Differential effects of somatostatin on circulating tissue factor procoagulant activity and protein.

PubMed

Boden, Guenther; Vaidyula, Vijender; Homko, Carol; Mozzoli, Maria; Rao, A Koneti

2007-05-01

The tissue factor (TF) pathway is the primary mechanism for initiation of blood coagulation. Circulating blood contains TF, which originates mainly from monocytes and is thrombogenic. The presence of somatostatin (SMS) receptors on monocytes suggests the possibility that SMS may regulate TF synthesis and/or release. Circulating TF procoagulant activity (TF-PCA), factor VIIa activity (FVIIa; clotting assays), TF antigen (TF-Ag; ELISA), prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complexes (ELISAs), CD40 ligand expression on platelets, and monocyte-platelet aggregates (flow cytometry) were determined in blood from normal volunteers undergoing 24 h of basal glucose/basal insulin (BG/BI) clamps and high-glucose/high-insulin (HG/HI) clamps with and without SMS. Infusions of SMS under basal conditions (BG/BI) raised TF-PCA 1.8-fold (P < 0.03), TF-Ag 2.3-fold (P < 0.001), and TF expression on monocytes by 36% (P < 0.001) and decreased plasma levels of FVIIa by 30% (P < 0.001). Infusion of SMS reduced the 8.6-fold HG/HI-induced increase in TF-Ag by 26% and the 8.6-fold increase in TF-PCA by 100%. SMS also prevented the 60% increase in TF expression on monocytes, the 2.2-fold increase in F1.2, the 40% increase in CD40L expression on platelets, and the 17% increase in monocyte-platelet aggregates seen during HG/HI. We conclude that SMS completely prevented HG/HI-induced TF activation in normal volunteers and may be of use to reduce the procoagulant state and acute vascular events in hyperinsulinemic insulin-resistant patients with type 2 diabetes.

Quantitative in vivo imaging of tissue factor expression in glioma using dynamic contrast-enhanced MRI derived parameters.

PubMed

Chen, Xiao; Xie, Tian; Fang, Jingqin; Xue, Wei; Tong, Haipeng; Kang, Houyi; Wang, Sumei; Yang, Yizeng; Xu, Minhui; Zhang, Weiguo

2017-08-01

Tissue Factor (TF) has been well established in angiogenesis, invasion, metastasis, and prognosis in glioma. A noninvasive assessment of TF expression status in glioma is therefore of obvious clinical relevance. Dynamic contrast-enhanced (DCE) MRI parameters have been used to evaluate microvascular characteristics and predict molecular expression status in tumors. Our aim is to investigate whether quantitative DCE-MRI parameters could assess TF expression in glioma. Thirty-two patients with histopathologically diagnosed supratentorial glioma who underwent DCE-MRI were retrospectively recruited. Extended Tofts linear model was used for DCE-MRI post-processing. Hot-spot, whole tumor cross-sectional approaches, and histogram were used for analysis of model based parameters. Four serial paraffin sections of each case were stained with TF, CD105, CD34 and α-Sooth Muscle Actin, respectively for evaluating the association of TF and microvascular properties. Pearson correlation was performed between percentage of TF expression area and DCE-MRI parameters, multiple microvascular indexes. Volume transfer constant (K trans ) hot-spot value best correlated with TF (r=0.886, p<0.001), followed by 90th percentile K trans value (r=0.801, p<0.001). Moreover, histogram analysis of K trans value demonstrated that weak TF expression was associated with less heterogeneous and positively skewed distribution. Finally, pathology analysis revealed TF was associated with glioma grade and significantly correlated with these two dynamic angiogenic indexes which could be used to explain the strong correlation between K trans and TF expression. Our results indicate that K trans may serve as a potential clinical imaging biomarker to predict TF expression status preoperatively in gliomas. Copyright © 2017 Elsevier B.V. All rights reserved.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption.

PubMed

Lockwood, C J; Paidas, M; Murk, W K; Kayisli, U A; Gopinath, A; Huang, S J; Krikun, G; Schatz, F

2009-11-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Among the cell types at the maternal fetal interface at term, TF expression is highest in decidual cells indicating that this TF meets the hemostatic demands of labor and delivery. TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggesting that the maintenance of elevated circulating progesterone provides hemostatic protection and that abruption-generated thrombin acts in an autocrine/paracrine fashion on decidual cells to promote hemostasis via enhanced TF expression.

