhibit IGF actions, whereas IGFBP-5 may enhance these actions under some circumstances. IGFBP-6 is unique among the IGFBPs for its marked IGF-II binding preference. IGFBPs 4-6 are found in the circulation as binary complexes with IGFs that can enter tissues. Additionally, about half of the circulating IGFBP-5 is found in ternary complexes with IGFs and an acid labile subunit; this high molecular complex cannot leave the circulation and acts as an IGF reservoir. IGFBPs 4-6 also have IGF-independent actions. These IGFBPs are regulated in a cell-specific manner and their dysregulation may play a role in a range of diseases including cancer. However, there is no clear clinical indication for measuring serum levels of these IGFBPs at present.

  2. Spurious transcription factor binding: Non-functional or genetically redundant?

    PubMed Central

    Spivakov, Mikhail

    2014-01-01

    Transcription factor binding sites (TFBSs) on the DNA are generally accepted as the key nodes of gene control. However, the multitudes of TFBSs identified in genome-wide studies, some of them seemingly unconstrained in evolution, have prompted the view that in many cases TF binding may serve no biological function. Yet, insights from transcriptional biochemistry, population genetics and functional genomics suggest that rather than segregating into ‘functional’ or ‘non-functional’, TFBS inputs to their target genes may be generally cumulative, with varying degrees of potency and redundancy. As TFBS redundancy can be diminished by mutations and environmental stress, some of the apparently ‘spurious’ sites may turn out to be important for maintaining adequate transcriptional regulation under these conditions. This has significant implications for interpreting the phenotypic effects of TFBS mutations, particularly in the context of genome-wide association studies for complex traits. PMID:24888900

  3. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  4. Estimating binding properties of transcription factors from genome-wide binding profiles.

    PubMed

    Zabet, Nicolae Radu; Adryan, Boris

    2015-01-01

    The binding of transcription factors (TFs) is essential for gene expression. One important characteristic is the actual occupancy of a putative binding site in the genome. In this study, we propose an analytical model to predict genomic occupancy that incorporates the preferred target sequence of a TF in the form of a position weight matrix (PWM), DNA accessibility data (in the case of eukaryotes), the number of TF molecules expected to be bound specifically to the DNA and a parameter that modulates the specificity of the TF. Given actual occupancy data in the form of ChIP-seq profiles, we backwards inferred copy number and specificity for five Drosophila TFs during early embryonic development: Bicoid, Caudal, Giant, Hunchback and Kruppel. Our results suggest that these TFs display thousands of molecules that are specifically bound to the DNA and that whilst Bicoid and Caudal display a higher specificity, the other three TFs (Giant, Hunchback and Kruppel) display lower specificity in their binding (despite having PWMs with higher information content). This study gives further weight to earlier investigations into TF copy numbers that suggest a significant proportion of molecules are not bound specifically to the DNA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

    PubMed

    Claes, J; Liesenborghs, L; Peetermans, M; Veloso, T R; Missiakas, D; Schneewind, O; Mancini, S; Entenza, J M; Hoylaerts, M F; Heying, R; Verhamme, P; Vanassche, T

    2017-02-09

    Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress.

  6. The role of Octamer binding transcription factors in glioblastoma multiforme

    PubMed Central

    Rooj, AK.; Bronisz, A.

    2016-01-01

    A group of transcription factors (TF) that are master developmental regulators of the establishment and maintenance of pluripotency during embryogenesis play additional roles to control tissue homeostasis and regeneration in adults. Among these TFs, members of the Octamer-binding transcription factor (OCT) gene family are well documented as major regulators controlling the self-renewal and pluripotency of stem cells isolated from different adult organs including the brain. In the last few years a large number of studies show the aberrant expression and dysfunction of OCT in different types of cancers including glioblastoma multiforme (GBM). GBM is the most common malignant primary brain tumor, and contains a subpopulation of undifferentiated stem cells (GSCs), with self-renewal and tumorigenic potential that contribute to tumor initiation, invasion, recurrence, and therapeutic resistance. In this review, we have summarized the current knowledge about OCT family in GBM and their crucial role in the initiation, maintenance and drug resistance properties of GSCs. PMID:26968235

  7. Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    PubMed Central

    Yarian, Fatemeh; Bandehpour, Mojgan; Seyed, Negar; Kazemi, Bahram

    2016-01-01

    Background and Objective: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. Materials and Methods: A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. Results and Conclusions: SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein. PMID:27092222

  8. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

    PubMed

    Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

    2017-02-07

    High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

  9. Identifying combinatorial regulation of transcription factors and binding motifs

    PubMed Central

    Kato, Mamoru; Hata, Naoya; Banerjee, Nilanjana; Futcher, Bruce; Zhang, Michael Q

    2004-01-01

    Background Combinatorial interaction of transcription factors (TFs) is important for gene regulation. Although various genomic datasets are relevant to this issue, each dataset provides relatively weak evidence on its own. Developing methods that can integrate different sequence, expression and localization data have become important. Results Here we use a novel method that integrates chromatin immunoprecipitation (ChIP) data with microarray expression data and with combinatorial TF-motif analysis. We systematically identify combinations of transcription factors and of motifs. The various combinations of TFs involved multiple binding mechanisms. We reconstruct a new combinatorial regulatory map of the yeast cell cycle in which cell-cycle regulation can be drawn as a chain of extended TF modules. We find that the pairwise combination of a TF for an early cell-cycle phase and a TF for a later phase is often used to control gene expression at intermediate times. Thus the number of distinct times of gene expression is greater than the number of transcription factors. We also see that some TF modules control branch points (cell-cycle entry and exit), and in the presence of appropriate signals they can allow progress along alternative pathways. Conclusions Combining different data sources can increase statistical power as demonstrated by detecting TF interactions and composite TF-binding motifs. The original picture of a chain of simple cell-cycle regulators can be extended to a chain of composite regulatory modules: different modules may share a common TF component in the same pathway or a TF component cross-talking to other pathways. PMID:15287978

  10. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    SciTech Connect

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang . E-mail: xudg@nic.bmi.ac.cn

    2007-06-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies.

  11. Regulation factors of corticosteroid-binding globulin: lesson from ontogenesis.

    PubMed

    Séralini, G E

    1996-01-01

    This short review summarizes recent data on corticosteroid-binding globulin (CBG), especially enlightening results on regulation factors of CBG gene expression during ontogenesis. The role of CBG as a specific steroid carrier, a structurally conserved glycoprotein of 50-60 kD in vertebrate species, is well documented, but this knowledge has often been limited to the young or adult life since CBG levels are low in the neonate. However, CBG and CBG mRNA have been recently detected, sometimes, in relatively high amounts, in various fetal tissues of mammals including liver, lung, pancreas, adrenal and kidney. CBG can thus participate in glucocorticoid-inducible events crucial for maturation. Moreover, its original molecular cloning, followed by its chromosomal localization, has shed a new light on the CBG role, as a member of the serine protease inhibitors and substrates (SERPINS) superfamily. This evidenced a special and unexpected way of steroid hormones delivery to their sites of action. Additionally, two classes of CBG receptors have been characterized, and an adenylate cyclase activity has been measured when the CBG-glucocorticoid complex binds to cell membranes.

  12. Atrial natriuretic factor binding sites in experimental congestive heart failure

    SciTech Connect

    Bianchi, C.; Thibault, G.; Wrobel-Konrad, E.; De Lean, A.; Genest, J.; Cantin, M. )

    1989-10-01

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor ((Ser99, Tyr126)ANF) binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF (des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2) (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease.

  13. Computational modeling reveals molecular details of epidermal growth factor binding

    PubMed Central

    Mayawala, Kapil; Vlachos, Dionisios G; Edwards, Jeremy S

    2005-01-01

    Background The ErbB family of receptors are dysregulated in a number of cancers, and the signaling pathway of this receptor family is a critical target for several anti-cancer drugs. Therefore a detailed understanding of the mechanisms of receptor activation is critical. However, despite a plethora of biochemical studies and recent single particle tracking experiments, the early molecular mechanisms involving epidermal growth factor (EGF) binding and EGF receptor (EGFR) dimerization are not as well understood. Herein, we describe a spatially distributed Monte Carlo based simulation framework to enable the simulation of in vivo receptor diffusion and dimerization. Results Our simulation results are in agreement with the data from single particle tracking and biochemical experiments on EGFR. Furthermore, the simulations reveal that the sequence of receptor-receptor and ligand-receptor reaction events depends on the ligand concentration, receptor density and receptor mobility. Conclusion Our computer simulations reveal the mechanism of EGF binding on EGFR. Overall, we show that spatial simulation of receptor dynamics can be used to gain a mechanistic understanding of receptor activation which may in turn enable improved cancer treatments in the future. PMID:16318625

  14. Effects of cytosine methylation on transcription factor binding sites

    PubMed Central

    2014-01-01

    Background DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. Results We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines “traffic lights”. We observed a strong selection against CpG “traffic lights” within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. Conclusions Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. PMID:24669864

  15. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  16. The role of octamer binding transcription factors in glioblastoma multiforme.

    PubMed

    Rooj, A K; Bronisz, A; Godlewski, J

    2016-06-01

    A group of transcription factors (TF) that are master developmental regulators of the establishment and maintenance of pluripotency during embryogenesis play additional roles to control tissue homeostasis and regeneration in adults. Among these TFs, members of the octamer-binding transcription factor (OCT) gene family are well documented as major regulators controlling the self-renewal and pluripotency of stem cells isolated from different adult organs including the brain. In the last few years a large number of studies show the aberrant expression and dysfunction of OCT in different types of cancers including glioblastoma multiforme (GBM). GBM is the most common malignant primary brain tumor, and contains a subpopulation of undifferentiated stem cells (GSCs), with self-renewal and tumorigenic potential that contribute to tumor initiation, invasion, recurrence, and therapeutic resistance. In this review, we have summarized the current knowledge about OCT family in GBM and their crucial role in the initiation, maintenance and drug resistance properties of GSCs. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  18. Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

    PubMed

    Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

    1999-02-05

    We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

  19. BRAFV600E Mutation, RET/PTC1 and PAX8-PPAR Gamma Rearrangements in Follicular Epithelium Derived Thyroid Lesions - Institutional Experience and Literature Review

    PubMed Central

    Şahpaz, Ahmet; Önal, Binnur; Yeşilyurt, Ahmet; Han, Ünsal; Delibaşı, Tuncay

    2015-01-01

    Background: Thyroid cancers are the most frequently occurring endocrine malignancy worldwide. In Turkey, thyroid cancers are ranked 2nd on the incidence list in women, with a rate of 16.2%, but they are not included among the top 10 cancer types in men. Aims: To identify the contribution of the BRAFV600E mutation, and the RET/PTC1 and PAX8-PPARγ rearrangements in the diagnosis and differential diagnosis of follicular epithelial-derived thyroid lesions. Study Design: Retrospective clinical and molecular genetic study. Methods: A total of 86 thyroid cases diagnosed between 2001 and 2012 at the Department of Pathology were included in the retrospective study group. Samples best representing the lesion and comprising capsules were chosen in the selection of paraffin blocks pertaining to the cases. The BRAFV600E mutation, and the RET/PTC1 and PAX8-PPARγ rearrangements were investigated in all cases. Results: The BRAFV600E mutation was observed in 12 out of 37 papillary carcinoma cases (32.4%), in 1 out of 15 follicular carcinoma cases (6.6%), and in 1 out of 7 undifferentiated carcinoma cases (14.3%). No mutation was detected in benign lesions. The RET/PTC1 rearrangement was detected in 2 out of 7 undifferentiated carcinoma cases (28.6%), and in 1 out of 15 follicular carcinoma cases (6.6%). No gene rearrangement was detected in benign lesions. The PAX8-PPARγ rearrangement was detected in 5 out of 15 follicular thyroid carcinoma cases (33.3%) and in 1 out of 15 follicular adenoma cases (6.6%). Conclusion: The BRAFV600E mutation and RET/PTC1 rearrangement were effective in distinguishing the follicular epithelium-derived benign and malignant lesions of the thyroid in the resection materials. The BRAFV600E mutation was rather specific to papillary carcinoma in the thyroid, and in cases where the BRAFV600E mutation was detected, multi-centricity, lymph node metastasis and capsular invasion findings were observed more frequently compared to cases in which no mutation was

  20. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

    Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. Images PMID:3006062

  1. Gibbs Recursive Sampler: finding transcription factor binding sites.

    PubMed

    Thompson, William; Rouchka, Eric C; Lawrence, Charles E

    2003-07-01

    The Gibbs Motif Sampler is a software package for locating common elements in collections of biopolymer sequences. In this paper we describe a new variation of the Gibbs Motif Sampler, the Gibbs Recursive Sampler, which has been developed specifically for locating multiple transcription factor binding sites for multiple transcription factors simultaneously in unaligned DNA sequences that may be heterogeneous in DNA composition. Here we describe the basic operation of the web-based version of this sampler. The sampler may be acces-sed at http://bayesweb.wadsworth.org/gibbs/gibbs.html and at http://www.bioinfo.rpi.edu/applications/bayesian/gibbs/gibbs.html. An online user guide is available at http://bayesweb.wadsworth.org/gibbs/bernoulli.html and at http://www.bioinfo.rpi.edu/applications/bayesian/gibbs/manual/bernoulli.html. Solaris, Solaris.x86 and Linux versions of the sampler are available as stand-alone programs for academic and not-for-profit users. Commercial licenses are also available. The Gibbs Recursive Sampler is distributed in accordance with the ISCB level 0 guidelines and a requirement for citation of use in scientific publications.

  2. Mutations and Binding Sites of Human Transcription Factors

    PubMed Central

    Kamanu, Frederick Kinyua; Medvedeva, Yulia A.; Schaefer, Ulf; Jankovic, Boris R.; Archer, John A. C.; Bajic, Vladimir B.

    2012-01-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, “insertions” are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. PMID:22670148

  3. Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.

    PubMed Central

    Li, C; Lai, C F; Sigman, D S; Gaynor, R B

    1991-01-01

    Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements. Images PMID:1909027

  4. Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

    PubMed Central

    Goetz, Tamara L.; Gu, Ting-Lei; Speck, Nancy A.; Graves, Barbara J.

    2000-01-01

    Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor α2 (CBFα2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10- to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBFα2. Furthermore, two additional lines of evidence indicated that CBFα2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors. PMID:10594011

  5. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  6. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

    PubMed

    Lane, B Josh; Mutchler, Charla; Al Khodor, Souhaila; Grieshaber, Scott S; Carabeo, Rey A

    2008-03-01

    Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  7. TEMPLE: analysing population genetic variation at transcription factor binding sites.

    PubMed

    Litovchenko, Maria; Laurent, Stefan

    2016-11-01

    Genetic variation occurring at the level of regulatory sequences can affect phenotypes and fitness in natural populations. This variation can be analysed in a population genetic framework to study how genetic drift and selection affect the evolution of these functional elements. However, doing this requires a good understanding of the location and nature of regulatory regions and has long been a major hurdle. The current proliferation of genomewide profiling experiments of transcription factor occupancies greatly improves our ability to identify genomic regions involved in specific DNA-protein interactions. Although software exists for predicting transcription factor binding sites (TFBS), and the effects of genetic variants on TFBS specificity, there are no tools currently available for inferring this information jointly with the genetic variation at TFBS in natural populations. We developed the software Transcription Elements Mapping at the Population LEvel (TEMPLE), which predicts TFBS, evaluates the effects of genetic variants on TFBS specificity and summarizes the genetic variation occurring at TFBS in intraspecific sequence alignments. We demonstrate that TEMPLE's TFBS prediction algorithms gives identical results to PATSER, a software distribution commonly used in the field. We also illustrate the unique features of TEMPLE by analysing TFBS diversity for the TF Senseless (SENS) in one ancestral and one cosmopolitan population of the fruit fly Drosophila melanogaster. TEMPLE can be used to localize TFBS that are characterized by strong genetic differentiation across natural populations. This will be particularly useful for studies aiming to identify adaptive mutations. TEMPLE is a java-based cross-platform software that easily maps the genetic diversity at predicted TFBSs using a graphical interface, or from the Unix command line.

  8. Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

    PubMed

    Bittoun, P; Bagheri-Yarmand, R; Chaubet, F; Crépin, M; Jozefonvicz, J; Fermandjian, S

    1999-06-15

    CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

  9. Membrane-binding properties of the Factor VIII C2 domain

    PubMed Central

    Novakovic, Valerie A.; Cullinan, David B.; Wakabayashi, Hironao; Fay, Philip J.; Baleja, James D.; Gilbert, Gary E.

    2013-01-01

    Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is cooperative with other domains of the intact Factor VIII molecule. PMID:21210768

  10. Inhibition by Elongation Factor EF G of Aminoacyl-tRNA Binding to Ribosomes

    PubMed Central

    Cabrer, Bartolomé; Vázquez, David; Modolell, Juan

    1972-01-01

    Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg++ concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site. PMID:4551985

  11. Reliable scaling of position weight matrices for binding strength comparisons between transcription factors.

    PubMed

    Ma, Xiaoyan; Ezer, Daphne; Navarro, Carmen; Adryan, Boris

    2015-08-20

    Scoring DNA sequences against Position Weight Matrices (PWMs) is a widely adopted method to identify putative transcription factor binding sites. While common bioinformatics tools produce scores that can reflect the binding strength between a specific transcription factor and the DNA, these scores are not directly comparable between different transcription factors. Other methods, including p-value associated approaches (Touzet H, Varré J-S. Efficient and accurate p-value computation for position weight matrices. Algorithms Mol Biol. 2007;2(1510.1186):1748-7188), provide more rigorous ways to identify potential binding sites, but their results are difficult to interpret in terms of binding energy, which is essential for the modeling of transcription factor binding dynamics and enhancer activities. Here, we provide two different ways to find the scaling parameter λ that allows us to infer binding energy from a PWM score. The first approach uses a PWM and background genomic sequence as input to estimate λ for a specific transcription factor, which we applied to show that λ distributions for different transcription factor families correspond with their DNA binding properties. Our second method can reliably convert λ between different PWMs of the same transcription factor, which allows us to directly compare PWMs that were generated by different approaches. These two approaches provide computationally efficient ways to scale PWM scores and estimate the strength of transcription factor binding sites in quantitative studies of binding dynamics. Their results are consistent with each other and previous reports in most of cases.