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy.

PubMed

Jacobsen, Christine; Oechsle, Karin; Hauschild, Jessica; Steinemann, Gustav; Spath, Brigitte; Bokemeyer, Carsten; Ruf, Wolfram; Honecker, Friedemann; Langer, Florian

2015-09-01

Patients with germ cell tumors (GCTs) receiving cisplatin-based chemotherapy are at increased risk of thrombosis, but the underlying cellular and molecular mechanisms remain obscure. To study baseline tissue factor (TF) expression by GCT cell lines and its modulation by cisplatin treatment. TF expression was assessed by single-stage clotting and thrombin generation assay, flow cytometry, ELISA, and Western blot analysis. Cell cycle analysis and detection of phosphatidylserine (PS) membrane exposure were carried out by flow cytometry. TF mRNA was analyzed by quantitative RT-PCR. Significant expression of TF-specific procoagulant activity (PCA) was detected on three non-seminoma (NT2, 2102Ep, NCCIT) and one seminoma cell line (TCam-2). Treatment with 0.4μM cisplatin (corresponding to the IC50) for 48hrs increased TF PCA on NT2 cells 3-fold, an effect that was largely independent of PS exposure and that could not be explained by translocation of active TF from intracellular storage pools. Cisplatin-induced TF PCA expression in NT2 cells did not occur before 12hrs, but was steady thereafter and accompanied by a 2-fold increase in total and surface-located TF antigen. Importantly, increased TF gene transcription or production and release of an intermediate factor were not involved in this process. Cell cycle analysis suggested that cisplatin-induced G2/M arrest resulted in an accumulation of procoagulant TF on the membrane surface of NT2 cells. In addition to induction of apoptosis/necrosis with PS-mediated activation of preformed TF, cisplatin may alter the procoagulant phenotype of GCT cells through an increase in total cellular TF antigen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hypoxia and PGE2 Regulate MiTF-CX During Cervical Ripening

PubMed Central

Hari Kishore, Annavarapu; Li, Xiang-Hong

2012-01-01

The mechanisms by which the cervix remains closed during the massive uterine expansion of pregnancy are unknown. IL-8 is important for recruitment of immune cells into the cervical stroma, matrix remodeling, and dilation of the cervix during labor. Previously, we have shown that several cytokine genes transcriptionally repressed in the cervix during gestation are activated during cervical ripening and dilation. IL-8 gene expression is repressed in cervical stromal cells during pregnancy by the transcription factor microphthalmia-associated transcription factor (MiTF-CX). Here, we tested the hypothesis that hypoxia and the transcription factor hypoxia inducible factor-1α (HIF-1α) may regulate MiTF-CX and cervical ripening. Using tissues from women during pregnancy before and after cervical ripening, we show that, during cervical ripening, HIF-1α was stabilized and relocalized to the nucleus. Further, we found that hypoxia and two hypoxia mimetics that stabilize HIF-1α activated the transcriptional repressor differentiated embryo chondrocyte-expressed gene 1, which bound to sites in the MiTF-CX promoter crucial for its positive autoregulation. Ectopic overexpression of MiTF-CX abrogated hypoxia-induced up-regulation of IL-8 gene expression. We also show that activation of HIF-1α induced cyclooxygenase-2 and that prostaglandin E2 repressed MiTF-CX. We conclude that hypoxia and stabilization of the transcription factor HIF-1α result in up-regulation of differentiated embryo chondrocyte-expressed gene 1, loss of MiTF, and absence of MiTF binding to the IL-8 promoter, which in turn leads to up-regulation of IL-8 gene expression. Hypoxia also up-regulated cyclooxygenase-2, leading to prostaglandin E2-mediated loss of MiTF in cervical stromal cells. The results support a pivotal role for hypoxia and HIF-1α in the cervical ripening process during pregnancy. PMID:23144021

Identification and expression of the tig gene coding for trigger factor from psychrophilic bacteria with no information of genome sequence available.

PubMed

Lee, Kyunghee; Choi, Hyojung; Im, Hana

2009-08-01

Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis-trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15 degrees C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4 degrees C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption

PubMed Central

Lockwood, C.J.; Paidas, M.; Murk, W.K.; Kayisli, U.A.; Gopinath, A.; Krikun, G.; Huang, S.J.; Schatz, F.