  12. Using protein-binding microarrays to study transcription factor specificity: homologs, isoforms and complexes

    PubMed Central

    Andrilenas, Kellen K.; Penvose, Ashley

    2015-01-01

    Protein–DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)–DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein–DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes. PMID:25431149

  13. CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

    PubMed Central

    Wright, K L; Vilen, B J; Itoh-Lindstrom, Y; Moore, T L; Li, G; Criscitiello, M; Cogswell, P; Clarke, J B; Ting, J P

    1994-01-01

    NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element. Images PMID:8076600

  14. Genome-scale study of the importance of binding site context for transcription factor binding and gene regulation

    PubMed Central

    Westholm, Jakub Orzechowski; Xu, Feifei; Ronne, Hans; Komorowski, Jan

    2008-01-01

    Background The rate of mRNA transcription is controlled by transcription factors that bind to specific DNA motifs in promoter regions upstream of protein coding genes. Recent results indicate that not only the presence of a motif but also motif context (for example the orientation of a motif or its location relative to the coding sequence) is important for gene regulation. Results In this study we present ContextFinder, a tool that is specifically aimed at identifying cases where motif context is likely to affect gene regulation. We used ContextFinder to examine the role of motif context in S. cerevisiae both for DNA binding by transcription factors and for effects on gene expression. For DNA binding we found significant patterns of motif location bias, whereas motif orientations did not seem to matter. Motif context appears to affect gene expression even more than it affects DNA binding, as biases in both motif location and orientation were more frequent in promoters of co-expressed genes. We validated our results against data on nucleosome positioning, and found a negative correlation between preferred motif locations and nucleosome occupancy. Conclusion We conclude that the requirement for stable binding of transcription factors to DNA and their subsequent function in gene regulation can impose constraints on motif context. PMID:19014636

  15. Pleiotropic virulence factor - Streptococcus pyogenes fibronectin-binding proteins.

    PubMed

    Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

    2013-04-01

    Streptococcus pyogenes causes a broad spectrum of infectious diseases, including pharyngitis, skin infections and invasive necrotizing fasciitis. The initial phase of infection involves colonization, followed by intimate contact with the host cells, thus promoting bacterial uptake by them. S. pyogenes recognizes fibronectin (Fn) through its own Fn-binding proteins to obtain access to epithelial and endothelial cells in host tissue. Fn-binding proteins bind to Fn to form a bridge to α5 β1 -integrins, which leads to rearrangement of cytoskeletal actin in host cells and uptake of invading S. pyogenes. Recently, several structural analyses of the invasion mechanism showed molecular interactions by which Fn converts from a compact plasma protein to a fibrillar component of the extracellular matrix. After colonization, S. pyogenes must evade the host innate immune system to spread into blood vessels and deeper organs. Some Fn-binding proteins contribute to evasion of host innate immunity, such as the complement system and phagocytosis. In addition, Fn-binding proteins have received focus as non-M protein vaccine candidates, because of their localization and conservation among different M serotypes.Here, we review the roles of Fn-binding proteins in the pathogenesis and speculate regarding possible vaccine antigen candidates. © 2012 Blackwell Publishing Ltd.

  16. Probing the Informational and Regulatory Plasticity of a Transcription Factor DNA–Binding Domain

    PubMed Central

    Shultzaberger, Ryan K.; Maerkl, Sebastian J.; Kirsch, Jack F.; Eisen, Michael B.

    2012-01-01

    Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA–binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable. PMID:22496663

  17. Relaxed selection against accidental binding of transcription factors with conserved chromatin contexts.

    PubMed

    Babbitt, G A

    2010-10-15

    The spurious (or nonfunctional) binding of transcription factors (TF) to the wrong locations on DNA presents a formidable challenge to genomes given the relatively low ceiling for sequence complexity within the short lengths of most binding motifs. The high potential for the occurrence of random motifs and subsequent nonfunctional binding of many transcription factors should theoretically lead to natural selection against the occurrence of spurious motif throughout the genome. However, because of the active role that chromatin can influence over eukaryotic gene regulation, it may also be expected that many supposed spurious binding sites could escape purifying selection if (A) they simply occur in regions of high nucleosome occupancy or (B) their surrounding chromatin was dynamically involved in their identity and function. We compared nucleosome occupancy and the presence/absence of functionally conserved chromatin context to the strength of selection against spurious binding of various TF binding motifs in Saccharomyces yeast. While we find no direct relationship with nucleosome occupancy, we find strong evidence that transcription factors spatially associated with evolutionarily conserved chromatin states are under relaxed selection against accidental binding. Transcription factors (with/without) a conserved chromatin context were found to occur on average, (87.7%/49.3%) of their expected frequencies. Functional binding motifs with conserved chromatin contexts were also significantly shorter in length and more often clustered. These results indicate a role of chromatin context dependency in relaxing selection against spurious binding in nearly half of all TF binding motifs throughout the yeast genome.

  18. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites.

    PubMed

    Lelieveld, Stefan H; Schütte, Judith; Dijkstra, Maurits J J; Bawono, Punto; Kinston, Sarah J; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-05-05

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites

    PubMed Central

    Lelieveld, Stefan H.; Schütte, Judith; Dijkstra, Maurits J.J.; Bawono, Punto; Kinston, Sarah J.; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-01-01

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. PMID:26721389

  20. Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene.

    PubMed

    Schwachtgen, J L; Janel, N; Barek, L; Duterque-Coquillaud, M; Ghysdael, J; Meyer, D; Kerbiriou-Nabias, D

    1997-12-18

    von Willebrand factor (vWF) gene expression is restricted to endothelial cells and megakaryocytes. Previous results demonstrated that basal transcription of the human vWF gene is mediated through a promoter located between base pairs -89 and +19 (cap site: +1) which is functional in endothelial and non endothelial cells. Two DNA repeats TTTCCTTT correlating with inverted consensus binding sites for the Ets family of transcription factors are present in the -56/-36 sequence. In order to analyse whether these DNA elements are involved in transcription, human umbilical vein endothelial cells (HUVEC), bovine calf pulmonary endothelial cell line (CPAE), HeLa and COS cells were transfected with constructs containing deletions of the -89/+19 fragment, linked to the chloramphenicol acetyl transferase (CAT) reporter gene. The -60/+19 region exhibits significant promoter activity in HUVEC and CPAE cells only. The -42/+19 fragment is not active. Mutations of the -60/+19 promoter fragment in the 5' (-56/-49) Ets binding site abolish transcription in endothelial cells whereas mutations in the 3' (-43/-36) site does not. The -60/-33 fragment forms three complexes with proteins from HUVEC nuclear extracts in electrophoretic mobility shift assay which are dependent on the presence of the 5' Ets binding site. Binding of recombinant Ets-1 protein to the -60/-33 fragment gives a complex which also depends on the 5' site. The -60/+19 vWF gene core promoter is transactivated in HeLa cells by cotransfecting with Ets-1 or Erg (Ets-related gene) expression plasmids. In contrast to the wild type construct, transcription of the 5' site mutants is not increased by these expressed proteins. The results indicate that the promoter activity of the -60/+19 region of the vWF gene depends on transcription factors of the Ets family of which several members like Ets-1, Ets-2 and Erg are expressed in endothelium. Cotransfection of Ets-1 and Erg expression plasmids is sufficient to induce the -60/+19 v

  1. The Xenopus laevis ribosomal gene promoter contains a binding site for nuclear factor-1.

    PubMed Central

    Walker, P; Reeder, R H

    1988-01-01

    Nuclear Factor I (NF1) is a DNA binding protein that is known to function in the replication of Adeno virus and also binds to many promoters recognized by RNA polymerase II. We have found that there is also an NF1 binding site within the ribosomal gene promoter from Xenopus laevis as well as in several other promoters recognized by RNA polymerase I. The function of a binding site for a polymerase II transcription factor within a promoter recognized by polymerase I is not known. However, its presence suggests interesting regulatory possibilities. Images PMID:3205719

  2. An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription.

    PubMed

    Yokomori, K; Verrijzer, C P; Tjian, R

    1998-06-09

    The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

  3. Suppressor Mutations within the Core Binding Factor (CBF/AML1) Binding Site of a T-Cell Lymphomagenic Retrovirus

    PubMed Central

    Martiney, Marita J.; Levy, Laura S.; Lenz, Jack

    1999-01-01

    The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TGCGGTCAA or TGTGGTCTA, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity. PMID:9971797

  4. Factors affecting binding of galacto ligands to Actinomyces viscosus lectin.

    PubMed Central

    Heeb, M J; Marini, A M; Gabriel, O

    1985-01-01

    The specificity requirements for the binding of Actinomyces viscosus T14V were examined by testing simple sugars, oligopeptides, and glycoproteins as inhibitors of the aggregation of glycoprotein-coated latex beads and washed A. viscosus cells. Lactose was the most inhibitory simple sugar; D-fucose and D-galactose were equally inhibitory, methyl-alpha-D-fucoside was slightly less inhibitory, and L-fucose and raffinose were not inhibitory. The concentration of galactose residues required for 50% inhibition of aggregation was 15 times higher in the form of lactose than in the form of asialoglycoprotein, suggesting an enhancement of lectin binding when galactose residues are clustered. However, when the inhibitory power of bi-, tri-, and tetraantennary asialooligopeptides of alpha 1-acid glycoprotein was compared with that of equivalent concentrations of galactose in the form of lactose, the biantennary form was slightly less effective than lactose, the triantennary form was approximately as effective as lactose, and the tetraantennary form was slightly more effective than lactose. Steric interference may prevent this type of clustering from enhancing lectin binding. The O-linked asialooligopeptides of asialofetuin were 10 times more inhibitory than an equivalent concentration of galactose in the form of N-linked asialooligopeptides. Thus, galactose beta-1----3 linked to N-acetylgalactosamine exhibits greater specificity for the A. viscosus lectin than does galactose beta-1----4 linked to N-acetylglucosamine. These results, taken together with previously reported data, are consistent with a lectin of low affinity, binding enhanced by multivalency, and specificity for beta-linked galactose. PMID:2578122

  5. Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.

    PubMed

    Kobayashi, Yuki; Takahashi, Toshiaki; Shibata, Toshio; Ikeda, Shunsuke; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2015-07-31

    Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade*

    PubMed Central

    Kobayashi, Yuki; Takahashi, Toshiaki; Shibata, Toshio; Ikeda, Shunsuke; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2015-01-01

    Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C. PMID:26109069

  7. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  8. Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

    PubMed Central

    Haycocks, James R. J.; Grainger, David C.

    2016-01-01

    A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality. PMID:27258043

  9. The Soft Touch: Low-Affinity Transcription Factor Binding Sites in Development and Evolution.

    PubMed

    Crocker, Justin; Noon, Ella Preger-Ben; Stern, David L

    2016-01-01

    Transcription factor proteins regulate gene expression by binding to specific DNA regions. Most studies of transcription factor binding sites have focused on the highest affinity sites for each factor. There is abundant evidence, however, that binding sites with a range of affinities, including very low affinities, are critical to gene regulation. Here, we present the theoretical and experimental evidence for the importance of low-affinity sites in gene regulation and development. We also discuss the implications of the widespread use of low-affinity sites in eukaryotic genomes for robustness, precision, specificity, and evolution of gene regulation. © 2016 Elsevier Inc. All rights reserved.

  10. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    NASA Astrophysics Data System (ADS)

    Clifford, Jacob; Adami, Christoph

    2015-10-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

  11. Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

    PubMed

    Roulet, E; Bucher, P; Schneider, R; Wingender, E; Dusserre, Y; Werner, T; Mermod, N

    2000-04-07

    Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo. Copyright 2000 Academic Press.

  12. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  13. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    PubMed

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  14. Acetylation of interferon regulatory factor-7 by p300/CREB-binding protein (CBP)-associated factor (PCAF) impairs its DNA binding.

    PubMed

    Caillaud, Alexandre; Prakash, Arun; Smith, Eric; Masumi, Atsuko; Hovanessian, Ara G; Levy, David E; Marié, Isabelle

    2002-12-20

    Interferon regulatory factor 7 (IRF7) is an interferon-inducible transcription factor required for induction of delayed early interferon alpha genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain. Although it is likely that IRF7 is subjected to a cascade of events responsible for regulating its biological activity, to date no mechanism other than phosphorylation has been reported to modulate IRF7 activity. Here, we report that IRF7 is acetylated in vivo by the histone acetyltransferases p300/CBP-associated factor (PCAF) and GCN5. The single lysine residue target for acetylation, lysine 92, is located in the DNA-binding domain and is conserved throughout the entire IRF family. Mutation of lysine 92 resulted in complete abolition of DNA binding ability. However, a mutant that cannot be acetylated by PCAF due to a change in the surrounding amino acid context of lysine 92 showed increased DNA binding and activity compared with wild type IRF7. Conversely, we showed that acetylated IRF7 displayed impaired DNA binding capability and that over-expression of PCAF led to decreased IRF7 activity. Together, our results strongly suggest that acetylation of lysine 92 negatively modulates IRF7 DNA binding.

  15. Bacterial chitinases and chitin-binding proteins as virulence factors.

    PubMed

    Frederiksen, Rikki F; Paspaliari, Dafni K; Larsen, Tanja; Storgaard, Birgit G; Larsen, Marianne H; Ingmer, Hanne; Palcic, Monica M; Leisner, Jørgen J

    2013-05-01

    Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

  16. Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

    PubMed Central

    Wang, S W; Speck, N A

    1992-01-01

    The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes. Images PMID:1309596

  17. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models.

    PubMed

    Mehta, Pankaj; Schwab, David J; Sengupta, Anirvan M

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

  18. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models

    NASA Astrophysics Data System (ADS)

    Mehta, Pankaj; Schwab, David J.; Sengupta, Anirvan M.

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

  19. New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

    PubMed Central

    Zucchelli, Chiara; Ferrari, Elena; Blasi, Francesco; Musco, Giovanna; Bruckmann, Chiara

    2017-01-01

    PREP1 and PBX1 are homeodomain (HD) transcription factors that play crucial roles in embryonic development. Here, we present the first biophysical characterization of a PREP1 HD, and the NMR spectroscopic study of its DNA binding pocket. The data show that residues flanking the HD participate in DNA binding. The kinetic parameters for DNA binding of individual PREP1 and PBX1 HDs, and of their combination, show that isolated PREP1 and PBX1 HDs bind to DNA in a cooperative manner. A novel PREP1 motif, flanking the HD at the C-terminus, is required for cooperativity. PMID:28094776

  20. Position specific variation in the rate of evolution intranscription factor binding sites

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  1. The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

    PubMed Central

    Wu-Baer, F; Lane, W S; Gaynor, R B

    1995-01-01

    Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation. Images PMID:8846792

  2. DNA binding and transcription activation by chicken interferon regulatory factor-3 (chIRF-3)

    PubMed Central

    Grant, Caroline E.; May, Donna L.; Deeley, Roger G.

    2000-01-01

    Interferon regulatory factors (IRFs) are a family of transcription factors involved in the cellular response to interferons and viral infection. Previously we isolated an IRF from a chicken embryonic liver cDNA library. Using a PCR-based binding site selection assay, we have characterised the binding specificity of chIRF-3. The optimal binding site (OBS) fits within the consensus interferon-stimulated response element (ISRE) but the specificity of chIRF-3 binding allows less variation in nucleotides outside the core IRF-binding sequence. A comparison of IRF-1 and chIRF-3 binding to ISREs in electrophoretic mobility shift assays confirmed that the binding specificity of chIRF-3 was clearly distinguishable from IRF-1. The selection assay also showed that chIRF-3 is capable of binding an inverted repeat of two half OBSs separated by 10–13 nt. ChIRF-3 appears to bind both the OBS and inverted repeat sites as a dimer with the protein–protein interaction requiring a domain between amino acids 117 and 311. In transfection experiments expression of chIRF-3 strongly activated a promoter containing the OBS. The activation domain was mapped to between amino acids 138 and 221 and a domain inhibitory to activation was also mapped to the C-terminal portion of chIRF-3. PMID:11095692

  3. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    SciTech Connect

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-04-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.

  4. Survey of variation in human transcription factors reveals prevalent DNA binding changes

    PubMed Central

    Barrera, Luis A.; Rogers, Julia M.; Gisselbrecht, Stephen S.; Rossin, Elizabeth J.; Woodard, Jaie; Mariani, Luca; Kock, Kian Hong; Inukai, Sachi; Siggers, Trevor; Shokri, Leila; Gordân, Raluca; Sahni, Nidhi; Cotsapas, Chris; Hao, Tong; Yi, Song; Kellis, Manolis; Daly, Mark J.; Vidal, Marc; Hill, David E.; Bulyk, Martha L.

    2016-01-01

    Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA-binding activity and used universal protein binding microarrays to assay sequence-specific DNA-binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA-binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA-binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA-binding activities, which may contribute to phenotypic variation. PMID:27013732

  5. Survey of variation in human transcription factors reveals prevalent DNA binding changes.

    PubMed

    Barrera, Luis A; Vedenko, Anastasia; Kurland, Jesse V; Rogers, Julia M; Gisselbrecht, Stephen S; Rossin, Elizabeth J; Woodard, Jaie; Mariani, Luca; Kock, Kian Hong; Inukai, Sachi; Siggers, Trevor; Shokri, Leila; Gordân, Raluca; Sahni, Nidhi; Cotsapas, Chris; Hao, Tong; Yi, Song; Kellis, Manolis; Daly, Mark J; Vidal, Marc; Hill, David E; Bulyk, Martha L

    2016-03-25

    Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation. Copyright © 2016, American Association for the Advancement of Science.

  6. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  7. Human rheumatoid factors bear the internal image of the Fc binding region of staphylococcal protein A.

    PubMed

    Oppliger, I R; Nardella, F A; Stone, G C; Mannik, M

    1987-09-01

    The binding specificity of rheumatoid factors (RFs) to human Fc resembles that of some microbial Fc-binding proteins, suggesting conformational similarities in their Fc-binding regions. Using polyclonal chicken antibodies against SPA, we have detected a crossreactive determinant shared by human RFs from different individuals, but not by non-RF IgM and IgG. Chicken anti-SPA was shown to bind to 18 of 19 IgM RFs and 2 of 2 IgG RFs isolated from different individuals. This binding was inhibitable with SPA, fragment D of SPA, human IgG, and Fc fragment of IgG. The binding site for RF was located on the Fab' fragment of chicken anti-SPA. The antigenic mimicry of RFs by a protein of microbial origin suggests that the immune response to infectious agents could induce or modulate RF production through an internal image autoantiidiotype mechanism.

  8. Genomewide analysis of Drosophila GAGA factor target genes reveals context-dependent DNA binding

    PubMed Central

    van Steensel, Bas; Delrow, Jeffrey; Bussemaker, Harmen J.

    2003-01-01

    The association of sequence-specific DNA-binding factors with their cognate target sequences in vivo depends on the local molecular context, yet this context is poorly understood. To address this issue, we have performed genomewide mapping of in vivo target genes of Drosophila GAGA factor (GAF). The resulting list of ≈250 target genes indicates that GAF regulates many cellular pathways. We applied unbiased motif-based regression analysis to identify the sequence context that determines GAF binding. Our results confirm that GAF selectively associates with (GA)n repeat elements in vivo. GAF binding occurs in upstream regulatory regions, but less in downstream regions. Surprisingly, GAF binds abundantly to introns but is virtually absent from exons, even though the density of (GA)n is roughly the same. Intron binding occurs equally frequently in last introns compared with first introns, suggesting that GAF may not only regulate transcription initiation, but possibly also elongation. We provide evidence for cooperative binding of GAF to closely spaced (GA)n elements and explain the lack of GAF binding to exons by the absence of such closely spaced GA repeats. Our approach for revealing determinants of context-dependent DNA binding will be applicable to many other transcription factors. PMID:12601174

  9. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    SciTech Connect

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  10. An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

    PubMed Central

    Schanke, J T; Marcuzzi, A; Podzorski, R P; Van Ness, B

    1994-01-01

    Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells. Images PMID:7816634

  11. Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

    PubMed Central

    2013-01-01

    Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

  12. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-[alpha

    SciTech Connect

    Carter, Percy H.; Scherle, Peggy A.; Muckelbauer, Jodi K.; Voss, Matthew E.; Liu, Rui-qin; Thompson III, Lorin A.; Xu, Meizhong; Lo, Yvonne C.; Li, Zhong; Strzemienski, Paul; Yang, Gengjie; Falahatpishen, Nikoo; Farrow, Neil A.; Tebben, Andrew J.; Underwood, Denis; Trzaskos, James M.; Friedman, Steven M.; Newton, Robert C.; Decicco, Carl P.