2009-01-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Our recent observations that: 1) among the cell types at the maternal fetal interface at term TF expression is highest in decidual cells indicates that this TF meets the hemostatic demands of labor and delivery; 2) TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggest that maintenance of elevated circulating progesterone at term provides hemostatic protection, whereas abruption-generated thrombin can act in autocrine/paracrine fashion on DCs to promote hemostasis via enhanced TF expression. PMID:19720393

p27{sup Kip1} inhibits tissue factor expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Breitenstein, Alexander, E-mail: alexander.breitenstein@usz.ch; Cardiovascular Research, Physiology Institute, University of Zurich; Center for Integrative Human Physiology

2013-10-04

Highlights: •p27{sup Kip1}regulates the expression of tissue factor at the transcriptional level. •This inhibitory effect of p27{sup Kip1} is independently of its cell regulatory action. •The current study provides new insights into a pleiotrophic function of p27{sup Kip1}. -- Abstract: Background: The cyclin-dependent kinase inhibitor (CDKI) p27{sup Kip1} regulates cell proliferation and thus inhibits atherosclerosis and vascular remodeling. Expression of tissue factor (TF), the key initator of the coagulation cascade, is associated with atherosclerosis. Yet, it has not been studied whether p27{sup Kip1} influences the expression of TF. Methods and results: p27{sup Kip1} overexpression in human aortic endothelial cells wasmore » achieved by adenoviral transfection. Cells were rendered quiescent for 24 h in 0.5% fetal-calf serum. After stimulation with TNF-α (5 ng/ml), TF protein expression and activity was significantly reduced (n = 4; P < 0.001) in cells transfected with p27{sup Kip1}. In line with this, p27{sup Kip1} overexpression reduced cytokine-induced TF mRNA expression (n = 4; P < 0.01) and TF promotor activity (n = 4; P < 0.05). In contrast, activation of the MAP kinases p38, ERK and JNK was not affected by p27{sup Kip1} overexpression. Conclusion: This in vitro study suggests that p27{sup Kip1} inhibits TF expression at the transcriptional level. These data indicate an interaction between p27{sup Kip1} and TF in important pathological alterations such as atherosclerosis and vascular remodeling.« less

Targeting tissue factor as a novel therapeutic oncotarget for eradication of cancer stem cells isolated from tumor cell lines, tumor xenografts and patients of breast, lung and ovarian cancer.

PubMed

Hu, Zhiwei; Xu, Jie; Cheng, Jijun; McMichael, Elizabeth; Yu, Lianbo; Carson, William E

2017-01-03

Targeting cancer stem cell (CSC) represents a promising therapeutic approach as it can potentially fight cancer at its root. The challenge is to identify a surface therapeutic oncotarget on CSC. Tissue factor (TF) is known as a common yet specific surface target for cancer cells and tumor neovasculature in several solid cancers. However, it is unknown if TF is expressed by CSCs. Here we demonstrate that TF is constitutively expressed on CD133 positive (CD133+) or CD24-CD44+ CSCs isolated from human cancer cell lines, tumor xenografts from mice and breast tumor tissues from patients. TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy) and fVII-IgG1Fc (Immunoconjugate or ICON for immunotherapy), can eradicate CSC via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively. In conclusion, these results demonstrate that TF is a novel surface therapeutic oncotarget for CSC, in addition to cancer cell TF and tumor angiogenic vascular endothelial TF. Moreover, this research highlights that TF-targeting therapeutics can effectively eradicate CSCs, without drug resistance, isolated from breast, lung and ovarian cancer with potential to translate into other most commonly diagnosed solid cancer, in which TF is also highly expressed.

PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcγRI

PubMed Central

Glover, Sam L.; Jonas, William; McEachron, Troy; Pawlinski, Rafal; Arepally, Gowthami M.; Key, Nigel S.; Mackman, Nigel

2012-01-01

Heparin-induced thrombocytopenia (HIT) is a potentially devastating form of drug-induced thrombocytopenia that occurs in patients receiving heparin for prevention or treatment of thrombosis. Patients with HIT develop autoantibodies to the platelet factor 4 (PF4)/heparin complex, which is termed the HIT Ab complex. Despite a decrease in the platelet count, the most feared complication of HIT is thrombosis. The mechanism of thrombosis in HIT remains poorly understood. We investigated the effects of the HIT Ab complex on tissue factor (TF) expression and release of TF-positive microparticles in peripheral blood mononuclear cells and monocytes. To model these effects ex vivo, we used a murine mAb specific for the PF4/heparin complex (KKO), as well as plasma from patients with HIT. We found that the HIT Ab complex induced TF expression in monocytes and the release of TF-positive microparticles. Further, we found that induction of TF is mediated via engagement of the FcγRI receptor and activation of the MEK1-ERK1/2 signaling pathway. Our data suggest that monocyte TF may contribute to the development of thrombosis in patients with HIT. PMID:22394597

Tissue Factor Inflammatory Response Regulated by Promoter Genotype and p38 MAPK in Neonatal vs. Adult Microvascular Endothelial Cells

PubMed Central

Buzby, Jeffrey S.; Williams, Shirley A.; Imfeld, Karen L.; Kunicki, Thomas J.; Nugent, Diane J.

2014-01-01

Objective and design Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18 base pair deletion (D) or insertion (I) at -1208. We sought to determine the relationship between these haplotypes and interleukin-1 (IL-1α)-induced TF expression in neonatal versus adult HMVEC. Results IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed 3-fold higher levels of TF mRNA, 2-fold higher TF protein, and 4-fold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. Conclusions Up-regulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants. PMID:24385191

Micro-RNA-126 Reduces the Blood Thrombogenicity in Diabetes Mellitus via Targeting of Tissue Factor.

PubMed

Witkowski, Marco; Weithauser, Alice; Tabaraie, Termeh; Steffens, Daniel; Kränkel, Nicolle; Witkowski, Mario; Stratmann, Bernd; Tschoepe, Diethelm; Landmesser, Ulf; Rauch-Kroehnert, Ursula

2016-06-01

Diabetes mellitus involves vascular inflammatory processes and is a main contributor to cardiovascular mortality. Notably, heightened levels of circulating tissue factor (TF) account for the increased thrombogenicity and put those patients at risk for thromboembolic events. Here, we sought to investigate the role of micro-RNA (miR)-driven TF expression and thrombogenicity in diabetes mellitus. Plasma samples of patients with diabetes mellitus were analyzed for TF protein and activity as well as miR-126 expression before and after optimization of the antidiabetic treatment. We found low miR-126 levels to be associated with markedly increased TF protein and TF-mediated thrombogenicity. Reduced miR-126 expression was accompanied by increased vascular inflammation as evident from the levels of vascular adhesion molecule-1 and fibrinogen, as well as leukocyte counts. With optimization of the antidiabetic treatment miR-126 levels increased and thrombogenicity was reduced. Using a luciferase reporter system, we demonstrated miR-126 to directly bind to the F3-3'-untranslated region, thereby reducing TF expression both on mRNA and on protein levels in human microvascular endothelial cells as well as TF mRNA and activity in monocytes. Circulating miR-126 exhibits antithrombotic properties via regulating post-transcriptional TF expression, thereby impacting the hemostatic balance of the vasculature in diabetes mellitus. © 2016 The Authors.

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

PubMed

Lindholm, Paul F; Lu, Yi; Adley, Brian P; Vladislav, Tudor; Jovanovic, Borko; Sivapurapu, Neela; Yang, Ximing J; Kajdacsy-Balla, André

2010-11-01

Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

Transposon integration enhances expression of stress response genes.

PubMed

Feng, Gang; Leem, Young-Eun; Levin, Henry L

2013-01-01

Transposable elements possess specific patterns of integration. The biological impact of these integration profiles is not well understood. Tf1, a long-terminal repeat retrotransposon in Schizosaccharomyces pombe, integrates into promoters with a preference for the promoters of stress response genes. To determine the biological significance of Tf1 integration, we took advantage of saturated maps of insertion activity and studied how integration at hot spots affected the expression of the adjacent genes. Our study revealed that Tf1 integration did not reduce gene expression. Importantly, the insertions activated the expression of 6 of 32 genes tested. We found that Tf1 increased gene expression by inserting enhancer activity. Interestingly, the enhancer activity of Tf1 could be limited by Abp1, a host surveillance factor that sequesters transposon sequences into structures containing histone deacetylases. We found the Tf1 promoter was activated by heat treatment and, remarkably, only genes that themselves were induced by heat could be activated by Tf1 integration, suggesting a synergy of Tf1 enhancer sequence with the stress response elements of target promoters. We propose that the integration preference of Tf1 for the promoters of stress response genes and the ability of Tf1 to enhance the expression of these genes co-evolved to promote the survival of cells under stress.