    2010-03-05

    The binding of tumor necrosis factor alpha (TNF-{alpha}) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-{alpha} to TNFRc1 (IC{sub 50} = 50 nM) and also blocked TNF-stimulated phosphorylation of I{kappa}-B in Ramos cells (IC{sub 50} = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 {micro}M. Detailed evaluation of this and related molecules revealed that compounds in this class are 'photochemically enhanced' inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 mM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-{alpha} to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-{alpha}-TNFRc1 interaction.

  13. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

    PubMed

    Carter, P H; Scherle, P A; Muckelbauer, J K; Voss, M E; Liu, R Q; Thompson, L A; Tebben, A J; Solomon, K A; Lo, Y C; Li, Z; Strzemienski, P; Yang, G; Falahatpisheh, N; Xu, M; Wu, Z; Farrow, N A; Ramnarayan, K; Wang, J; Rideout, D; Yalamoori, V; Domaille, P; Underwood, D J; Trzaskos, J M; Friedman, S M; Newton, R C; Decicco, C P; Muckelbauer, J A

    2001-10-09

    The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

  14. Genome-wide inference of natural selection on human transcription factor binding sites.

    PubMed

    Arbiza, Leonardo; Gronau, Ilan; Aksoy, Bulent A; Hubisz, Melissa J; Gulko, Brad; Keinan, Alon; Siepel, Adam

    2013-07-01

    For decades, it has been hypothesized that gene regulation has had a central role in human evolution, yet much remains unknown about the genome-wide impact of regulatory mutations. Here we use whole-genome sequences and genome-wide chromatin immunoprecipitation and sequencing data to demonstrate that natural selection has profoundly influenced human transcription factor binding sites since the divergence of humans from chimpanzees 4-6 million years ago. Our analysis uses a new probabilistic method, called INSIGHT, for measuring the influence of selection on collections of short, interspersed noncoding elements. We find that, on average, transcription factor binding sites have experienced somewhat weaker selection than protein-coding genes. However, the binding sites of several transcription factors show clear evidence of adaptation. Several measures of selection are strongly correlated with predicted binding affinity. Overall, regulatory elements seem to contribute substantially to both adaptive substitutions and deleterious polymorphisms with key implications for human evolution and disease.

  15. Regulation of cell cycle transcription factor Swi4 through auto-inhibition of DNA binding.

    PubMed

    Baetz, K; Andrews, B

    1999-10-01

    In Saccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G(1)/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G(1) phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA in trans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA.

  16. Age-associated changes in basal c-fos transcription factor binding activity in rat hearts.

    PubMed

    Tsou, H; Azhar, G; Lu, X G; Kovacs, S; Peacocke, M; Wei, J Y

    1996-12-15

    The early response proto-oncogene c-fos is expressed at very low levels in the mammalian heart at baseline. To further investigate the mechanism of altered c-fos expression with age, we studied in the basal state the binding of five transcription proteins to their cognate sites in the c-fos promoter/enhancer region, in adult and old F344 rats. Our results show a reduced binding of E2F and AP1 proteins to the c-fos promoter in aging hearts. The major calcium/cyclic AMP response element (CRE) and SP1 binding was unchanged. The only increase seen with age was in the serum response element (SRE) binding proteins. SRE is the point of convergence of different signal transduction pathways (via MAP kinases and the Rho family of GTPases) at the c-fos promoter. Increased SRE binding may reflect a compensation for a decreased binding of other transcription proteins to the c-fos promoter, alteration in the phosphorylation status of SRF, or a change in the ternary complex factors Elk 1 or SAP 1. Other possibilities include defects in the signal transduction pathways with aging, which combine to produce an overall negative balance in the function of the c-fos promoter despite the increased SRE binding activity. Both in vitro and in vivo experiments have shown decreased c-fos expression with age. This may be due partly to alterations in the basal levels of transcription factor binding.

  17. Epidermal growth factor receptors on PC12 cells: alteration of binding properties by lectins

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1983-01-01

    The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of /sup 125/I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37 degrees C and 4 degrees C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylation of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with /sup 125/I-NGF binding, WGA but not Con A was found to increase, by severalfold, the proportion of /sup 125/I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.

  18. Decoupling of evolutionary changes in transcription factor binding and gene expression in mammals.

    PubMed

    Wong, Emily S; Thybert, David; Schmitt, Bianca M; Stefflova, Klara; Odom, Duncan T; Flicek, Paul

    2015-02-01

    To understand the evolutionary dynamics between transcription factor (TF) binding and gene expression in mammals, we compared transcriptional output and the binding intensities for three tissue-specific TFs in livers from four closely related mouse species. For each transcription factor, TF-dependent genes and the TF binding sites most likely to influence mRNA expression were identified by comparing mRNA expression levels between wild-type and TF knockout mice. Independent evolution was observed genome-wide between the rate of change in TF binding and the rate of change in mRNA expression across taxa, with the exception of a small number of TF-dependent genes. We also found that binding intensities are preferentially conserved near genes whose expression is dependent on the TF, and the conservation is shared among binding peaks in close proximity to each other near the TSS. Expression of TF-dependent genes typically showed an increased sensitivity to changes in binding levels as measured by mRNA abundance. Taken together, these results highlight a significant tolerance to evolutionary changes in TF binding intensity in mammalian transcriptional networks and suggest that some TF-dependent genes may be largely regulated by a single TF across evolution.

  19. Definition and prediction of the full range of transcription factor binding sites—the hepatocyte nuclear factor 1 dimeric site

    PubMed Central

    Locker, Joseph; Ghosh, David; Luc, Phuong-Van; Zheng, Jianhua

    2002-01-01

    In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding. PMID:12202766

  20. Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process.

    PubMed

    Jorissen, Robert N; Treutlein, Herbert R; Epa, V Chandana; Burgess, Antony W

    2002-06-01

    Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.

  1. Insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I

    SciTech Connect

    Elgin, R.G.; Busby, H.W. Jr.; Clemmons, D.R.

    1987-05-01

    The insulin-like growth factors IGF-I and IGF-II circulate in blood bound to carrier proteins. The higher molecular mass IGF-binding protein complex (150 kDa) is composed of subunits, and one subunits that forms this complex is growth hormone dependent. In addition, many cell types and tissues secrete another form of IGF binding protein that is not growth hormone dependent. Both forms of the IGF binding protein are believed to inactivate the IGFs and to function as delivery systems to tissues. This conclusion was based on studies that determined the effects of impure preparations of these binding proteins or that examined the effect of these proteins only on the insulin-like actions of the IGFs. The authors report here that a pure preparation of the extracellular form of the IGF binding protein (purified from human amniotic fluid) markedly potentiated replication of several cell types in response to human IGF-I. Secondary cultures of human, mouse, and chicken embryo fibroblasts as well as porcine aortic smooth muscle cells showed marked enhancement of their DNA synthesis response to IGF-I in the presence of this protein. The binding protein not only potentiated the DNA synthesis response but also enhanced the increase in cell number in response to IGF-I. This stimulation is specific for growth factors that bind to the binding protein since incubation with insulin, which binds to the type I IGF receptor but not to the binding protein, did not result in potentiation of this response. They conclude that a form of IGF binding protein that is present in extracellular fluids and is secreted by many types of cells can markedly potentiate the cellular response to IGF-I.

  2. Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

    PubMed

    Cameron, Dale M; Thompson, Jill; March, Paul E; Dahlberg, Albert E

    2002-05-24

    The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

  3. Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1).

    PubMed

    Günther, S; Huchzermeyer, B

    1998-12-01

    The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.

  4. Using competition assays to quantitatively model cooperative binding by transcription factors and other ligands.

    PubMed

    Peacock, Jacob; Jaynes, James B

    2017-08-01

    The affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations. We use standard computational and mathematical methods, and develop novel methods as described in Results. We explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive expressions for the equilibria among the various complexes, and explore the limitations of binding experiments that model the system using a single parameter. We describe how to use single-ligand binding and ternary complex formation in tandem to determine parameters that have thermodynamic relevance. We develop an improved method for finding both single-ligand dissociation constants and concentrations simultaneously. We show how the cooperativity factor can be found when only one of the single-ligand dissociation constants can be measured. The methods that we develop constitute an optimized approach to accurately model cooperative binding. The expressions and methods we develop for modeling and analyzing DNA binding and cooperativity are applicable to most cases where multiple ligands bind to distinct sites on a common substrate. The parameters determined using these methods can be fed into models of higher-order cooperativity to increase their predictive power. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  6. Analysis of transcription-factor binding-site evolution by using the Hamilton-Jacobi equations

    NASA Astrophysics Data System (ADS)

    Ancliff, Mark; Park, Jeong-Man

    2016-12-01

    We investigate a quasi-species mutation-selection model of transcription-factor binding-site evolution. By considering the mesa and the crater fitness landscapes designed to describe these binding sites and point mutations, we derive an evolution equation for the population distribution of binding sequences. In the long-length limit, the evolution equation is replaced by a Hamilton-Jacobi equation which we solve for the stationary state solution. From the stationary solution, we derive the population distributions and find that an error threshold, separating populations in which the binding site does or does not evolve, only exists for certain values of the fitness parameters. A phase diagram in this parameter space is derived and shows a critical line below which no error threshold exists. We also investigate the evolution of multiple binding sites for the same transcription factor. For two binding sites, we perform an analysis similar to that for a single site and determine a phase diagram showing different phases with both, one, or no binding sites selected. In the phase diagram, the phase boundary between the one-or-two selected site phases is qualitatively different for the mesa and the crater fitness landscapes. As fitness benefits for a second bound transcription factor tend to zero, the minimum mutation rate at which the two-site phase occurs diverges in the mesa landscape whereas the mutation rate at the phase boundary tends to a finite value for the crater landscape.

  7. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

    PubMed Central

    Jolma, Arttu; Kivioja, Teemu; Toivonen, Jarkko; Cheng, Lu; Wei, Gonghong; Enge, Martin; Taipale, Mikko; Vaquerizas, Juan M.; Yan, Jian; Sillanpää, Mikko J.; Bonke, Martin; Palin, Kimmo; Talukder, Shaheynoor; Hughes, Timothy R.; Luscombe, Nicholas M.; Ukkonen, Esko; Taipale, Jussi

    2010-01-01

    The genetic code—the binding specificity of all transfer-RNAs—defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the ∼1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers. PMID:20378718

  8. Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development.

    PubMed

    Kazemian, Majid; Pham, Hannah; Wolfe, Scot A; Brodsky, Michael H; Sinha, Saurabh

    2013-09-01

    Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein-protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.

  9. Molecular docking studies in factor XIa binding site

    NASA Astrophysics Data System (ADS)

    Balaji, Govardhan A.; Balaji, Vitukudi N.; Rao, Shashidhar N.

    2016-03-01

    Factor XIa inhibitors have been identified to have potential as anticoagulants with robust efficacy and low bleeding risks. In light of their significance and the availability of their 3-D X-ray data in the PDB, we present molecular docking studies carried out with a view to obtain docking protocols that will successfully reproduce the experimentally observed protein-ligand interactions in the case of various X-ray ligands. In this context, we have specifically investigated the efficacy of various cross-docking protocols in reproducing experimental data. Our studies demonstrate that an ensemble of the three apo proteins is capable of accurately docking a majority of the X-ray ligands accurately without invoking any additional conformational flexibility than that present in their experimental structures. Further, we demonstrate that such an ensemble is successfully able to enrich a collection of known active factor XIa inhibitors embedded in a decoy database of drug-like molecules.

  10. The Yeast Candida albicans Binds Complement Regulators Factor H and FHL-1

    PubMed Central

    Meri, T.; Hartmann, A.; Lenk, D.; Eck, R.; Würzner, R.; Hellwage, J.; Meri, S.; Zipfel, P. F.

    2002-01-01

    The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products. PMID:12183569

  11. The yeast Candida albicans binds complement regulators factor H and FHL-1.

    PubMed

    Meri, T; Hartmann, A; Lenk, D; Eck, R; Würzner, R; Hellwage, J; Meri, S; Zipfel, P F

    2002-09-01

    The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.

  12. Binding of Transcription Factors Adapts to Resolve Information-Energy Tradeoff

    NASA Astrophysics Data System (ADS)

    Savir, Yonatan; Kagan, Jacob; Tlusty, Tsvi

    2016-03-01

    We examine the binding of transcription factors to DNA in terms of an information transfer problem. The input of the noisy channel is the biophysical signal of a factor bound to a DNA site, and the output is a distribution of probable DNA sequences at this site. This task involves an inherent tradeoff between the information gain and the energetics of the binding interaction—high binding energies provide higher information gain but hinder the dynamics of the system as factors are bound too tightly. We show that adaptation of the binding interaction towards increasing information transfer under a general energy constraint implies that the information gain per specific binding energy at each base-pair is maximized. We analyze hundreds of prokaryote and eukaryote transcription factors from various organisms to evaluate the discrimination energies. We find that, in accordance with our theoretical argument, binding energies nearly maximize the information gain per energy. This work suggests the adaptation of information gain as a generic design principle of molecular recognition systems.

  13. Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.

    PubMed

    Bendetowicz, A V; Morris, J A; Wise, R J; Gilbert, G E; Kaufman, R J

    1998-07-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

  14. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    PubMed Central

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  15. Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA

    SciTech Connect

    Greco-Stewart, Valerie S.; Thibault, Catherine St-Laurent; Pelchat, Martin . E-mail: mpelchat@uottawa.ca

    2006-12-20

    The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle.

  16. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae

    PubMed Central

    Lang, Michael; Juan, Elvira

    2010-01-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities. PMID:20542916

  17. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  18. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  19. A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor

    PubMed Central

    Sayou, Camille; Nanao, Max H.; Jamin, Marc; Posé, David; Thévenon, Emmanuel; Grégoire, Laura; Tichtinsky, Gabrielle; Denay, Grégoire; Ott, Felix; Peirats Llobet, Marta; Schmid, Markus; Dumas, Renaud; Parcy, François

    2016-01-01

    Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF. PMID:27097556

  20. De Novo Identification and Biophysical Characterization of Transcription Factor Binding Sites with Microfluidic Affinity Analysis

    PubMed Central

    Fordyce, Polly M.; Gerber, Doron; Tran, Danh; Zheng, Jiashun; Li, Hao; DeRisi, Joseph L.; Quake, Stephen R.

    2010-01-01

    Gene expression is regulated in part by protein transcription factors (TFs) that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link TFs to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 S. cerevisiae TFs with a variety of DNA binding domains, including several that have proven difficult to study via other techniques. For each TF, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for 4 TFs, we also determined absolute affinities. We anticipate that these data and future use of this technique will provide information essential for understanding TF specificity, improving identification of regulatory sites, and reconstructing regulatory interactions. PMID:20802496

  1. A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor.

    PubMed

    Sayou, Camille; Nanao, Max H; Jamin, Marc; Posé, David; Thévenon, Emmanuel; Grégoire, Laura; Tichtinsky, Gabrielle; Denay, Grégoire; Ott, Felix; Peirats Llobet, Marta; Schmid, Markus; Dumas, Renaud; Parcy, François

    2016-04-21

    Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF.

  2. Toward an atomistic model for predicting transcription-factor binding sites.

    PubMed

    Endres, Robert G; Schulthess, Thomas C; Wingreen, Ned S

    2004-11-01

    Identifying the specific DNA-binding sites of transcription-factor proteins is essential to understanding the regulation of gene expression in the cell. Bioinformatics approaches are fast compared to experiments, but require prior knowledge of multiple binding sites for each protein. Here, we present an atomistic force-field method to predict binding sites based only on the X-ray structure of a related bound complex. Specific flexible contacts between the protein and DNA are modeled by a library of amino acid side-chain rotamers. Using the example of the mouse transcription factor, Zif268, a well-studied zinc-finger protein, we show that the protein sequence alone, without the detailed experimental structure, gives a strong bias toward the consensus binding site.

  3. Growth factors induce monocyte binding to vascular smooth muscle cells: implications for monocyte retention in atherosclerosis.

    PubMed

    Cai, Qiangjun; Lanting, Linda; Natarajan, Rama

    2004-09-01

    Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte beta(1)- and beta(2)-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

  4. Malaria parasites possess a telomere repeat-binding protein that shares ancestry with transcription factor IIIA.

    PubMed

    Bertschi, Nicole L; Toenhake, Christa G; Zou, Angela; Niederwieser, Igor; Henderson, Rob; Moes, Suzette; Jenoe, Paul; Parkinson, John; Bartfai, Richard; Voss, Till S

    2017-03-13

    Telomere repeat-binding factors (TRFs) are essential components of the molecular machinery that regulates telomere function. TRFs are widely conserved across eukaryotes and bind duplex telomere repeats via a characteristic MYB-type domain. Here, we identified the telomere repeat-binding protein PfTRZ in the malaria parasite Plasmodium falciparum, a member of the Alveolate phylum for which TRFs have not been described so far. PfTRZ lacks an MYB domain and binds telomere repeats via a C2H2-type zinc finger domain instead. In vivo, PfTRZ binds with high specificity to the telomeric tract and to interstitial telomere repeats upstream of subtelomeric virulence genes. Conditional depletion experiments revealed that PfTRZ regulates telomere length homeostasis and is required for efficient cell cycle progression. Intriguingly, we found that PfTRZ also binds to and regulates the expression of 5S rDNA genes. Combined with detailed phylogenetic analyses, our findings identified PfTRZ as a remote functional homologue of the basic transcription factor TFIIIA, which acquired a new function in telomere maintenance early in the apicomplexan lineage. Our work sheds unexpected new light on the evolution of telomere repeat-binding proteins and paves the way for dissecting the presumably divergent mechanisms regulating telomere functionality in one of the most deadly human pathogens.

  5. Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

    PubMed Central

    Singh, Appu Kumar; Mondal, Alok K.; Kumaran, S.

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. PMID:23049810

  6. Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

    PubMed

    Biswas, Dipanwita; Pandya, Vaibhav; Singh, Appu Kumar; Mondal, Alok K; Kumaran, S

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

  7. An information transmission model for transcription factor binding at regulatory DNA sites.

    PubMed

    Tan, Mingfeng; Yu, Dong; Jin, Yuan; Dou, Lei; Li, Beiping; Wang, Yuelan; Yue, Junjie; Liang, Long

    2012-06-06

    Computational identification of transcription factor binding sites (TFBSs) is a rapid, cost-efficient way to locate unknown regulatory elements. With increased potential for high-throughput genome sequencing, the availability of accurate computational methods for TFBS prediction has never been as important as it currently is. To date, identifying TFBSs with high sensitivity and specificity is still an open challenge, necessitating the development of novel models for predicting transcription factor-binding regulatory DNA elements. Based on the information theory, we propose a model for transcription factor binding of regulatory DNA sites. Our model incorporates position interdependencies in effective ways. The model computes the information transferred (TI) between the transcription factor and the TFBS during the binding process and uses TI as the criterion to determine whether the sequence motif is a possible TFBS. Based on this model, we developed a computational method to identify TFBSs. By theoretically proving and testing our model using both real and artificial data, we found that our model provides highly accurate predictive results. In this study, we present a novel model for transcription factor binding regulatory DNA sites. The model can provide an increased ability to detect TFBSs.