Transposon integration enhances expression of stress response genes

PubMed Central

Feng, Gang; Leem, Young-Eun; Levin, Henry L.

2013-01-01

Transposable elements possess specific patterns of integration. The biological impact of these integration profiles is not well understood. Tf1, a long-terminal repeat retrotransposon in Schizosaccharomyces pombe, integrates into promoters with a preference for the promoters of stress response genes. To determine the biological significance of Tf1 integration, we took advantage of saturated maps of insertion activity and studied how integration at hot spots affected the expression of the adjacent genes. Our study revealed that Tf1 integration did not reduce gene expression. Importantly, the insertions activated the expression of 6 of 32 genes tested. We found that Tf1 increased gene expression by inserting enhancer activity. Interestingly, the enhancer activity of Tf1 could be limited by Abp1, a host surveillance factor that sequesters transposon sequences into structures containing histone deacetylases. We found the Tf1 promoter was activated by heat treatment and, remarkably, only genes that themselves were induced by heat could be activated by Tf1 integration, suggesting a synergy of Tf1 enhancer sequence with the stress response elements of target promoters. We propose that the integration preference of Tf1 for the promoters of stress response genes and the ability of Tf1 to enhance the expression of these genes co-evolved to promote the survival of cells under stress. PMID:23193295

Absence of tissue factor is characteristic of lymphoid malignancies of both T- and B-cell origin

PubMed Central

Cesarman-Maus, Gabriela; Braggio, Esteban; Lome-Maldonado, Carmen; Morales-Leyte, Ana Lilia; Fonseca, Rafael

2014-01-01

Summary Background Thrombosis is a marker of poor prognosis in individuals with solid tumors. The expression of tissue factor (TF) on the cell surface membrane of malignant cells is a pivotal molecular link between activation of coagulation, angiogenesis, metastasis, aggressive tumor behavior and poor survival. Interestingly, thrombosis is associated with shortened survival in solid, but not in lymphoid neoplasias. Objectives We sought to study whether the lack of impact of thrombosis on survival in lymphoid neoplasias could be due to a lack of tumor-derived TF expression. Methods We analyzed TF gene (F3) expression in lymphoid (N=114), myeloid (N=49) and solid tumor (N=856) cell lines using the publicly available dataset from the Broad-Novartis Cancer Cell Line Encyclopedia (http://www.broadinstitute.org/ccle/home), and in 90 patient-derived lymphoma samples. TF protein expression was studied by immunohistochemistry (IHC). Results In sharp contrast to wide F3 expression in solid tumors (74.2%), F3 was absent in all low and high grade T- and B-cell lymphomas, and in most myeloid tumors, except for select acute myeloid leukemias with monocytic component. IHC confirmed the absence of TF protein in all indolent and high-grade B-cell (0/90) and T-cell (0/20) lymphomas, and acute leukemias (0/11). Conclusions We show that TF in lymphomas does not derive from the malignant cells, since these do not express either F3 or TF protein. Therefore, it is unlikely that thrombosis in patients with lymphoid neoplasms is secondary to tumor-derived tissue factor. PMID:24491425

Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

PubMed

Amirkhosravi, A; Meyer, T; Warnes, G; Amaya, M; Malik, Z; Biggerstaff, J P; Siddiqui, F A; Sherman, P; Francis, J L

1998-10-01

Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear.

Self-production of tissue factor-coagulation factor VII complex by ovarian cancer cells.

PubMed

Yokota, N; Koizume, S; Miyagi, E; Hirahara, F; Nakamura, Y; Kikuchi, K; Ruf, W; Sakuma, Y; Tsuchiya, E; Miyagi, Y

2009-12-15

Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis. In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT-PCR, western blotting and flow cytometry. Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia. These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.

Tissue Factor-Factor VIIa Complex Triggers Protease Activated Receptor 2-Dependent Growth Factor Release and Migration in Ovarian Cancer

PubMed Central

Chanakira, Alice; Westmark, Pamela R.; Ong, Irene M.; Sheehan, John P.

2017-01-01

Objective Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC. Methods TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expression were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists. Results Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4–10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present. Conclusions Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation. PMID:28148395

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

PubMed

Luna-Zurita, Luis; Stirnimann, Christian U; Glatt, Sebastian; Kaynak, Bogac L; Thomas, Sean; Baudin, Florence; Samee, Md Abul Hassan; He, Daniel; Small, Eric M; Mileikovsky, Maria; Nagy, Andras; Holloway, Alisha K; Pollard, Katherine S; Müller, Christoph W; Bruneau, Benoit G

2016-02-25

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Evening primrose oil or forskolin ameliorates celecoxib-enhanced upregulation of tissue factor expression in mice subjected to lipopolysaccharide-induced endotoxemia.

PubMed

Mosaad, Sarah M; Zaitone, Sawsan A; Ahmed, Amal A M; Abo-Elmatty, Dina M; El-Baz, Amani A; Moustafa, Yasser M

2017-05-01

Celecoxib, a selective cyclooxygenase-2 inhibitor, produces thrombotic events in patients predisposed to cardiovascular risk factors. One theory reported an increase in endothelial expression of tissue factor (TF) as a predisposing factor. This work explored the effect of evening primrose oil (EPO), a source of prostaglandin E1, and forskolin (a cyclic adenosine monophosphate stimulator) against the prothrombotic effect of celecoxib in mice. Lipopolysaccharide mouse model of endotoxemia was used to induce an upregulation of TF activity. Male mice received celecoxib (25 mg/kg), celecoxib plus EPO, or celecoxib plus forskolin for 4 weeks and then subjected to a prothrombotic challenge in the form of an intraperitoneal injection of lipopolysaccharide. Results showed an increase in plasma TF activity, endothelial TF expression, and thrombin-antithrombin (TAT) but lower antithrombin III (ATIII) level in mice that received celecoxib in comparison to those that received the vehicle. Adding EPO or forskolin to celecoxib regimen significantly decreased the prothrombotic effect of celecoxib. A positive correlation (r = 0.8501) was found between TF activity and TAT. Co-administration of EPO or forskolin decreased the activity of TF and mitigated the prothrombotic effect of celecoxib. Therefore, these combinations may have the utility to abrogate the prothrombotic adverse effect of celecoxib in clinical setting.

The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay.

PubMed

Amirkhosravi, A; Alexander, M; May, K; Francis, D A; Warnes, G; Biggerstaff, J; Francis, J L

1996-01-01

Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean +/- SD) expression of monocyte TF in normal subjects was very low (1.1 +/- 0.95%, Mean Fluorescence [Mean FL] 0.20 +/- 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 +/- 11.2% (Mean FL 0.32 +/- 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

PubMed Central

Gong, Lan-Bo; He, Li; Liu, Yang; Chen, Xue-Qing; Jiang, Bo

2005-01-01

AIM: To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. RESULTS: After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF. PMID:16124058

Substance P - Neurokinin-1 Receptor Interaction Upregulates Monocyte Tissue Factor

PubMed Central

Khan, Mohammad M; Douglas, Steven D; Benton, Tami D

2011-01-01

Monocytes play an important role in hemostasis. In this study, the prothrombotic effects of the neuropeptide substance P (SP) on human monocytes through neurokinin-1 receptor (NK1-R) were characterized. SP upregulated monocyte tissue factor (TF), the major coagulation cascade stimulator, in a concentration and time dependent manner. Specific inhibition of NK1-R completely blocked TF expression. Monocytes stimulated by SP released cytokines and chemokines. When monocytes were stimulated with cytokines or chemokines, TF was expressed by the cytokines (GM-CSF, IFN-γ and TNF-α). Cytokines may play a major role in the mechanism of SP induced monocyte TF expression. NK1-R antagonists (NK1-RA) may have a role in developing novel therapeutic approaches to patients vulnerable to vaso-occlusive disorders. PMID:22115773

Molecular Screening Tools to Study Arabidopsis Transcription Factors

PubMed Central

Wehner, Nora; Weiste, Christoph; Dröge-Laser, Wolfgang

2011-01-01

In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta. PMID:22645547

Tissue factor expression in rheumatoid synovium: a potential role in pannus invasion of rheumatoid arthritis.