  8. An information transmission model for transcription factor binding at regulatory DNA sites

    PubMed Central

    2012-01-01

    Background Computational identification of transcription factor binding sites (TFBSs) is a rapid, cost-efficient way to locate unknown regulatory elements. With increased potential for high-throughput genome sequencing, the availability of accurate computational methods for TFBS prediction has never been as important as it currently is. To date, identifying TFBSs with high sensitivity and specificity is still an open challenge, necessitating the development of novel models for predicting transcription factor-binding regulatory DNA elements. Results Based on the information theory, we propose a model for transcription factor binding of regulatory DNA sites. Our model incorporates position interdependencies in effective ways. The model computes the information transferred (TI) between the transcription factor and the TFBS during the binding process and uses TI as the criterion to determine whether the sequence motif is a possible TFBS. Based on this model, we developed a computational method to identify TFBSs. By theoretically proving and testing our model using both real and artificial data, we found that our model provides highly accurate predictive results. Conclusions In this study, we present a novel model for transcription factor binding regulatory DNA sites. The model can provide an increased ability to detect TFBSs. PMID:22672438

  9. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    PubMed

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

  10. Systematic dissection of genomic features determining transcription factor binding and enhancer function

    PubMed Central

    Grossman, Sharon R.; Zhang, Xiaolan; Wang, Li; Engreitz, Jesse; Melnikov, Alexandre; Rogov, Peter; Tewhey, Ryan; Isakova, Alina; Deplancke, Bart; Bernstein, Bradley E.; Mikkelsen, Tarjei S.; Lander, Eric S.

    2017-01-01

    Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation. PMID:28137873

  11. Systematic dissection of genomic features determining transcription factor binding and enhancer function.

    PubMed

    Grossman, Sharon R; Zhang, Xiaolan; Wang, Li; Engreitz, Jesse; Melnikov, Alexandre; Rogov, Peter; Tewhey, Ryan; Isakova, Alina; Deplancke, Bart; Bernstein, Bradley E; Mikkelsen, Tarjei S; Lander, Eric S

    2017-02-14

    Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function-including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

  12. Evaluating the impact of single nucleotide variants on transcription factor binding

    PubMed Central

    Shi, Wenqiang; Fornes, Oriol; Mathelier, Anthony; Wasserman, Wyeth W.

    2016-01-01

    Diseases and phenotypes caused by disrupted transcription factor (TF) binding are being identified, but progress is hampered by our limited capacity to predict such functional alterations. Improving predictions may be dependent on expanding the set of bona fide TF binding alterations. Allele-specific binding (ASB) events, where TFs preferentially bind to one of the two alleles at heterozygous sites, reveal the impact of sequence variations in altered TF binding. Here, we present the largest ASB compilation to our knowledge, 10 765 ASB events retrieved from 45 ENCODE ChIP-Seq data sets. Our analysis showed that ASB events were frequently associated with motif alterations of the ChIP'ed TF and potential partner TFs, allelic difference of DNase I hypersensitivity and allelic difference of histone modifications. For TF dimers bound symmetrically to DNA, ASB data revealed that central positions of the TF binding motifs were disproportionately important for binding. Lastly, the impact of variation on TF binding was predicted by a classification model incorporating all the investigated features of ASB events. Classification models using only DNase I hypersensitivity and sequence data exhibited predictive accuracy approaching the models with substantially more features. Taken together, the combination of ASB data and the classification model represents an important step toward elucidating regulatory variants across the human genome. PMID:27492288

  13. Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

    PubMed Central

    Wong, Tuck Seng; Rajagopalan, Sridharan; Freund, Stefan M.; Rutherford, Trevor J.; Andreeva, Antonina; Townsley, Fiona M.; Petrovich, Miriana; Fersht, Alan R.

    2009-01-01

    Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a Kd of 1.95 ± 0.19 μM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM–p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices α1 and α2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM–p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM–p53 interaction are discussed. PMID:19755502

  14. Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

    SciTech Connect

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. )

    1988-09-01

    To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific binding of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.

  15. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    PubMed Central

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

  16. Inclusion of neighboring base interdependencies substantially improves genome-wide prokaryotic transcription factor binding site prediction

    PubMed Central

    Salama, Rafik A.; Stekel, Dov J.

    2010-01-01

    Prediction of transcription factor binding sites is an important challenge in genome analysis. The advent of next generation genome sequencing technologies makes the development of effective computational approaches particularly imperative. We have developed a novel training-based methodology intended for prokaryotic transcription factor binding site prediction. Our methodology extends existing models by taking into account base interdependencies between neighbouring positions using conditional probabilities and includes genomic background weighting. This has been tested against other existing and novel methodologies including position-specific weight matrices, first-order Hidden Markov Models and joint probability models. We have also tested the use of gapped and ungapped alignments and the inclusion or exclusion of background weighting. We show that our best method enhances binding site prediction for all of the 22 Escherichia coli transcription factors with at least 20 known binding sites, with many showing substantial improvements. We highlight the advantage of using block alignments of binding sites over gapped alignments to capture neighbouring position interdependencies. We also show that combining these methods with ChIP-on-chip data has the potential to further improve binding site prediction. Finally we have developed the ungapped likelihood under positional background platform: a user friendly website that gives access to the prediction method devised in this work. PMID:20439311

  17. Romulus: robust multi-state identification of transcription factor binding sites from DNase-seq data

    PubMed Central

    Jankowski, Aleksander; Tiuryn, Jerzy; Prabhakar, Shyam

    2016-01-01

    Motivation: Computational prediction of transcription factor (TF) binding sites in the genome remains a challenging task. Here, we present Romulus, a novel computational method for identifying individual TF binding sites from genome sequence information and cell-type–specific experimental data, such as DNase-seq. It combines the strengths of previous approaches, and improves robustness by reducing the number of free parameters in the model by an order of magnitude. Results: We show that Romulus significantly outperforms existing methods across three sources of DNase-seq data, by assessing the performance of these tools against ChIP-seq profiles. The difference was particularly significant when applied to binding site prediction for low-information-content motifs. Our method is capable of inferring multiple binding modes for a single TF, which differ in their DNase I cut profile. Finally, using the model learned by Romulus and ChIP-seq data, we introduce Binding in Closed Chromatin (BCC) as a quantitative measure of TF pioneer factor activity. Uniquely, our measure quantifies a defining feature of pioneer factors, namely their ability to bind closed chromatin. Availability and Implementation: Romulus is freely available as an R package at http://github.com/ajank/Romulus. Contact: ajank@mimuw.edu.pl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153645

  18. Romulus: robust multi-state identification of transcription factor binding sites from DNase-seq data.

    PubMed

    Jankowski, Aleksander; Tiuryn, Jerzy; Prabhakar, Shyam

    2016-08-15

    Computational prediction of transcription factor (TF) binding sites in the genome remains a challenging task. Here, we present Romulus, a novel computational method for identifying individual TF binding sites from genome sequence information and cell-type-specific experimental data, such as DNase-seq. It combines the strengths of previous approaches, and improves robustness by reducing the number of free parameters in the model by an order of magnitude. We show that Romulus significantly outperforms existing methods across three sources of DNase-seq data, by assessing the performance of these tools against ChIP-seq profiles. The difference was particularly significant when applied to binding site prediction for low-information-content motifs. Our method is capable of inferring multiple binding modes for a single TF, which differ in their DNase I cut profile. Finally, using the model learned by Romulus and ChIP-seq data, we introduce Binding in Closed Chromatin (BCC) as a quantitative measure of TF pioneer factor activity. Uniquely, our measure quantifies a defining feature of pioneer factors, namely their ability to bind closed chromatin. Romulus is freely available as an R package at http://github.com/ajank/Romulus ajank@mimuw.edu.pl Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  19. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints.

    PubMed

    He, Qiye; Johnston, Jeff; Zeitlinger, Julia

    2015-04-01

    Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins--human TBP and Drosophila NFkB, Twist and Max--shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.

  20. Effect of oxidative DNA damage in promoter elements on transcription factor binding.

    PubMed

    Ghosh, R; Mitchell, D L

    1999-08-01

    Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters.

  1. Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.

    PubMed

    Wang, Man; Zhang, Yan; Li, Benqiang; Zhu, Jianguo

    2015-06-01

    Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. The role of the DNA-binding One Zinc Finger (DOF) transcription factor family in plants.

    PubMed

    Noguero, Mélanie; Atif, Rana Muhammad; Ochatt, Sergio; Thompson, Richard D

    2013-08-01

    The DOF (DNA-binding One Zinc Finger) family of transcription factors is involved in many fundamental processes in higher plants, including responses to light and phytohormones as well as roles in seed maturation and germination. DOF transcription factor genes are restricted in their distribution to plants, where they are in many copies in both gymnosperms and angiosperms and also present in lower plants such as the moss Physcomitrella patens and in the alga Chlamydomonas reinhardtii which possesses a single DOF gene. DOF transcription factors bind to their promoter targets at the consensus sequence AAAG. This binding depends upon the presence of the highly conserved DOF domain in the protein. Depending on the target gene, DOF factor binding may activate or repress transcription. DOF factors are expressed in most if not all tissues of higher plants, but frequently appear to be functionally redundant. Recent next-generation sequencing data provide a more comprehensive survey of the distribution of DOF sequence classes among plant species and within tissue types, and clues as to the evolution of functions assumed by this transcription factor family. DOFs do not appear to be implicated in the initial differentiation of the plant body plan into organs via the resolution of meristematic zones, in contrast to MADS-box and homeobox transcription factors, which are found in other non-plant eukaryotes, and this may reflect a more recent evolutionary origin.

  3. The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding

    PubMed Central

    2011-01-01

    Background In Drosophila embryos, many biochemically and functionally unrelated transcription factors bind quantitatively to highly overlapping sets of genomic regions, with much of the lowest levels of binding being incidental, non-functional interactions on DNA. The primary biochemical mechanisms that drive these genome-wide occupancy patterns have yet to be established. Results Here we use data resulting from the DNaseI digestion of isolated embryo nuclei to provide a biophysical measure of the degree to which proteins can access different regions of the genome. We show that the in vivo binding patterns of 21 developmental regulators are quantitatively correlated with DNA accessibility in chromatin. Furthermore, we find that levels of factor occupancy in vivo correlate much more with the degree of chromatin accessibility than with occupancy predicted from in vitro affinity measurements using purified protein and naked DNA. Within accessible regions, however, the intrinsic affinity of the factor for DNA does play a role in determining net occupancy, with even weak affinity recognition sites contributing. Finally, we show that programmed changes in chromatin accessibility between different developmental stages correlate with quantitative alterations in factor binding. Conclusions Based on these and other results, we propose a general mechanism to explain the widespread, overlapping DNA binding by animal transcription factors. In this view, transcription factors are expressed at sufficiently high concentrations in cells such that they can occupy their recognition sequences in highly accessible chromatin without the aid of physical cooperative interactions with other proteins, leading to highly overlapping, graded binding of unrelated factors. PMID:21473766

  4. Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI.

    PubMed

    Baglia, F A; Jameson, B A; Walsh, P N

    1993-02-25

    Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with

  5. MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

    PubMed

    Xu, Meixiang; Cross, Courtney E; Speidel, Jordan T; Abdel-Rahman, Sherif Z

    2016-10-01

    The O(6)-methylguanine-DNA methyltransferase (MGMT) protein removes O(6)-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity. In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

  6. Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding.

    PubMed Central

    Laskin, J D; Lee, E; Laskin, D L; Gallo, M A

    1986-01-01

    The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin. PMID:3490664

  7. Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding

    SciTech Connect

    Laskin, J.D.; Lee, E.; Laskin, D.L.; Gallo, M.A.

    1986-11-01

    The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin.

  8. Activin inhibits binding of transcription factor Pit-1 to the growth hormone promoter.

    PubMed Central

    Struthers, R S; Gaddy-Kurten, D; Vale, W W

    1992-01-01

    Activin A is a potent growth and differentiation factor related to transforming growth factor beta. In somatotrophs, activin suppresses the biosynthesis and secretion of growth hormone (GH) and cellular proliferation. We report here that, in MtTW15 somatotrophic tumor cells, activin decreased GH mRNA levels and inhibited expression of transfected GH promoter--chloramphenicol acetyltransferase fusion genes. Deletion mapping of nucleotide sequences mediating this inhibition led to the identification of a region that has previously been characterized as binding the pituitary-specific transcription factor Pit-1/GHF-1. Characterization of nuclear factor binding to this region demonstrated that binding of Pit-1 to the GH promoter is lost on activin treatment. These results indicate that activin-induced repression of GH biosynthesis is mediated by the loss of tissue-specific transcription factor binding to the GH promoter and suggest a possible general mechanism for other activin responses, whereby activin regulates the function of other POU- or homeodomain-containing transcription factors. Images PMID:1454833

  9. Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

    PubMed Central

    Vignais, M L; Woudt, L P; Wassenaar, G M; Mager, W H; Sentenac, A; Planta, R J

    1987-01-01

    Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:3301327

  10. Auto-Inhibition and Partner Proteins, Core-Binding Factor β (CBFβ) and Ets-1, Modulate DNA Binding by CBFα2 (AML1)

    PubMed Central

    Gu, Ting-Lei; Goetz, Tamara L.; Graves, Barbara J.; Speck, Nancy A.

    2000-01-01

    Core-binding factor α2 (CBFα2; otherwise known as AML1 or PEBP2αB) is a DNA-binding subunit in the family of core-binding factors (CBFs), heterodimeric transcription factors that play pivotal roles in multiple developmental processes in mammals, including hematopoiesis and bone development. The Runt domain in CBFα2 (amino acids 51 to 178) mediates DNA binding and heterodimerization with the non-DNA-binding CBFβ subunit. Both the CBFβ subunit and the DNA-binding protein Ets-1 stimulate DNA binding by the CBFα2 protein. Here we quantify and compare the extent of cooperativity between CBFα2, CBFβ, and Ets-1. We also identify auto-inhibitory sequences within CBFα2 and sequences that modulate its interactions with CBFβ and Ets-1. We show that sequences in the CBFα2 Runt domain and sequences C terminal to amino acid 214 inhibit DNA binding. Sequences C terminal to amino acid 214 also inhibit heterodimerization with the non-DNA-binding CBFβ subunit, particularly heterodimerization off DNA. CBFβ rescinds the intramolecular inhibition of CBFα2, stimulating DNA binding approximately 40-fold. In comparison, Ets-1 stimulates CBFα2 DNA binding 7- to 10-fold. Although the Runt domain alone is sufficient for heterodimerization with CBFβ, sequences N terminal to amino acid 41 and between amino acids 190 and 214 are required for cooperative DNA binding with Ets-1. Cooperative DNA binding with Ets-1 is less pronounced with the CBFα2-CBFβ heterodimer than with CBFα2 alone. These analyses demonstrate that CBFα2 is subject to both negative regulation by intramolecular interactions, and positive regulation by two alternative partnerships. PMID:10594012

  11. DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

    PubMed Central

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

    2013-01-01

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

  12. DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

    PubMed

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

    2013-03-29

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

  13. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    PubMed

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  14. A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment*

    PubMed Central

    Donovan, Jesse; Caban, Kelvin; Ranaweera, Ruchira; Gonzalez-Flores, Jonathan N.; Copeland, Paul R.

    2008-01-01

    Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) element in their 3′-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNASec, and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site. PMID:18948268

  15. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network

    PubMed Central

    Orian, Amir; van Steensel, Bas; Delrow, Jeffrey; Bussemaker, Harmen J.; Li, Ling; Sawado, Tomoyuki; Williams, Eleanor; Loo, Lenora W.M.; Cowley, Shaun M.; Yost, Cynthia; Pierce, Sarah; Edgar, Bruce A.; Parkhurst, Susan M.; Eisenman, Robert N.

    2003-01-01

    The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci (∼15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression. PMID:12695332

  16. Global genome analysis of the downstream binding targets of testis determining factor SRY and SOX9.

    PubMed

    Bhandari, Ramji K; Haque, Md M; Skinner, Michael K

    2012-01-01

    A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.

  17. Global Genome Analysis of the Downstream Binding Targets of Testis Determining Factor SRY and SOX9

    PubMed Central

    Bhandari, Ramji K.; Haque, Md. M.; Skinner, Michael K.

    2012-01-01

    A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination. PMID:22984422

  18. Binding and functional effects of atrial natriuretic factor in isolated rat kidney

    SciTech Connect

    Suzuki, M.; Almeida, F.A.; Nussenzveig, D.R.; Sawyer, D.; Maack, T. )

    1987-11-01

    A new methodological approach was developed to study the relationship between specific binding and dose-response curves of the renal effects of atrial natriuretic factor (ANF) in isolated perfused rat kidneys (IK). IK were perfused with {sup 125}I-labeled and unlabeled ANF 1-28 to determine the following: (1) distribution, capacity (C{sub max}), and apparent affinity (S{sub 50}) of specific binding of ANF 1-28 in cortex, outer medulla, and papilla and (2) dose-response curves of the effects of ANF 1-28 on renal hemodynamics and excretion of fluid and electrolytes. The kidney had a very high density of high-affinity binding sites for ANF. Cortex had >90% of total binding sites whereas papilla had <2% of total binding sites with a 10-fold lower apparent affinity than in cortex. ANF-induced increases in glomerular filtration rate and excretion of fluid and electrolytes were detectable at 10-100 pM and maximal effects occurred at 1-10 nM ANF. Below 1 nM there was no dissociation between the renal hemodynamic and natriuretic effects of ANF. There was a close agreement between dose-response and binding curves of ANF to cortex. Results demonstrates that binding site occupancy in kidney cortex and renal effects of ANF occur at near physiological concentrations of the hormone.

  19. Nucleotide-binding properties of kinase-deficient epidermal-growth-factor-receptor mutants.

    PubMed

    Cheng, K; Koland, J G

    1998-02-15

    The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. Binding affinities for ATP and Mn.ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn.ATP binding, although the binding affinity for the Mn.ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction.

  20. Nucleotide-binding properties of kinase-deficient epidermal-growth-factor-receptor mutants.

    PubMed Central

    Cheng, K; Koland, J G

    1998-01-01

    The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.Binding affinities for ATP and Mn.ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn.ATP binding, although the binding affinity for the Mn.ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction. PMID:9461530

  1. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    PubMed

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  2. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    PubMed

    Bauer, Amy L; Hlavacek, William S; Unkefer, Pat J; Mu, Fangping

    2010-11-18

    An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF). Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  3. Chromatin Architecture and Transcription Factor Binding Regulate Expression of Erythrocyte Membrane Protein Genes▿ †

    PubMed Central

    Steiner, Laurie A.; Maksimova, Yelena; Schulz, Vincent; Wong, Clara; Raha, Debasish; Mahajan, Milind C.; Weissman, Sherman M.; Gallagher, Patrick G.

    2009-01-01

    Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. PMID:19687298

  4. CCAAT-box binding transcription factors in plants: Y so many?

    PubMed

    Laloum, Tom; De Mita, Stéphane; Gamas, Pascal; Baudin, Maël; Niebel, Andreas

    2013-03-01

    Transcription factors belonging to the CCAAT-box binding factor family (also known as the Nuclear Factor Y) are present in all higher eukaryotes. Studies in plants have revealed that each subunit of this heterotrimeric transcription factor is encoded by a gene belonging to a multigene family allowing a considerable modularity. In this review, we focus on recent findings concerning the expression patterns and potential functions of different members of these NF-Y protein families using a phylogenetic approach. During the course of evolution plant CCAAT-box binding factors seem to have diversified into at least two main groups. The first group has more general expression patterns and/or functions whereas the second group has acquired more specific expression patterns and/or functions and could play key roles in specific pathways. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Decreased Transcription Factor Binding Levels Nearby Primate Pseudogenes Suggest Regulatory Degeneration

    PubMed Central

    Douglas, Gavin M.; Wilson, Michael D.; Moses, Alan M.

    2016-01-01

    Characteristics of pseudogene degeneration at the coding level are well-known, such as a shift toward neutral rates of nonsynonymous substitutions and gain of frameshift mutations. In contrast, degeneration of pseudogene transcriptional regulation is not well understood. Here, we test two predictions of regulatory degeneration along a pseudogenized lineage: 1) Decreased transcription factor (TF) binding and 2) accelerated evolution in putative cis-regulatory regions. We find evidence for decreased TF binding levels nearby two primate pseudogenes compared with functional liver genes. However, the majority of TF-bound sequences nearby pseudogenes do not show evidence for lineage-specific accelerated rates of evolution. We conclude that decreases in TF binding level could be a marker for regulatory degeneration, while sequence degeneration in primate cis-regulatory modules may be obscured by background rates of TF binding site turnover. PMID:26882985

  6. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

    PubMed Central

    2010-01-01

    Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation. PMID:20875111

  7. Identification of two new hydrophobic residues on basic fibroblast growth factor important for fibroblast growth factor receptor binding.

    PubMed

    Zhu, H; Ramnarayan, K; Menzel, P; Miao, Y; Zheng, J; Mong, S

    1998-10-01

    Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitopes required for receptor binding of the growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor binding have already been identified [Springer, B.A., Pantoliano, M.W., Barberal, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Rosenfeld, S.A. and Book, G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu, H.Y., Ramnarayan, K., Anchin, J., Miao, Y., Sereno, A., Millman, L., Zheng, J., Balaji, V.N. and Wolff, M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu, H.Y., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one receptor binding site includes two polar residues Glu96 and Asn104 on bFGF whereas the other includes four hydrophobic residues Tyr24, Tyr103, Leu140 and Met142. Using a protein modelling technique, we report here the identification of a new hydrophobic patch on bFGF which includes residues Tyr73, Val88 and Phe93. The role of this area on receptor binding affinity was evaluated by mutating each of these residues individually and determining the mutated protein's (mutein's) receptor binding affinity. In addition, we examined the role of two other hydrophobic residues, Phe30 and Leu138, on bFGF for high-affinity receptor binding. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with alanine reduced the receptor binding affinity about 10- and 80-fold, respectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding affinities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93

  8. Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

    PubMed Central

    Gao, Rong

    2015-01-01

    ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

  9. A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.

    PubMed Central

    Onaka, H; Sugiyama, M; Horinouchi, S

    1997-01-01

    A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA. PMID:9098075

  10. Phosphorylation states of translational initiation factors affect mRNA cap binding in wheat.

    PubMed

    Khan, Mateen A; Goss, Dixie J

    2004-07-20

    Phosphorylation of eukaryotic translational initiation factors (eIFs) has been shown to be an important means of regulating protein synthesis. Plant initiation factors undergo phosphorylation/dephosphorylation under a variety of stress and growth conditions. We have shown that recombinant wheat cap-binding protein, eIF(iso)4E, produced from E. coli can be phosphorylated in vitro. Phosphorylation of eIF(iso)4E has effects on m(7)G cap-binding affinity similar to those of phosphorylation of mammalian eIF4E even though eIF(iso)4E lacks an amino acid that can be phosphorylated at the residue corresponding to Ser-209, the phosphorylation site in mammalian eIF4E. The cap-binding affinity was reduced 1.2-2.6-fold when eIF(iso)4E was phosphorylated. The in vitro phosphorylation site for wheat eIF(iso)4E was identified as Ser-207. Addition of eIF(iso)4G and eIF4B that had also been phosphorylated in vitro further reduced cap-binding affinity. Temperature-dependent studies showed that DeltaH(degrees) was favorable for cap binding regardless of the phosphorylation state of the initiation factors. The entropy, however, was unfavorable (negative) except when eIF(iso)4E was phosphorylated and interacting with eIF(iso)4G. Phosphorylation may modulate not only cap-binding activity, but other functions of eukaryotic initiation factors as well.

  11. Jasmonate Regulates the INDUCER OF CBF EXPRESSION–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis[W

    PubMed Central

    Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

    2013-01-01

    The INDUCER OF CBF EXPRESSION (ICE)–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance. PMID:23933884

  12. Metal and DNA binding properties of a two-domain fragment of neural zinc finger factor 1, a CCHC-type zinc binding protein.

    PubMed

    Berkovits, H J; Berg, J M

    1999-12-21

    Neural zinc finger factor 1 (NZF-1) is a member of a family of neural-specific transcription factors that contain multiple copies of a relatively uncharacterized zinc binding motif. We have studied the metal binding and DNA binding properties of a fragment of NZF-1 containing two adjacent zinc binding domains. Partial proteolysis with endoproteinase Lys-C identified metal-stabilized fragments containing either one or both of the zinc binding domains. Both domains were required for specific DNA binding to the beta-retinoic acid receptor element, producing a DNase I footprint covering predominantly one strand. The metal binding site was probed via cobalt(II) substitution. The visible absorption spectrum of the cobalt(II) complex is consistent with Cys-Cys-His-Cys coordination of the metal. The two domains appear to have similar affinities for metal and bind cobalt(II) and zinc(II) with dissociation constants of 4 (+/- 2) x 10(-)(7) M and 1.4 (+/- 0.8) x 10(-)(10) M, respectively. The domains fold upon the addition of zinc, as observed by (1)H NMR. However, an additional weak binding site causes line broadening in the presence of excess zinc, presumably due to aggregation.

  13. Identification of candidate transcription factor binding sites in the cattle genome

    USDA-ARS?s Scientific Manuscript database

    A resource that provides candidate transcription factor binding sites does not currently exist for cattle. Such data is necessary, as predicted sites may serve as excellent starting locations for future 'omics studies to develop transcriptional regulation hypotheses. In order to generate this resour...

  14. Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II*

    PubMed Central

    Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A.; McDonald, W. Hayes; Koganitsky, Anna; Eisenbach, Michael; Iverson, Tina M.; Cecchini, Gary

    2016-01-01

    Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation. PMID:26644464

  15. Regulation of the Sre1 hypoxic transcription factor by oxygen-dependent control of DNA binding

    PubMed Central

    Lee, Chih-Yung S.; Yeh, Tzu-Lan; Hughes, Bridget T.; Espenshade, Peter J.

    2011-01-01

    Summary Regulation of gene expression plays an integral role in adaptation of cells to hypoxic stress. In mammals, prolyl hydroxylases control levels of the central transcription factor hypoxia inducible factor (HIF) through regulation of HIFα subunit stability. Here, we report that the hydroxylase Ofd1 regulates the Sre1 hypoxic transcription factor in fission yeast by controlling DNA binding. Prolyl hydroxylases require oxygen as a substrate and the activity of Ofd1 regulates Sre1-dependent transcription. In the presence of oxygen, Ofd1 binds the Sre1 N-terminal transcription factor domain (Sre1N) and inhibits Sre1-dependent transcription by blocking DNA binding. In the absence of oxygen, the inhibitor Nro1 binds Ofd1, thereby releasing Sre1N and leading to activation of genes required for hypoxic growth. In contrast to the HIF system where proline hydroxylation is essential for regulation, Ofd1 inhibition of Sre1N does not require hydroxylation, and thus defines a new mechanism for hypoxic gene regulation. PMID:22017871

  16. Asap: A Framework for Over-Representation Statistics for Transcription Factor Binding Sites

    PubMed Central

    Marstrand, Troels T.; Frellsen, Jes; Moltke, Ida; Thiim, Martin; Valen, Eivind; Retelska, Dorota; Krogh, Anders

    2008-01-01

    Background In studies of gene regulation the efficient computational detection of over-represented transcription factor binding sites is an increasingly important aspect. Several published methods can be used for testing whether a set of hypothesised co-regulated genes share a common regulatory regime based on the occurrence of the modelled transcription factor binding sites. However there is little or no information available for guiding the end users choice of method. Furthermore it would be necessary to obtain several different software programs from various sources to make a well-founded choice. Methodology We introduce a software package, Asap, for fast searching with position weight matrices that include several standard methods for assessing over-representation. We have compared the ability of these methods to detect over-represented transcription factor binding sites in artificial promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. Conclusions We show that our implementation is significantly faster than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server is available at http://servers.binf.ku.dk/asap/. PMID:18286180

  17. Analysis of variation at transcription factor binding sites in Drosophila and humans

    PubMed Central

    2012-01-01

    Background Advances in sequencing technology have boosted population genomics and made it possible to map the positions of transcription factor binding sites (TFBSs) with high precision. Here we investigate TFBS variability by combining transcription factor binding maps generated by ENCODE, modENCODE, our previously published data and other sources with genomic variation data for human individuals and Drosophila isogenic lines. Results We introduce a metric of TFBS variability that takes into account changes in motif match associated with mutation and makes it possible to investigate TFBS functional constraints instance-by-instance as well as in sets that share common biological properties. We also take advantage of the emerging per-individual transcription factor binding data to show evidence that TFBS mutations, particularly at evolutionarily conserved sites, can be efficiently buffered to ensure coherent levels of transcription factor binding. Conclusions Our analyses provide insights into the relationship between individual and interspecies variation and show evidence for the functional buffering of TFBS mutations in both humans and flies. In a broad perspective, these results demonstrate the potential of combining functional genomics and population genetics approaches for understanding gene regulation. PMID:22950968

  18. Temporal hierarchy of gene expression mediated by transcription factor binding affinity and activation dynamics.

    PubMed

    Gao, Rong; Stock, Ann M

    2015-05-26

    Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. A single TF often orchestrates the expression of multiple genes in response to environmental stimuli. It is not clear how different TF-binding sites within the regulon dictate the expression profile. Our studies of Escherichia coli PhoB, a response

  19. Dynamic localization and persistent stimulation of factor-dependent cells by a stem cell factor / cellulose binding domain fusion protein.

    PubMed

    Jervis, Eric J; Guarna, M Marta; Doheny, J Greg; Haynes, Charles A; Kilburn, Douglas G

    2005-08-05

    The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.

  20. The genome-wide binding profile of the Sulfolobus solfataricus transcription factor Ss-LrpB shows binding events beyond direct transcription regulation.

    PubMed

    Nguyen-Duc, Trong; van Oeffelen, Liesbeth; Song, Ningning; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge; Charlier, Daniel; Peeters, Eveline

    2013-11-25

    Gene regulatory processes are largely resulting from binding of transcription factors to specific genomic targets. Leucine-responsive Regulatory Protein (Lrp) is a prevalent transcription factor family in prokaryotes, however, little information is available on biological functions of these proteins in archaea. Here, we study genome-wide binding of the Lrp-like transcription factor Ss-LrpB from Sulfolobus solfataricus. Chromatin immunoprecipitation in combination with DNA microarray analysis (ChIP-chip) has revealed that Ss-LrpB interacts with 36 additional loci besides the four previously identified local targets. Only a subset of the newly identified binding targets, concentrated in a highly variable IS-dense genomic region, is also bound in vitro by pure Ss-LrpB. There is no clear relationship between the in vitro measured DNA-binding specificity of Ss-LrpB and the in vivo association suggesting a limited permissivity of the crenarchaeal chromatin for transcription factor binding. Of 37 identified binding regions, 29 are co-bound by LysM, another Lrp-like transcription factor in S. solfataricus. Comparative gene expression analysis in an Ss-lrpB mutant strain shows no significant Ss-LrpB-mediated regulation for most targeted genes, with exception of the CRISPR B cluster, which is activated by Ss-LrpB through binding to a specific motif in the leader region. The genome-wide binding profile presented here implies that Ss-LrpB is associated at additional genomic binding sites besides the local gene targets, but acts as a specific transcription regulator in the tested growth conditions. Moreover, we have provided evidence that two Lrp-like transcription factors in S. solfataricus, Ss-LrpB and LysM, interact in vivo.

  1. A Potential Structural Switch for Regulating DNA-Binding by TEAD Transcription Factors.

    PubMed

    Lee, Dong-Sun; Vonrhein, Clemens; Albarado, Diana; Raman, C S; Veeraraghavan, Sudha

    2016-06-19

    TEA domain (TEAD) transcription factors are essential for the normal development of eukaryotes and are the downstream effectors of the Hippo tumor suppressor pathway. Whereas our earlier work established the three-dimensional structure of the highly conserved DNA-binding domain using solution NMR spectroscopy, the structural basis for regulating the DNA-binding activity remains unknown. Here, we present the X-ray crystallographic structure and activity of a TEAD mutant containing a truncated L1 loop, ΔL1 TEAD DBD. Unexpectedly, the three-dimensional structure of the ΔL1 TEAD DBD reveals a helix-swapped homodimer wherein helix 1 is swapped between monomers. Furthermore, each three-helix bundle in the domain-swapped dimer is a structural homolog of MYB-like domains. Our investigations of the DNA-binding activity reveal that although the formation of the three-helix bundle by the ΔL1 TEAD DBD is sufficient for binding to an isolated M-CAT-like DNA element, multimeric forms are deficient for cooperative binding to tandemly duplicated elements, indicating that the L1 loop contributes to the DNA-binding activity of TEAD. These results suggest that switching between monomeric and domain-swapped forms may regulate DNA selectivity of TEAD proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    SciTech Connect

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.; Tamás, Markus J.

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  3. Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

    SciTech Connect

    Fayein, N.A.; Courtois, Y.; Jeanny, J.C. )

    1990-05-01

    Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.

  4. Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

    SciTech Connect

    Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R.

    2010-02-08

    The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

  5. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8.

    PubMed

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L; Rosen, Barry P; Tamás, Markus J

    2015-12-28

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

  6. Specific binding of nerve growth factor (NGF) by murine C 1300 neuroblastoma cells.

    PubMed

    Revoltella, R; Bertolini, L; Pediconi, M; Vigneti, E

    1974-08-01

    Murine C 1300 neuroblastoma cells bind with high avidity on their membrane surface the nerve growth factor (NGF), a protein capable of inducing differentiation of sympathetic nerve cells. The total binding capacity of NGF by the cells was quantitatively measured by a radioimmunoassay technique, using (125)I-labeled NGF. An average number of about 10(6) molecules of NGF could be bound, at saturation, by each cell with an average relative association constant of about 10(7) liters/mol. Using synchronized cells, it was found, however, that either the number of molecules of ligand bound or the avidity of the binding interaction between NGF and cells varied depending upon their growth cycle, the maximal-binding occurring during the G(1) and early S phase. Binding of [(125)I]NGF was suppressed by trypsin treatment of the cells, however new receptor sites were rapidly replaced onto the membrane surface within 1-2 h. Cells exposed to 3 M KCl released into the supernate a protein product exhibiting similar high avidity for NGF. Acrylamide gel electrophoresis suggested a restricted molecular heterogeneity of this product, with a major component in the 52,000 mol wt region. Antibodies made specific to this protein were capable, in the absence of the complement, of inhibiting the binding of [(125)I]NGF by the cells and in the presence of the complement they killed them.

  7. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    PubMed Central

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  8. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  9. Sequence2Vec: A novel embedding approach for modeling transcription factor binding affinity landscape.

    PubMed

    Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

    2017-07-27

    An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model (HMM) which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these HMMs into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA data sets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec .

  10. Identification of amino acid residues of transcription factor AP-2 involved in DNA binding.

    PubMed

    García, M A; Campillos, M; Ogueta, S; Valdivieso, F; Vázquez, J

    2000-08-25

    AP-2 is a cell-type specific, developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identified, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. Here, we describe the identification of a region of AP-2 which was protected by an oligonucleotide probe containing its binding site from trypsin digestion, monitored by peptide mapping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependent APOE promoter, as well as the ability to inhibit the effect of the wild-type protein of each one of a set of single-site substitution AP-2 mutants spanning the identified region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential for its DNA-binding activity. Particularly, the individual substitution in any of the residues 253, 254, 255, 257 or 260 is sufficient for completely abolishing the interaction with DNA and the stimulation of APOE promoter activity. These results indicate a crucial role of this region in the formation of an active DNA-binding domain and strongly suggest that these residues provide direct contacts with the DNA structure at the AP-2 binding site. Copyright 2000 Academic Press.

  11. Nonmyogenic factors bind nicotinic acetylcholine receptor promoter elements required for response to denervation.

    PubMed

    Bessereau, J L; Laudenbach, V; Le Poupon, C; Changeux, J P

    1998-05-22

    Nicotinic acetylcholine receptors (AChRs) belong to a class of muscle proteins whose expression is regulated by muscle electrical activity. In innervated muscle fiber, AChR genes are transcriptionally repressed outside of the synapse, while after denervation they become reexpressed throughout the fiber. The myogenic determination factors (MDFs) of the MyoD family have been shown to play a central role in this innervation-dependent regulation. In the chicken AChR alpha-subunit gene promoter, two E-boxes that bind MDFs are necessary to achieve the enhancement of transcription following muscle denervation. However, the deletion of promoter sequences located upstream to these E-boxes greatly impairs the response to denervation (Bessereau, J. L., Stratford- Perricaudet, L. D., Piette, J., Le Poupon, C. and Changeux, J. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1304-1308). Here we identified two additional cis-regulatory elements of the alpha-subunit gene promoter that cooperate with the E-boxes in the denervation response. One region binds the Sp1 and Sp3 zinc finger transcription factors. The second region binds at least three distinct factors, among which we identified an upstream stimulatory factor, a b-ZIP-HLH transcription factor. We propose that among MDF-responsive muscle promoters, a specific combination between myogenic and nonmyogenic factors specify innervation-dependent versus innervation-independent promoters.

  12. Comparative thermodynamic analysis of zinc binding to the His/Cys motif in virion infectivity factor.

    PubMed

    Ghimire-Rijal, Sudipa; Maynard, Ernest L

    2014-05-05

    HIV-1 virion infectivity factor (Vif) is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). Degradation of APOBEC3G requires the interaction of Vif with Cul5, the scaffold for an E3 ubiquitin ligase. A highly conserved region in HIV-1 Vif termed the HCCH motif binds zinc and is critical for recruitment of Cul5 and degradation of APOBEC3G. To gain thermodynamic and mechanistic insight into zinc binding to diverse Vif proteins, we have employed a combination of isothermal titration calorimetry, analytical ultracentrifugation, and Cul5 pull down assays. The proton linkage of zinc binding to HIV-1 Vif was analyzed under different buffer conditions and consistent with the release of two Cys-thiol protons upon zinc binding, supporting earlier EXAFS studies. Zinc binding to Vif proteins from HIV-1, SIVAgm, HIV-2, and SIVMac followed a trend in which the enthalpy of zinc binding became less favorable and the entropy of zinc binding became more favorable. Using AUC, we determined that zinc induced oligomerization of Vif proteins from HIV-1 and SIVAgm but had little or no effect on the oligomeric properties of Vif proteins from HIV-2 and SIVMac. The zinc dependence of Cul5 recruitment by Vif was investigated. All Vif proteins except HIV-2 Vif required zinc to stabilize the interaction with Cul5. The trends in enthalpy-entropy compensation, zinc-induced oligomerization, and Cul5 recruitment are discussed in terms of the apo conformation of the HCCH motif and the role of zinc in stabilizing the structure of Vif.