PubMed

Chen, Lujun; Lu, Yahua; Chu, Yang; Xie, Jun; Ding, Wen'ge; Wang, Fengming

2013-09-01

Angiogenesis, as well as pannus formation within the joint, plays an important role in the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). Tissue factor (TF), an essential initiator of the extrinsic pathway of blood coagulation, is also involved in the angiogenesis and the pannus formation of RA progression. In the present study, we used immunofluorescence and confocal scanning methods to characterize TF immunolocalization in RA synovium. We showed that positive staining of TF could be immunolocalized in synoviocytes, CD19(+) B cells and CD68(+) macrophages, whereas weak or negative staining of tissue factor could be found in CD34(+) endothelial cells of neo-vessels, CD3(+) T cells and CD14(+) monocytes in RA synovium tissues. Our study demonstrates a detailed local expression of TF in the rheumatoid synovium, and supports the notion that TF, expressed not only by the synoviocytes themselves, but also the infiltrating CD19(+) B cells and CD68(+) macrophages, is involved in the pannus invasion in the progression of rheumatoid arthritis. Copyright © 2013 Elsevier GmbH. All rights reserved.

Expression of Tissue Factor by Melanoma Cells Promotes Efficient Hematogenous Metastasis

NASA Astrophysics Data System (ADS)

Mueller, Barbara M.; Reisfeld, Ralph A.; Edgington, Thomas S.; Ruf, Wolfram

1992-12-01

Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher levels of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence is insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

PubMed

Barbon, Elena; Pignani, Silvia; Branchini, Alessio; Bernardi, Francesco; Pinotti, Mirko; Bovolenta, Matteo

2016-06-24

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

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

Simultaneous expression of tissue factor and tissue factor pathway inhibitor by human monocytes. A potential mechanism for localized control of blood coagulation

PubMed Central

1994-01-01

Cells of monocytic lineage can initiate extravascular fibrin deposition via expression of blood coagulation mediators. This report is about experiments on three mechanisms with the potential to modulate monocyte- initiated coagulation. Monocyte procoagulant activity was examined as a function of lipid cofactor, protein cofactor, and specific inhibitor expression during short-term culture in vitro. Lipid cofactor activity was measured as the initial rate of factor X activation by intrinsic- pathway components, the assembly of which depends on this cofactor. Lipid cofactor activity levels changed by < 30% during 48-h culture. Protein cofactor, i.e., tissue factor (TF) antigen was measured by enzyme immunoassay. It increased from 461 pg/ml to a maximum value of 3,550 pg/ml at 24 h and remained at 70% of this value. Specific TF activity, measured as factor VII-dependent factor X activation rate, decreased from 54 to 18 nM FXa/min between 24 and 48 h. TF activity did not correlate well with either lipid cofactor or TF protein levels. In contrast, the decrease in TF activity coincided in time with maximal expression of tissue factor pathway inhibitor (TFPI) mRNA, which was determined using reverse transcriptase polymerase chain reaction (RT- PCR), and with maximal TFPI protein levels measured by immunoassay. The number of mRNA copies coding for TFPI and TF in freshly isolated blood monocytes were 46 and 20 copies/cells, respectively. These values increased to 220 and 63 copies/cell during short-term cell culture in the presence of endotoxin. Results demonstrate concomitant expression by monocytes of genes coding for both the essential protein cofactor and the specific inhibitor of the extrinsic coagulation pathway. Together with functional and antigenic analyses, they also imply that the initiation of blood clotting by extravascular monocyte/macrophages can be modulated locally by TFPI independently of plasma sources of the inhibitor. PMID:8195712

CisMapper: predicting regulatory interactions from transcription factor ChIP-seq data

PubMed Central

O'Connor, Timothy; Bodén, Mikael

2017-01-01

Abstract Identifying the genomic regions and regulatory factors that control the transcription of genes is an important, unsolved problem. The current method of choice predicts transcription factor (TF) binding sites using chromatin immunoprecipitation followed by sequencing (ChIP-seq), and then links the binding sites to putative target genes solely on the basis of the genomic distance between them. Evidence from chromatin conformation capture experiments shows that this approach is inadequate due to long-distance regulation via chromatin looping. We present CisMapper, which predicts the regulatory targets of a TF using the correlation between a histone mark at the TF's bound sites and the expression of each gene across a panel of tissues. Using both chromatin conformation capture and differential expression data, we show that CisMapper is more accurate at predicting the target genes of a TF than the distance-based approaches currently used, and is particularly advantageous for predicting the long-range regulatory interactions typical of tissue-specific gene expression. CisMapper also predicts which TF binding sites regulate a given gene more accurately than using genomic distance. Unlike distance-based methods, CisMapper can predict which transcription start site of a gene is regulated by a particular binding site of the TF. PMID:28204599

Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expression in vascular cells : A possible mechanism for the cardiovascular benefits associated with moderate consumption of wine.