  13. Determining the DNA sequence elements required for binding integration host factor to two different target sites.

    PubMed Central

    Hales, L M; Gumport, R I; Gardner, J F

    1994-01-01

    Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding. PMID:8188600

  14. A systematic, large-scale comparison of transcription factor binding site models.

    PubMed

    Hombach, Daniela; Schwarz, Jana Marie; Robinson, Peter N; Schuelke, Markus; Seelow, Dominik

    2016-05-21

    The modelling of gene regulation is a major challenge in biomedical research. This process is dominated by transcription factors (TFs) and mutations in their binding sites (TFBSs) may cause the misregulation of genes, eventually leading to disease. The consequences of DNA variants on TF binding are modelled in silico using binding matrices, but it remains unclear whether these are capable of accurately representing in vivo binding. In this study, we present a systematic comparison of binding models for 82 human TFs from three freely available sources: JASPAR matrices, HT-SELEX-generated models and matrices derived from protein binding microarrays (PBMs). We determined their ability to detect experimentally verified "real" in vivo TFBSs derived from ENCODE ChIP-seq data. As negative controls we chose random downstream exonic sequences, which are unlikely to harbour TFBS. All models were assessed by receiver operating characteristics (ROC) analysis. While the area-under-curve was low for most of the tested models with only 47 % reaching a score of 0.7 or higher, we noticed strong differences between the various position-specific scoring matrices with JASPAR and HT-SELEX models showing higher success rates than PBM-derived models. In addition, we found that while TFBS sequences showed a higher degree of conservation than randomly chosen sequences, there was a high variability between individual TFBSs. Our results show that only few of the matrix-based models used to predict potential TFBS are able to reliably detect experimentally confirmed TFBS. We compiled our findings in a freely accessible web application called ePOSSUM ( http:/mutationtaster.charite.de/ePOSSUM/ ) which uses a Bayes classifier to assess the impact of genetic alterations on TF binding in user-defined sequences. Additionally, ePOSSUM provides information on the reliability of the prediction using our test set of experimentally confirmed binding sites.

  15. Insulin-like growth factor-I and insulin-like growth factor-binding proteins in the nephrotic syndrome.

    PubMed

    Feld, S M; Hirschberg, R

    1996-06-01

    Similar to findings in the nephrotic syndrome in humans, rats with the doxorubicin-induced nephrotic syndrome (which resembles minimal change disease) have reduced serum levels of insulin-like growth factor-I (IGF-I). This is mainly caused by glomerular ultrafiltration of IGF-I-containing binding protein complexes, primarily of a molecular weight of approximately 50 kilodaltons, and urinary losses of the peptide. Despite urinary excretion of IGF-binding protein (IGFBP)-2, serum levels are increased more than twofold in the nephrotic syndrome compared with controls, because of increased synthesis of this binding protein by the liver. In contrast, the liver synthesis of IGFBP-3, the predominant binding protein in normal serum, is unchanged in the nephrotic syndrome. However, binding and serum levels of IGFBP-3 are reduced in nephrotic rat serum, apparently due to proteolytic degradation of IGFBP-3. The glomerular ultrafiltration of IGF-I, which leads to biologically significant IGF-I concentrations of about 1.35 nM in proximal tubule fluid, may have metabolic consequences, such as increased tubular phosphate absorption. Hypothetically, tubule fluid IGF-I may also contribute to progressive tubulointerstitial fibrosis which is sometimes present in protractive nephrotic glomerulopathies. The profound changes in the IGF-I/IGFBP system in the nephrotic syndrome may also contribute to systemic metabolic abnormalities and growth failure.

  16. Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

    PubMed Central

    Stuiver, M H; van der Vliet, P C

    1990-01-01

    The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI. Images PMID:2293667

  17. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    PubMed

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  18. Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.

    PubMed

    Shoji, Tsubasa; Mishima, Masaki; Hashimoto, Takashi

    2013-06-01

    Transcription factors (TFs) recognize target DNA sequences with distinct DNA-binding domains (DBDs). The DBD of Arabidopsis (Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (AtERF1) uses three consecutive β-strands to recognize a GCC-containing sequence, but tobacco (Nicotiana tabacum) ERF189 and periwinkle (Catharanthus roseus) Octadecanoid-derivative Responsive Catharanthus AP2-domain protein3 (ORCA3) of the same TF subgroup appear to target similar but divergent DNA sequences. Here, we examined how DNA-binding specificities of these TFs have diverged in each plant lineage to regulate distinct defense metabolisms. Extensive mutational analyses of these DBDs suggest that two modes of protein-DNA interactions independently contribute to binding specificity and affinity. Substitution of a conserved arginine to lysine in the first β-strand of ERF189 relaxes its interaction with the second GC pair of the GCC DNA sequence. By contrast, an increased number of basic amino acids in the first two β-strands of ORCA3 allows this TF to recognize more than one GCC-related target, presumably via increased electrostatic interactions with the negatively charged phosphate backbone of DNA. Divergent DNA-binding specificities of the ERFs may have arisen through mutational changes of these amino acid residues.

  19. Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

    PubMed Central

    Lin, Fubao; Ren, Xiang-Dong; Pan, Zhi; Macri, Lauren; Zong, Wei-Xing; Tonnesen, Marcia G.; Rafailovich, Miriam; Bar-Sagi, Dafna; Clark, Richard A.F.

    2011-01-01

    Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg–Gly–Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10–100 nm. FN-null cells cultured on recombinant CCBD (FNIII8–11) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII8–11 contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII8–11 and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration. PMID:20811396

  20. Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

    PubMed Central

    Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

    2016-01-01

    Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

  1. Energy-dependent fitness: a quantitative model for the evolution of yeast transcription factor binding sites.

    PubMed

    Mustonen, Ville; Kinney, Justin; Callan, Curtis G; Lässig, Michael

    2008-08-26

    We present a genomewide cross-species analysis of regulation for broad-acting transcription factors in yeast. Our model for binding site evolution is founded on biophysics: the binding energy between transcription factor and site is a quantitative phenotype of regulatory function, and selection is given by a fitness landscape that depends on this phenotype. The model quantifies conservation, as well as loss and gain, of functional binding sites in a coherent way. Its predictions are supported by direct cross-species comparison between four yeast species. We find ubiquitous compensatory mutations within functional sites, such that the energy phenotype and the function of a site evolve in a significantly more constrained way than does its sequence. We also find evidence for substantial evolution of regulatory function involving point mutations as well as sequence insertions and deletions within binding sites. Genes lose their regulatory link to a given transcription factor at a rate similar to the neutral point mutation rate, from which we infer a moderate average fitness advantage of functional over nonfunctional sites. In a wider context, this study provides an example of inference of selection acting on a quantitative molecular trait.

  2. Histidine 197 in Release Factor 1 is Essential for A Site Binding and Peptide Release

    PubMed Central

    Field, Andrew; Hetrick, Byron; Mathew, Merrill; Joseph, Simpson

    2010-01-01

    Class I peptide release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (197 in E. coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1•ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue. PMID:20873815

  3. Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

    PubMed Central

    Roberts, E. Claire; Deed, Richard W.; Inoue, Toshiaki; Norton, John D.; Sharrocks, Andrew D.

    2001-01-01

    The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family. PMID:11134340

  4. G =  MAT: linking transcription factor expression and DNA binding data.

    PubMed

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-31

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

  5. G = MAT: Linking Transcription Factor Expression and DNA Binding Data

    PubMed Central

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-01

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

  6. Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

    PubMed Central

    Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

    2015-01-01

    Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

  7. Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

    PubMed Central

    Mazzoni, Esteban O; Mahony, Shaun; Closser, Michael; Morrison, Carolyn A; Nedelec, Stephane; Williams, Damian J; An, Disi; Gifford, David K; Wichterle, Hynek

    2013-01-01

    Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types. PMID:23872598

  8. Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

    PubMed

    Tian, Erming; Børset, Magne; Sawyer, Jeffrey R; Brede, Gaute; Våtsveen, Thea K; Hov, Håkon; Waage, Anders; Barlogie, Bart; Shaughnessy, John D; Epstein, Joshua; Sundan, Anders

    2015-11-01

    The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single-nucleotide polymorphism (SNP) and crucial single-nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele-specific mutations functionally reassembled wild-type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa-B), MZF1 (myeloid zinc finger 1), and NRF-2 (nuclear factor erythroid 2-related factor 2). In vitro, a mutant allele that gained novel NFKB-binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome-wide sequencing (GWS) of numerous non-coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild-type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells.

  9. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    SciTech Connect

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  10. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site.

    PubMed

    Al-Zyoud, Walid A; Hynson, Robert M G; Ganuelas, Lorraine A; Coster, Adelle C F; Duff, Anthony P; Baker, Matthew A B; Stewart, Alastair G; Giannoulatou, Eleni; Ho, Joshua W K; Gaus, Katharina; Liu, Dali; Lee, Lawrence K; Böcking, Till

    2016-02-18

    Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR-DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The repertoire of DNA-binding transcription factors in prokaryotes: functional and evolutionary lessons.

    PubMed

    Perez-Rueda, Ernesto; Martinez-Nuñez, Mario Alberto

    2012-01-01

    The capabilities of organisms to contend with environmental changes depend on their genes and their ability to regulate their expression. DNA-binding transcription factors (TFs) play a central role in this process, because they regulate gene expression positively and/or negatively, depending on the operator context and ligand-binding status. In this review, we summarise recent findings regarding the function and evolution of TFs in prokaryotes. We consider the abundance of TFs in bacteria and archaea, the role of DNA-binding domains and their partner domains, and the effects of duplication events in the evolution of regulatory networks. Finally, a comprehensive picture for how regulatory networks have evolved in prokaryotes is provided.

  12. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    SciTech Connect

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  13. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Das, Theerthankar; Kutty, Samuel K.; Tavallaie, Roya; Ibugo, Amaye I.; Panchompoo, Janjira; Sehar, Shama; Aldous, Leigh; Yeung, Amanda W. S.; Thomas, Shane R.; Kumar, Naresh; Gooding, J. Justin; Manefield, Mike

    2015-01-01

    Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation. PMID:25669133

  14. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    SciTech Connect

    Lilic,M.; Vujanac, M.; Stebbins, C.

    2006-01-01

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

  15. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  16. Formation of STAT5-containing DNA binding complexes in response to colony-stimulating factor-1 and platelet-derived growth factor.

    PubMed

    Novak, U; Mui, A; Miyajima, A; Paradiso, L

    1996-08-02

    Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

  17. Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development

    PubMed Central

    Kaplan, Tommy; Li, Xiao-Yong; Sabo, Peter J.; Thomas, Sean; Stamatoyannopoulos, John A.; Biggin, Mark D.; Eisen, Michael B.

    2011-01-01

    Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be

  18. Recent Insights into Insulin-Like Growth Factor Binding Protein 2 Transcriptional Regulation

    PubMed Central

    Park, Jae-Hyung; Bae, Jae-Hoon; Song, Dae-Kyu

    2017-01-01

    Insulin-like growth factor binding proteins (IGFBPs) are major regulators of insulin-like growth factor bioavailability and activity in metabolic signaling. Seven IGFBP family isoforms have been identified. Recent studies have shown that IGFBPs play a pivotal role in metabolic signaling and disease, including the pathogenesis of obesity, diabetes, and cancer. Although many studies have documented the various roles played by IGFBPs, transcriptional regulation of IGFBPs is not well understood. In this review, we focus on the regulatory mechanisms of IGFBP gene expression, and we summarize the findings of transcription factor activity in the IGFBP promoter region. PMID:28116872

  19. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  20. Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions.

    PubMed

    O'Seaghdha, Maghnus; van Schooten, Carina J; Kerrigan, Steven W; Emsley, Jonas; Silverman, Gregg J; Cox, Dermot; Lenting, Peter J; Foster, Timothy J

    2006-11-01

    Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D'-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D'-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.

  1. Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

    PubMed Central

    Sanders, Jeffrey M.; Wampole, Matthew E.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF

  2. Genome-scale Analysis of Escherichia coli FNR Reveals Complex Features of Transcription Factor Binding

    PubMed Central

    Myers, Kevin S.; Yan, Huihuang; Ong, Irene M.; Chung, Dongjun; Liang, Kun; Tran, Frances; Keleş, Sündüz; Landick, Robert; Kiley, Patricia J.

    2013-01-01

    FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut. PMID:23818864

  3. A graphical modelling approach to the dissection of highly correlated transcription factor binding site profiles.

    PubMed

    Stojnic, Robert; Fu, Audrey Qiuyan; Adryan, Boris

    2012-01-01

    Inferring the combinatorial regulatory code of transcription factors (TFs) from genome-wide TF binding profiles is challenging. A major reason is that TF binding profiles significantly overlap and are therefore highly correlated. Clustered occurrence of multiple TFs at genomic sites may arise from chromatin accessibility and local cooperation between TFs, or binding sites may simply appear clustered if the profiles are generated from diverse cell populations. Overlaps in TF binding profiles may also result from measurements taken at closely related time intervals. It is thus of great interest to distinguish TFs that directly regulate gene expression from those that are indirectly associated with gene expression. Graphical models, in particular Bayesian networks, provide a powerful mathematical framework to infer different types of dependencies. However, existing methods do not perform well when the features (here: TF binding profiles) are highly correlated, when their association with the biological outcome is weak, and when the sample size is small. Here, we develop a novel computational method, the Neighbourhood Consistent PC (NCPC) algorithms, which deal with these scenarios much more effectively than existing methods do. We further present a novel graphical representation, the Direct Dependence Graph (DDGraph), to better display the complex interactions among variables. NCPC and DDGraph can also be applied to other problems involving highly correlated biological features. Both methods are implemented in the R package ddgraph, available as part of Bioconductor (http://bioconductor.org/packages/2.11/bioc/html/ddgraph.html). Applied to real data, our method identified TFs that specify different classes of cis-regulatory modules (CRMs) in Drosophila mesoderm differentiation. Our analysis also found depletion of the early transcription factor Twist binding at the CRMs regulating expression in visceral and somatic muscle cells at later stages, which suggests a CRM

  4. A Graphical Modelling Approach to the Dissection of Highly Correlated Transcription Factor Binding Site Profiles

    PubMed Central

    Stojnic, Robert; Fu, Audrey Qiuyan; Adryan, Boris

    2012-01-01

    Inferring the combinatorial regulatory code of transcription factors (TFs) from genome-wide TF binding profiles is challenging. A major reason is that TF binding profiles significantly overlap and are therefore highly correlated. Clustered occurrence of multiple TFs at genomic sites may arise from chromatin accessibility and local cooperation between TFs, or binding sites may simply appear clustered if the profiles are generated from diverse cell populations. Overlaps in TF binding profiles may also result from measurements taken at closely related time intervals. It is thus of great interest to distinguish TFs that directly regulate gene expression from those that are indirectly associated with gene expression. Graphical models, in particular Bayesian networks, provide a powerful mathematical framework to infer different types of dependencies. However, existing methods do not perform well when the features (here: TF binding profiles) are highly correlated, when their association with the biological outcome is weak, and when the sample size is small. Here, we develop a novel computational method, the Neighbourhood Consistent PC (NCPC) algorithms, which deal with these scenarios much more effectively than existing methods do. We further present a novel graphical representation, the Direct Dependence Graph (DDGraph), to better display the complex interactions among variables. NCPC and DDGraph can also be applied to other problems involving highly correlated biological features. Both methods are implemented in the R package ddgraph, available as part of Bioconductor (http://bioconductor.org/packages/2.11/bioc/html/ddgraph.html). Applied to real data, our method identified TFs that specify different classes of cis-regulatory modules (CRMs) in Drosophila mesoderm differentiation. Our analysis also found depletion of the early transcription factor Twist binding at the CRMs regulating expression in visceral and somatic muscle cells at later stages, which suggests a CRM

  5. Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

    PubMed

    Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

    2017-08-01

    Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

  6. Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

    PubMed Central

    Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

    2014-01-01

    The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

  7. Measurement of immunotargeted plasmonic nanoparticles' cellular binding: a key factor in optimizing diagnostic efficacy

    NASA Astrophysics Data System (ADS)

    Fu, Kun; Sun, Jiantang; Bickford, Lissett R.; Lin, Alex W. H.; Halas, Naomi J.; Yu, Tse-Kuan; Drezek, Rebekah A.

    2008-01-01

    In this study, we use polarized light scattering to study immunotargeted plasmonic nanoparticles which bind to live SK-BR-3 human breast carcinoma cells. Gold nanoparticles can be conjugated to various biomolecules in order to target specific molecular signatures of disease. This specific targeting provides enhanced contrast in scattering-based optical imaging techniques. While there are papers which report the number of antibodies that bind per nanoparticle, there are almost no reports of the key factor which influences diagnostic or therapeutic efficacy using nanoparticles: the number of targeted nanoparticles that bind per cell. To achieve this goal, we have developed a 'negative' method of determining the binding concentration of those antibody/nanoparticle bioconjugates which are targeted specifically to breast cancer cells. Unlike previously reported methods, we collected unbound nanoparticle bioconjugates and measured the light scattering from dilute solutions of these particles so that quantitative binding information can be obtained. By following this process, the interaction effects of adjacent bound nanoparticles on the cell membrane can be avoided simply by measuring the light scattering from the unbound nanoparticles. Specifically, using nanoshells of two different sizes, we compared the binding concentrations of anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates targeted to HER2-positive SK-BR-3 breast cancer cells. The results indicate that, for anti-HER2/nanoshell bioconjugates, there are approximately 800-1600 nanoshells bound per cell; for anti-IgG/nanoshell bioconjugates, the binding concentration is significantly lower at nearly 100 nanoshells bound per cell. These results are also supported by dark-field microscopy images of the cells labeled with anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates.

  8. Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

    PubMed Central

    Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

    2016-01-01

    A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

  9. SMiLE-seq identifies binding motifs of single and dimeric transcription factors.

    PubMed

    Isakova, Alina; Groux, Romain; Imbeault, Michael; Rainer, Pernille; Alpern, Daniel; Dainese, Riccardo; Ambrosini, Giovanna; Trono, Didier; Bucher, Philipp; Deplancke, Bart

    2017-03-01

    Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes. All tested TFs yielded DNA-binding models with predictive power comparable to or greater than that of other in vitro assays. De novo motif discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes provided novel insights into partner-specific heterodimer DNA-binding preferences. We also successfully analyzed the DNA-binding properties of uncharacterized human C2H2 zinc-finger proteins and validated several using ChIP-exo.

  10. Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

    PubMed Central

    Jiang, Chao; Wilkinson, Mark C.

    2016-01-01

    The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans. PMID:27030175

  11. Identification of transcription factor binding sites from ChIP-seq data at high resolution

    PubMed Central

    Knoblich, Juergen A.; Zeitlinger, Julia; Stark, Alexander

    2013-01-01

    Motivation: Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) is widely used to study the in vivo binding sites of transcription factors (TFs) and their regulatory targets. Recent improvements to ChIP-seq, such as increased resolution, promise deeper insights into transcriptional regulation, yet require novel computational tools to fully leverage their advantages. Results: To this aim, we have developed peakzilla, which can identify closely spaced TF binding sites at high resolution (i.e. resolves individual binding sites even if spaced closely), as we demonstrate using semisynthetic datasets, performing ChIP-seq for the TF Twist in Drosophila embryos with different experimental fragment sizes, and analyzing ChIP-exo datasets. We show that the increased resolution reached by peakzilla is highly relevant, as closely spaced Twist binding sites are strongly enriched in transcriptional enhancers, suggesting a signature to discriminate functional from abundant non-functional or neutral TF binding. Peakzilla is easy to use, as it estimates all the necessary parameters from the data and is freely available. Availability and implementation: The peakzilla program is available from https://github.com/steinmann/peakzilla or http://www.starklab.org/data/peakzilla/. Contact: stark@starklab.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23980024

  12. Comparative study of binding of ovine complement factor H with different Borrelia genospecies.

    PubMed

    Kišová-Vargová, Lucia; Cerňanská, Dana; Bhide, Mangesh

    2012-03-01

    This study presents the binding of ovine factor H (fH) by various serotypes of Borrelia and simultaneously correlates their complement resistance to sheep serum. Affinity ligand binding assay was employed to study the binding of borrelial proteins to ovine recombinant fH and its truncated forms (short consensus repeat, SCR 7 and SCRs 19-20). From a repertoire of 17 borrelial strains, only two strains showed affinity to sheep fH. A ~28-kDa protein of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s., strain SKT-2) bound full-length fH as well as SCRs 19-20. This fH-binding protein was further identified as complement regulator-acquiring surface protein of B. burgdorferi (BbCRASP-1) by MALDI-TOF analysis. Surprisingly, a ~26-kDa protein of Borrelia bissettii (DN127) showed affinity to full-length fH but not to SCR 7 and SCRs19-20. In complement sensitivity assay, both strains-SKT-2 and DN127-were resistant to normal sheep serum. Significant complement resistance of two Borrelia garinii strains (G117 and T25) was also observed; however, none of those strains was able to bind sheep fH. Our study underscores the need of further exploration of fH-mediated evasion of complement system by Borrelia in domestic animals.

  13. Flanking sequence context-dependent transcription factor binding in early Drosophila development

    PubMed Central

    2013-01-01

    Background Gene expression in the Drosophila embryo is controlled by functional interactions between a large network of protein transcription factors (TFs) and specific sequences in DNA cis-regulatory modules (CRMs). The binding site sequences for any TF can be experimentally determined and represented in a position weight matrix (PWM). PWMs can then be used to predict the location of TF binding sites in other regions of the genome, although there are limitations to this approach as currently implemented. Results In this proof-of-principle study, we analyze 127 CRMs and focus on four TFs that control transcription of target genes along the anterio-posterior axis of the embryo early in development. For all four of these TFs, there is some degree of conserved flanking sequence that extends beyond the predicted binding regions. A potential role for these conserved flanking sequences may be to enhance the specificity of TF binding, as the abundance of these sequences is greatly diminished when we examine only predicted high-affinity binding sites. Conclusions Expanding PWMs to include sequence context-dependence will increase the information content in PWMs and facilitate a more efficient functional identification and dissection of CRMs. PMID:24093548

  14. Structural basis for the regulation of insulin-like growth factors by IGF binding proteins.

    PubMed

    Siwanowicz, Igor; Popowicz, Grzegorz M; Wisniewska, Magdalena; Huber, Robert; Kuenkele, Klaus-Peter; Lang, Kurt; Engh, Richard A; Holak, Tad A

    2005-01-01

    Insulin-like growth factor binding proteins (IGFBPs) control the extracellular distribution, function, and activity of IGFs. Here, we report an X-ray structure of the binary complex of IGF-I and the N-terminal domain of IGFBP-4 (NBP-4, residues 3-82) and a model of the ternary complex of IGF-I, NBP-4, and the C-terminal domain (CBP-4, residues 151-232) derived from diffraction data with weak definition of the C-terminal domain. These structures show how the IGFBPs regulate IGF signaling. Key features of the structures include (1) a disulphide bond ladder that binds to IGF and partially masks the IGF residues responsible for type 1 IGF receptor (IGF-IR) binding, (2) the high-affinity IGF-I interaction site formed by residues 39-82 in a globular fold, and (3) CBP-4 interactions. Although CBP-4 does not bind individually to either IGF-I or NBP-4, in the ternary complex, CBP-4 contacts both and also blocks the IGF-IR binding region of IGF-I.

  15. Conservation of transcription factor binding specificities across 600 million years of bilateria evolution

    PubMed Central

    Nitta, Kazuhiro R; Jolma, Arttu; Yin, Yimeng; Morgunova, Ekaterina; Kivioja, Teemu; Akhtar, Junaid; Hens, Korneel; Toivonen, Jarkko; Deplancke, Bart; Furlong, Eileen E M; Taipale, Jussi

    2015-01-01

    Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells. DOI: http://dx.doi.org/10.7554/eLife.04837.001 PMID:25779349

  16. GAGA factor binding to DNA via a single trinucleotide sequence element.

    PubMed Central

    Wilkins, R C; Lis, J T

    1998-01-01

    GAGA transcription factor (GAF) is an essential protein in Drosophila , important for the transcriptional regulation of numerous genes. GAF binds to GA repeats in the promoters of these genes via a DNA-binding domain containing a single zinc finger. While GAF binding sites are typically composed of 3.5 GA repeats, the Drosophila hsp70 gene contains much smaller elements, some of which are as little as three bases (GAG) in length. Interestingly, the binding of GAF to more distant trinucleotide elements is relatively strong and not appreciably affected by the removal of larger GA arrays in the promoter. Moreover, a simple synthetic GAG sequence is sufficient to bind GAF in vitro . Here we directly compare the affinity of GAF for different sequence elements by immunoprecipitation and gel mobility shift analysis. Furthermore, our measures of the concentration of GAF in vivo indicate that it is a highly abundant nuclear protein, prevalent enough to occupy a sizable fraction of correspondingly abundant trinucleotide sites. PMID:9592153

  17. Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor.

    PubMed

    Van Loy, Cristina P; Sokurenko, Evgeni V; Samudrala, Ram; Moseley, Steve L

    2002-07-01

    Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.

  18. Heparin-Binding Epidermal Growth Factor-like Growth Factor/Diphtheria Toxin Receptor in Normal and Neoplastic Hematopoiesis

    PubMed Central

    Vinante, Fabrizio; Rigo, Antonella

    2013-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant physiological and pathological processes spanning from proliferation and apoptosis to morphogenesis. We outline here the main activities of HB-EGF in connection with normal or neoplastic differentiative or proliferative events taking place primitively in the hematopoietic microenvironment. PMID:23888518

  19. MORPHEUS, a Webtool for Transcription Factor Binding Analysis Using Position Weight Matrices with Dependency.

    PubMed

    Minguet, Eugenio Gómez; Segard, Stéphane; Charavay, Céline; Parcy, François

    2015-01-01

    Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools (called Morpheus) based on the proven Position Weight Matrices (PWM) formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene APETALA1 during the divergence of Brassicales clade.

  20. MORPHEUS, a Webtool for Transcription Factor Binding Analysis Using Position Weight Matrices with Dependency

    PubMed Central

    Minguet, Eugenio Gómez; Segard, Stéphane; Charavay, Céline; Parcy, François

    2015-01-01

    Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools (called Morpheus) based on the proven Position Weight Matrices (PWM) formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene APETALA1 during the divergence of Brassicales clade. PMID:26285209

  1. Orthogonal matrix factorization enables integrative analysis of multiple RNA binding proteins

    PubMed Central

    Stražar, Martin; Žitnik, Marinka; Zupan, Blaž; Ule, Jernej; Curk, Tomaž

    2016-01-01

    Motivation: RNA binding proteins (RBPs) play important roles in post-transcriptional control of gene expression, including splicing, transport, polyadenylation and RNA stability. To model protein–RNA interactions by considering all available sources of information, it is necessary to integrate the rapidly growing RBP experimental data with the latest genome annotation, gene function, RNA sequence and structure. Such integration is possible by matrix factorization, where current approaches have an undesired tendency to identify only a small number of the strongest patterns with overlapping features. Because protein–RNA interactions are orchestrated by multiple factors, methods that identify discriminative patterns of varying strengths are needed. Results: We have developed an integrative orthogonality-regularized nonnegative matrix factorization (iONMF) to integrate multiple data sources and discover non-overlapping, class-specific RNA binding patterns of varying strengths. The orthogonality constraint halves the effective size of the factor model and outperforms other NMF models in predicting RBP interaction sites on RNA. We have integrated the largest data compendium to date, which includes 31 CLIP experiments on 19 RBPs involved in splicing (such as hnRNPs, U2AF2, ELAVL1, TDP-43 and FUS) and processing of 3’UTR (Ago, IGF2BP). We show that the integration of multiple data sources improves the predictive accuracy of retrieval of RNA binding sites. In our study the key predictive factors of protein–RNA interactions were the position of RNA structure and sequence motifs, RBP co-binding and gene region type. We report on a number of protein-specific patterns, many of which are consistent with experimentally determined properties of RBPs. Availability and implementation: The iONMF implementation and example datasets are available at https://github.com/mstrazar/ionmf. Contact: tomaz.curk@fri.uni-lj.si Supplementary information: Supplementary data are available

  2. Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

    PubMed Central

    Forsten-Williams, Kimberly; Zhang, Jun; Fannon, Michael

    2010-01-01

    Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove

  3. Exploiting ancestral mammalian genomes for the prediction of human transcription factor binding sites

    PubMed Central

    2012-01-01

    Background The computational prediction of Transcription Factor Binding Sites (TFBS) remains a challenge due to their short length and low information content. Comparative genomics approaches that simultaneously consider several related species and favor sites that have been conserved throughout evolution improve the accuracy (specificity) of the predictions but are limited due to a phenomenon called binding site turnover, where sequence evolution causes one TFBS to replace another in the same region. In parallel to this development, an increasing number of mammalian genomes are now sequenced and it is becoming possible to infer, to a surprisingly high degree of accuracy, ancestral mammalian sequences. Results We propose a TFBS prediction approach that makes use of the availability of inferred ancestral mammalian genomes to improve its accuracy. This method aims to identify binding loci, which are regions of a few hundred base pairs that have preserved their potential to bind a given transcription factor over evolutionary time. After proposing a neutral evolutionary model of predicted TFBS counts in a DNA region of a given length, we use it to identify regions that have preserved the number of predicted TFBS they contain to an unexpected degree given their divergence. The approach is applied to human chromosome 1 and shows significant gains in accuracy as compared to both existing single-species and multi-species TFBS prediction approaches, in particular for transcription factors that are subject to high turnover rates. Availability The source code and predictions made by the program are available at http://www.cs.mcgill.ca/~blanchem/bindingLoci. PMID:23281809

  4. Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

    PubMed Central

    Quitschke, W W; DePonti-Zilli, L; Lin, Z Y; Paterson, B M

    1989-01-01

    The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis. Images PMID:2552286

  5. SNP2TFBS – a database of regulatory SNPs affecting predicted transcription factor binding site affinity

    PubMed Central

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-01

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/. PMID:27899579

  6. Effects of binding factors on structural elements in F-actin.

    PubMed

    Scoville, Damon; Stamm, John D; Altenbach, Christian; Shvetsov, Alexander; Kokabi, Kaveh; Rubenstein, Peter A; Hubbell, Wayne L; Reisler, Emil

    2009-01-20

    Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

  7. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  8. Solution structure of the first HMG box domain in human upstream binding factor.

    PubMed

    Xu, Yingqi; Yang, Wulin; Wu, Jihui; Shi, Yunyu

    2002-04-30

    Human upstream binding factor is a nucleolar transcription factor involved in transcription by RNA polymerase I. It contains six HMG box domains; the HMG box is a minor groove DNA-binding domain that has been found in hundreds of proteins with different functions. Among the six HMG box domains in hUBF, the first one can bind to the ribosomal promoter specifically by itself and is essential for the whole protein's DNA binding specificity. Here we report the three-dimensional structure of this first HMG box free in solution determined by multidimensional NMR using (13)C,(15)N-labeled protein. Like the previously determined HMG box structures, hUBF HMG box 1 adopts a twisted L-shape consisting of three alpha-helices: helix 1 (17-30) and helix 2 (38-51) pack onto each other to form the short arm, while helix 3 (57-76) is associated with an extended strand N-terminal to helix 1 and forms the long arm. A cluster of conserved residues, in particular the aromatic residues F21, Y49, and Y60, is important to maintain the fold. The short arm is rigid due to extensive hydrophobic interaction between helix 1 and helix 2, while the long arm is less rigid.

  9. SNP2TFBS - a database of regulatory SNPs affecting predicted transcription factor binding site affinity.

    PubMed

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-04

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/.

  10. The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

    PubMed Central

    Lyonnais, Sébastien; Tarrés-Soler, Aleix; Rubio-Cosials, Anna; Cuppari, Anna; Brito, Reicy; Jaumot, Joaquim; Gargallo, Raimundo; Vilaseca, Marta; Silva, Cristina; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Eritja, Ramon; Solà, Maria

    2017-01-01

    The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

  11. The MAPPER2 Database: a multi-genome catalog of putative transcription factor binding sites

    PubMed Central

    Riva, Alberto

    2012-01-01

    The mapper2 Database (http://genome.ufl.edu/mapperdb) is a component of mapper2, a web-based system for the analysis of transcription factor binding sites in multiple genomes. The database contains predicted binding sites identified in the promoters of all human, mouse and Drosophila genes using 1017 probabilistic models representing over 600 different transcription factors. In this article we outline the current contents of the database and we describe its web-based user interface in detail. We then discuss ongoing work to extend the database contents to experimental data and to add analysis capabilities. Finally, we provide information about recent improvements to the hardware and software platform that mapper2 is based on. PMID:22121218

  12. CompleteMOTIFs: DNA motif discovery platform for transcription factor binding experiments.

    PubMed

    Kuttippurathu, Lakshmi; Hsing, Michael; Liu, Yongchao; Schmidt, Bertil; Maskell, Douglas L; Lee, Kyungjoon; He, Aibin; Pu, William T; Kong, Sek Won

    2011-03-01

    CompleteMOTIFs (cMOTIFs) is an integrated web tool developed to facilitate systematic discovery of overrepresented transcription factor binding motifs from high-throughput chromatin immunoprecipitation experiments. Comprehensive annotations and Boolean logic operations on multiple peak locations enable users to focus on genomic regions of interest for de novo motif discovery using tools such as MEME, Weeder and ChIPMunk. The pipeline incorporates a scanning tool for known motifs from TRANSFAC and JASPAR databases, and performs an enrichment test using local or precalculated background models that significantly improve the motif scanning result. Furthermore, using the cMOTIFs pipeline, we demonstrated that multiple transcription factors could cooperatively bind to the upstream of important stem cell differentiation regulators. http://cmotifs.tchlab.org.

  13. The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.

    PubMed

    Yahata, T; de Caestecker, M P; Lechleider, R J; Andriole, S; Roberts, A B; Isselbacher, K J; Shioda, T

    2000-03-24

    The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.

  14. Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody

    PubMed Central

    Maeda, Atsuhiko; Iwayanagi, Yuki; Haraya, Kenta; Tachibana, Tatsuhiko; Nakamura, Genki; Nambu, Takeru; Esaki, Keiko; Hattori, Kunihiro; Igawa, Tomoyuki

    2017-01-01

    ABSTRACT Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases. PMID:28387635

  15. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    PubMed

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  16. Insights into structural and functional diversity of Dof (DNA binding with one finger) transcription factor.

    PubMed

    Gupta, S; Malviya, N; Kushwaha, H; Nasim, J; Bisht, N C; Singh, V K; Yadav, D

    2015-03-01

    The structural, functional and in-silico studies of Dof transcription factor attempted so far reveals immense opportunity to analyze the plant genomes in terms of number of Dof genes and discuss in light of the evolution. The multiple functions of Dof genes needs to explored for crop improvement. Transcription factors play a very vital role in gene regulation at transcriptional level and are being extensively studied across phylas. In recent years, sequencing of plant genomes has led to genome-wide identification and characterizations of diverse types of plant-specific transcription factor gene family providing key insights into their structural and functional diversity. The DNA binding with one finger (Dof), a class belonging to C2H2-type zinc finger family proteins, is a plant-specific transcription factor having multiple roles such as seed maturation and germination, phytohormone and light-mediated regulation and plant responses to biotic and abiotic stresses. Dof proteins are present across plant lineage, from green algae to higher angiosperm, and represent a unique class of transcription factor having bifunctional binding activities, with both DNA and proteins, to regulate the complex transcriptional machinery in plant cells. The structural and functional diversity of the Dof transcription factor family along with the bioinformatics analysis highlighting the phylogeny of Dof families is reviewed in light of its importance in plant biotechnology for crop improvement.

  17. Sperm and spermatids contain different proteins and bind distinct egg factors.

    PubMed

    Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J; Gurdon, John B; Jullien, Jerome

    2014-09-19

    Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

  18. Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

    PubMed Central

    Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

    1971-01-01

    Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

  19. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

    PubMed

    Gerold, Gisa; Meissner, Felix; Bruening, Janina; Welsch, Kathrin; Perin, Paula M; Baumert, Thomas F; Vondran, Florian W; Kaderali, Lars; Marcotrigiano, Joseph; Khan, Abdul G; Mann, Matthias; Rice, Charles M; Pietschmann, Thomas

    2015-08-04

    Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Compound hierarchical correlated beta mixture with an application to cluster mouse transcription factor DNA binding data.

    PubMed

    Dai, Hongying; Charnigo, Richard

    2015-10-01

    Modeling correlation structures is a challenge in bioinformatics, especially when dealing with high throughput genomic data. A compound hierarchical correlated beta mixture (CBM) with an exchangeable correlation structure is proposed to cluster genetic vectors into mixture components. The correlation coefficient, [Formula: see text], is homogenous within a mixture component and heterogeneous between mixture components. A random CBM with [Formula: see text] brings more flexibility in explaining correlation variations among genetic variables. Expectation-Maximization (EM) algorithm and Stochastic Expectation-Maximization (SEM) algorithm are used to estimate parameters of CBM. The number of mixture components can be determined using model selection criteria such as AIC, BIC and ICL-BIC. Extensive simulation studies were conducted to compare EM, SEM and model selection criteria. Simulation results suggest that CBM outperforms the traditional beta mixture model with lower estimation bias and higher classification accuracy. The proposed method is applied to cluster transcription factor-DNA binding probability in mouse genome data generated by Lahdesmaki and others (2008, Probabilistic inference of transcription factor binding from multiple data sources. PLoS One, 3: , e1820). The results reveal distinct clusters of transcription factors when binding to promoter regions of genes in JAK-STAT, MAPK and other two pathways. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Structural basis of human transcription factor Sry-related box 17 binding to DNA.

    PubMed

    Gao, Nana; Jiang, Wei; Gao, Hai; Cheng, Zhong; Qian, Huolian; Si, Shuyi; Xie, Yong

    2013-04-01

    Sry-related box (Sox) transcription factors share a conserved high-mobility-group box domain (HMG-domain) that binds DNA in the minor groove and bends DNA for further assembly of transcriptional machineries. During organogenesis, each member of the Sox family triggers a specific cell lineage differentiation, indicating that their interactions with DNA are different from each other. Therefore, investigating structural rearrangement of each Sox transcription factor HMG-domain upon binding to DNA would help to elucidate the distinctive molecular mechanism by which they interact with DNA. Previous studies have determined the crystal structures of Sox2 HMG-domain/DNA, Sox4 HMGdomain/ DNA, Sox9 HMG-domain/DNA and Sox17 HMG-domain/DNA complexes. However, major gaps remain in the structural information on the Sox transcription factor HMG-domains. Here, we report the crystal structure of the human Sox17 HMG-domain alone at 2.4 A resolution. Comparing this structure and the structure of the mouse Sox17 HMGdomain/ DNA complex provides structural understanding of the mechanism of Sox17 binding to DNA. Specifically, after electrostatic interactions attract Sox17 to DNA, Asn73, Ser99, and Trp106 form hydrogen bonds with DNA, Arg70, Lys80, Arg83, His94, and Asn95 on Sox17 undergo conformational changes and form hydrogen bonds with DNA, contributing to the electrostatic interaction between Sox17 and DNA.

  2. Engineering transcription factors with novel DNA-binding specificity using comparative genomics

    PubMed Central

    Desai, Tasha A.; Rodionov, Dmitry A.; Gelfand, Mikhail S.; Alm, Eric J.; Rao, Christopher V.

    2009-01-01

    The transcriptional program for a gene consists of the promoter necessary for recruiting RNA polymerase along with neighboring operator sites that bind different activators and repressors. From a synthetic biology perspective, if the DNA-binding specificity of these proteins can be changed, then they can be used to reprogram gene expression in cells. While many experimental methods exist for generating such specificity-altering mutations, few computational approaches are available, particularly in the case of bacterial transcription factors. In a previously published computational study of nitrogen oxide metabolism in bacteria, a small number of amino-acid residues were found to determine the specificity within the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors. By analyzing how these amino acids vary in different regulators, a simple relationship between the identity of these residues and their target DNA-binding sequence was constructed. In this article, we experimentally tested whether this relationship could be used to engineer novel DNA–protein interactions. Using Escherichia coli CRP as a template, we tested eight designs based on this relationship and found that four worked as predicted. Collectively, these results in this work demonstrate that comparative genomics can inform the design of bacterial transcription factors. PMID:19264798

  3. Conformational stability and DNA binding specificity of the cardiac T-box transcription factor Tbx20.

    PubMed

    Macindoe, Ingrid; Glockner, Laura; Vukasin, Paul; Stennard, Fiona A; Costa, Mauro W; Harvey, Richard P; Mackay, Joel P; Sunde, Margaret

    2009-06-12

    The transcription factor Tbx20 acts within a hierarchy of T-box factors in lineage specification and morphogenesis in the mammalian heart and is mutated in congenital heart disease. T-box family members share a approximately 20-kDa DNA-binding domain termed the T-box. The question of how highly homologous T-box proteins achieve differential transcriptional control in heart development, while apparently binding to the same DNA sequence, remains unresolved. Here we show that the optimal DNA recognition sequence for the T-box of Tbx20 corresponds to a T-half-site. Furthermore, we demonstrate using purified recombinant domains that distinct T-boxes show significant differences in the affinity and kinetics of binding and in conformational stability, with the T-box of Tbx20 displaying molten globule character. Our data highlight unique features of Tbx20 and suggest mechanistic ways in which cardiac T-box factors might interact synergistically and/or competitively within the cardiac regulatory network.

  4. Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

    PubMed

    Hirose, F; Yamaguchi, M; Matsukage, A

    1999-09-01

    The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

  5. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors

    PubMed Central

    Radulović, Željko M.; Porter, Lindsay M.; Kim, Tae K.; Bakshi, Mariam; Mulenga, Albert

    2015-01-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in series of assays to obtain further insight into role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both, males and females, secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick-feeding site. Majority of tick-borne pathogens are transmitted 48 hours after the tick has attached. Thus, demonstrated antigenicity and secretion into the host within 24–48 hours of the tick starting to feed makes AamIGFBP-rP1 an attractive target for anti-tick vaccine development. PMID:26108887

  6. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

    PubMed

    Radulović, Ž M; Porter, L M; Kim, T K; Bakshi, M; Mulenga, A

    2015-10-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development. © 2015 The Royal Entomological Society.

  7. Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein‡

    PubMed Central

    Zhang, Lei; Andersen, Eskil M.E.; Khajo, Abdelahad; Magliozzo, Richard S.; Koder, Ronald L.

    2013-01-01

    We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins. PMID:23249163

  8. Binding characteristics of radioiodinated crystal - induced chemotactic factor to human neutrophils

    SciTech Connect

    Spilberg, I.; Mehta, J.

    1984-12-01

    The binding of radiolabeled crystal-induced chemotactic factor (CCF) to human neutrophils is characterized. Binding of /sup 125/I-CCF to the cells was higher at 4/sup 0/C than at 24/sup 0/ or 37/sup 0/C and was found to be independent of Ca/sup 2 +/ and Mg/sup 2 +/ ion concentration. The binding showed a pH optimum of 6.0. Tosylamido phenylethyl chloromethyl ketone at 100 ..mu..mol/L concentration inhibited 20% of /sup 125/I-CCF binding, but phenylmethylsulfonyl fluoride at 200 ..mu..mol/L had no effect. Approximately 50% of the cell-associated /sup 125/I-CCF was released after treatment with proteases. The nonspecific uptake by the cells, as measured by the uptake of /sup 3/H-sucrose and /sup 14/C-inulin in the presence of CCF, was negligible. After the steady-state binding of /sup 125/I-CCF to the cells, approx. 15% of the cell-associated radioactivity at 4/sup 0/C and 40% to 50% at 24/sup 0/ and 37/sup 0/C was released into the medium after 60 minutes of incubation in medium alone. Dissociation of the radioactive material was not affected by the presence of tosylamido phenylethyl chloromethyl ketone or phenanthroline in the media. The dissociated material was determined to be degraded /sup 125/I-CCF, suggesting that degradation of /sup 125/I-CCF occurs after binding to its specific receptor on the human neutrophil. 22 references, 3 figures, 2 tables.

  9. Discovering transcription factor regulatory targets using gene expression and binding data.

    PubMed

    Maienschein-Cline, Mark; Zhou, Jie; White, Kevin P; Sciammas, Roger; Dinner, Aaron R

    2012-01-15

    Identifying the target genes regulated by transcription factors (TFs) is the most basic step in understanding gene regulation. Recent advances in high-throughput sequencing technology, together with chromatin immunoprecipitation (ChIP), enable mapping TF binding sites genome wide, but it is not possible to infer function from binding alone. This is especially true in mammalian systems, where regulation often occurs through long-range enhancers in gene-rich neighborhoods, rather than proximal promoters, preventing straightforward assignment of a binding site to a target gene. We present EMBER (Expectation Maximization of Binding and Expression pRofiles), a method that integrates high-throughput binding data (e.g. ChIP-chip or ChIP-seq) with gene expression data (e.g. DNA microarray) via an unsupervised machine learning algorithm for inferring the gene targets of sets of TF binding sites. Genes selected are those that match overrepresented expression patterns, which can be used to provide information about multiple TF regulatory modes. We apply the method to genome-wide human breast cancer data and demonstrate that EMBER confirms a role for the TFs estrogen receptor alpha, retinoic acid receptors alpha and gamma in breast cancer development, whereas the conventional approach of assigning regulatory targets based on proximity does not. Additionally, we compare several predicted target genes from EMBER to interactions inferred previously, examine combinatorial effects of TFs on gene regulation and illustrate the ability of EMBER to discover multiple modes of regulation. All code used for this work is available at http://dinner-group.uchicago.edu/downloads.html.

  10. Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

    PubMed Central

    Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

    2014-01-01

    Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

  11. Sprouty 2 binds ESCRT-II factor Eap20 and facilitates HIV-1 gag release.

    PubMed

    Medina, G N; Ehrlich, L S; Chen, M H; Khan, M B; Powell, M D; Carter, C A

    2011-07-01

    The four ESCRT (endocytic sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) normally operate sequentially in the trafficking of cellular cargo. HIV-1 Gag trafficking and release as virus-like particles (VLPs) require the participation of ESCRTs; however, its use of ESCRTs is selective and nonsequential. Specifically, Gag trafficking to release sites on the plasma membrane does not require ESCRT-0 or -II. It is known that a bypass of ESCRT-0 is achieved by the direct linkage of the ESCRT-I component, Tsg101, to the primary L domain motif (PTAP) in Gag and that bypass of ESCRT-II is achieved by the linkage of Gag to ESCRT-III through the adaptor protein Alix. However, the mechanism by which Gag suppresses the interaction of bound ESCRT-I with ESCRT-II is unknown. Here we show (i) that VLP release requires the steady-state level of Sprouty 2 (Spry2) in COS-1 cells, (ii) that Spry2 binds the ESCRT-II component Eap20, (iii) that binding Eap20 permits Spry2 to disrupt ESCRT-I interaction with ESCRT-II, and (iv) that coexpression of Gag with a Spry2 fragment that binds Eap20 increases VLP release. Spry2 also facilitated release of P7L-Gag (i.e., release in the absence of Tsg101 binding). In this case, rescue required the secondary L domain (YPX(n)L) in HIV-1 Gag that binds Alix and the region in Spry2 that binds Eap20. The results identify Spry2 as a novel cellular factor that facilitates release driven by the primary and secondary HIV-1 Gag L domains.

  12. Transcription factor binding site analysis identifies FOXO transcription factors as regulators of the cutaneous wound healing process.

    PubMed

    Roupé, Karl Markus; Veerla, Srinivas; Olson, Joshua; Stone, Erica L; Sørensen, Ole E; Hedrick, Stephen M; Nizet, Victor

    2014-01-01

    The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.

  13. Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone:Receptor Binding Specificity.

    PubMed

    Křížková, Květoslava; Chrudinová, Martina; Povalová, Anna; Selicharová, Irena; Collinsová, Michaela; Vaněk, Václav; Brzozowski, Andrzej M; Jiráček, Jiří; Žáková, Lenka

    2016-05-31

    Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain.

  14. Atrial natriuretic factor mRNA and binding sites in the adrenal gland.

    PubMed Central

    Nunez, D J; Davenport, A P; Brown, M J

    1990-01-01

    The factor inhibiting aldosterone secretion produced by the adrenal medulla may be atrial natriuretic factor (ANF), since the latter abolishes aldosterone release in response to a number of secretagogues, including angiotensin II and K+. In this study we have shown that cells in the adrenal medulla contain ANF mRNA and therefore have the potential to synthesize this peptide. The presence of binding sites for ANF predominantly in the adrenal zona glomerulosa suggests that, if ANF is synthesized in the medulla and transferred to the cortex, it may affect mineralocorticoid status. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2146954

  15. αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

    PubMed Central

    Hansen, Scott D.; Kwiatkowski, Adam V.; Ouyang, Chung-Yueh; Liu, HongJun; Pokutta, Sabine; Watkins, Simon C.; Volkmann, Niels; Hanein, Dorit; Weis, William I.; Mullins, R. Dyche; Nelson, W. James

    2013-01-01

    The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays. PMID:24068324

  16. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50)--DNA binding.

    PubMed

    Pande, Vineet; Sharma, Rakesh K; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J

    2003-12-01

    Nuclear Factor-kappa B (NF-kappaB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IkappaB family of proteins. NF-kappaB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-kappaB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IkappaB, leading to the release of free NF-kappaB. This free NF-kappaB then binds to its target sites (KB sites in the DNA) to initiate transcription. These kappaB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-kappaB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-kappaB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we

  17. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50) DNA binding

    NASA Astrophysics Data System (ADS)

    Pande, Vineet; Sharma, Rakesh K.; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J.

    2003-12-01

    Nuclear Factor-kappa B (NF-κB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IκB family of proteins. NF-κB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-κB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IκB, leading to the release of free NF-κB. This free NF-κB then binds to its target sites (κB sites in the DNA) to initiate transcription. These κB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-κB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-κB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we conclude that the principal

  18. Evolution of DNA-Binding Sites of a Floral Master Regulatory Transcription Factor

    PubMed Central

    Muiño, Jose M.; de Bruijn, Suzanne; Pajoro, Alice; Geuten, Koen; Vingron, Martin; Angenent, Gerco C.; Kaufmann, Kerstin

    2016-01-01

    Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BSs) among plant species have not yet been experimentally determined. Here, we performed comparative ChIP (chromatin immunoprecipitation)-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: Arabidopsis thaliana and A. lyrata which have very similar floral organ morphology. We found that BS conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor and explores factors affecting this phenomenon. PMID:26429922

  19. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

    PubMed

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-09-15

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.

  20. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

    PubMed Central

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-01-01

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

  1. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    NASA Astrophysics Data System (ADS)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-07-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

  2. Mapping genome-wide transcription-factor binding sites using DAP-seq.

    PubMed

    Bartlett, Anna; O'Malley, Ronan C; Huang, Shao-Shan Carol; Galli, Mary; Nery, Joseph R; Gallavotti, Andrea; Ecker, Joseph R

    2017-08-01

    To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF-DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.

  3. Detection of active transcription factor binding sites with the combination of DNase hypersensitivity and histone modifications.

    PubMed

    Gusmao, Eduardo G; Dieterich, Christoph; Zenke, Martin; Costa, Ivan G

    2014-11-15

    The identification of active transcriptional regulatory elements is crucial to understand regulatory networks driving cellular processes such as cell development and the onset of diseases. It has recently been shown that chromatin structure information, such as DNase I hypersensitivity (DHS) or histone modifications, significantly improves cell-specific predictions of transcription factor binding sites. However, no method has so far successfully combined both DHS and histone modification data to perform active binding site prediction. We propose here a method based on hidden Markov models to integrate DHS and histone modifications occupancy for the detection of open chromatin regions and active binding sites. We have created a framework that includes treatment of genomic signals, model training and genome-wide application. In a comparative analysis, our method obtained a good trade-off between sensitivity versus specificity and superior area under the curve statistics than competing methods. Moreover, our technique does not require further training or sequence information to generate binding location predictions. Therefore, the method can be easily applied on new cell types and allow flexible downstream analysis such as de novo motif finding. Our framework is available as part of the Regulatory Genomics Toolbox. The software information and all benchmarking data are available at http://costalab.org/wp/dh-hmm. ivan.costa@rwth-aachen.de or eduardo.gusmao@rwth-aachen.de Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Peptide Chain Termination: Effect of Protein S on Ribosomal Binding of Release Factors

    PubMed Central

    Goldstein, J. L.; Caskey, C. T.

    1970-01-01

    The protein factor S, previously shown to stimulate polypeptide chain termination in bacterial extracts, has two effects upon the complex formed between ribosomes, release factor, and terminator (trinucleotide) codon: (1) in the absence of GTP or GDP, S stimulates formation of an [R·UAA·ribosome] intermediate, and (2) in the presence of GTP or GDP, S participates in dissociation of this intermediate. Factor S can stimulate fMet release from [fMet-tRNAf·AUG·ribosome] intermediates in either the presence or absence of GTP or GDP. A model is proposed which relates the in vitro effects of S ± GTP (or GDP) on fMet release to the effects of S ± GTP (or GDP) on the binding and dissociation of R factor from ribosomes. PMID:5289007

  5. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    SciTech Connect

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  6. Arabidopsis Sigma Factor Binding Proteins Are Activators of the WRKY33 Transcription Factor in Plant Defense[W

    PubMed Central

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-01-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif–containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens. PMID:21990940

  7. ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-COP to Golgi membranes.

    PubMed Central

    Donaldson, J G; Cassel, D; Kahn, R A; Klausner, R D

    1992-01-01

    The coatomer is a cytosolic protein complex that reversibly associates with Golgi membranes and is implicated in modulating Golgi membrane transport. The association of beta-COP, a component of coatomer, with Golgi membranes is enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable analogue of GTP, and by a mixture of aluminum and fluoride ions (Al/F). Here we show that the ADP-ribosylation factor (ARF) is required for the binding of beta-COP. Thus, beta-COP contained in a coatomer fraction that has been resolved from ARF does not bind to Golgi membranes, whereas binding can be reconstituted by the addition of recombinant ARF. Furthermore, an N-terminal peptide of ARF, which blocks ARF binding to Golgi membranes, inhibits GTP[gamma S]- as well as the Al/F-enhanced binding of beta-COP. We show that Golgi coat protein binding involves a sequential reaction where an initial interaction of ARF and GTP[gamma S] with the membrane allows subsequent binding of beta-COP to take place in the absence of free ARF and GTP[gamma S]. The fungal metabolite brefeldin A, which is known to prevent the association of coat proteins with Golgi membrane, is shown to exert this effect by interfering with the initial ARF-membrane interaction step. Images PMID:1631136

  8. Phytochrome B inhibits binding of Phytochrome-Interacting Factors to their target promoters

    PubMed Central

    Park, Eunae; Park, Jeongmoo; Kim, Junghyun; Nagatani, Akira; Lagarias, J. Clark; Choi, Giltsu

    2012-01-01

    Summary Phytochromes are red and far-red light receptors in plants that mediate critical responses to light throughout the life cycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome-interacting factors (PIFs) for degradation within the nucleus. It is not known, however, if protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use ChIP analysis to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N-terminal fragment of phyB (NG-GUS-NLS; NGB) also inhibits the binding of PIF3 to its target promoters. Unlike the full-length phyB, however, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIFs’ binding to DNA. We further show that Pfr forms of both full-length phyB and NGB inhibit the DNA binding of PIF1 and PIF3 in vitro. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes, sequestration and protein degradation. PMID:22849408

  9. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells

    PubMed Central

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation. PMID:26941778

  10. Biophysical Models of Evolution: Application to Transcription Factor Binding Sites in Yeast

    NASA Astrophysics Data System (ADS)

    Manhart, Michael; Haldane, Allan; Morozov, Alexandre

    2012-02-01

    There has been growing interest in understanding the physical driving forces of evolution at the molecular scale, in particular how biophysics determines the fitness landscapes that shape the evolution of DNA and proteins. To that end we study a model of molecular evolution that explicitly incorporates the underlying biophysics. Using population genetics, we derive a steady-state distribution of monomorphic populations evolving on an arbitrary fitness landscape. Compared to previous studies, we find this result is universal for a large class of population models and fully incorporates both stochastic effects and strong natural selection. This distribution can then be used to infer the underlying fitness landscape from genomic data. This model can be applied to a variety of systems, but we focus on transcription factor binding sites, which play a crucial role in gene regulatory networks. Since these sites must be bound for successful gene regulation, we consider a simple thermodynamic model of fitness as a function of the free energy for binding a transcription factor at the site. Using empirical energy matrices and genome-wide sets of binding sites from the yeast Saccharomyces cerevisiae, we use this model to infer the role of DNA-protein interaction physics in evolution.

  11. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

    PubMed

    Tabib-Salazar, Aline; Liu, Bing; Doughty, Philip; Lewis, Richard A; Ghosh, Somadri; Parsy, Marie-Laure; Simpson, Peter J; O'Dwyer, Kathleen; Matthews, Steve J; Paget, Mark S

    2013-06-01

    RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

  12. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

    PubMed Central

    Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  13. Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

    PubMed

    Avin, Brittany A; Umbricht, Christopher B; Zeiger, Martha A

    2016-12-01

    The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.

  14. Evans Blue is an inhibitor of nuclear factor-kappa B (NF-kappaB)-DNA binding</