PubMed

Pendurthi, U R; Williams, J T; Rao, L V

1999-02-01

A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNFalpha mRNA in endothelial cells and of TNFalpha and IL-1beta mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription. However, resveratrol did not significantly alter the binding of the transcription factors c-Fos/c-Jun and c-Rel/p65, the transcription factors required for the induction of TF promoter in both endothelial cells and monocytes. Similarly, resveratrol had no significant effect on the binding of NF-kappaB in endothelial cells stimulated with IL-1beta, TNFalpha, and LPS. Overall, our data show that resveratrol could effectively suppress the aberrant expression of TF and cytokines in vascular cells, but it requires further investigation to understand how resveratrol exerts its inhibitory effect.

Repressor- and Activator-Type Ethylene Response Factors Functioning in Jasmonate Signaling and Disease Resistance Identified via a Genome-Wide Screen of Arabidopsis Transcription Factor Gene Expression[w

PubMed Central

McGrath, Ken C.; Dombrecht, Bruno; Manners, John M.; Schenk, Peer M.; Edgar, Cameron I.; Maclean, Donald J.; Scheible, Wolf-Rüdiger; Udvardi, Michael K.; Kazan, Kemal

2005-01-01

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NAC TF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance. PMID:16183832

Transcriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha

PubMed Central

2013-01-01

Background Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. Results In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. Conclusions The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants. PMID:24365221

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schechter, Melissa E.; Andrade, Bruno B.; He, Tianyu

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. Thus, a better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidencemore » of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor–α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)–related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.« less

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

DOE PAGES

Schechter, Melissa E.; Andrade, Bruno B.; He, Tianyu; ...

2017-08-30

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. Thus, a better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidencemore » of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor–α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)–related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.« less

Coup-TF1 and Coup-TF2 control subtype and laminar identity of MGE-derived neocortical interneurons.

PubMed

Hu, Jia Sheng; Vogt, Daniel; Lindtner, Susan; Sandberg, Magnus; Silberberg, Shanni N; Rubenstein, John L R

2017-08-01

Distinct cortical interneuron (CIN) subtypes have unique circuit functions; dysfunction in specific subtypes is implicated in neuropsychiatric disorders. Somatostatin- and parvalbumin-expressing (SST + and PV + ) interneurons are the two major subtypes generated by medial ganglionic eminence (MGE) progenitors. Spatial and temporal mechanisms governing their cell-fate specification and differential integration into cortical layers are largely unknown. We provide evidence that Coup-TF1 and Coup-TF2 ( Nr2f1 and Nr2f2 ) transcription factor expression in an arc-shaped progenitor domain within the MGE promotes time-dependent survival of this neuroepithelium and the time-dependent specification of layer V SST + CINs. Coup-TF1 and Coup-TF2 autonomously repress PV + fate in MGE progenitors, in part through directly driving Sox6 expression. These results have identified, in mouse, a transcriptional pathway that controls SST-PV fate. © 2017. Published by The Company of Biologists Ltd.

Quantitative PET Imaging of Tissue Factor Expression Using 18F-Labeled Active Site-Inhibited Factor VII.

PubMed

Nielsen, Carsten H; Erlandsson, Maria; Jeppesen, Troels E; Jensen, Mette M; Kristensen, Lotte K; Madsen, Jacob; Petersen, Lars C; Kjaer, Andreas

2016-01-01

Tissue factor (TF) is upregulated in many solid tumors, and its expression is linked to tumor angiogenesis, invasion, metastasis, and prognosis. A noninvasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII is the natural ligand to TF. Here we report the development of a new PET tracer for specific imaging of TF using an (18)F-labeled derivative of factor VII. Active site-inhibited factor VIIa (FVIIai) was obtained by inactivation with phenylalanine-phenylalanine-arginine-chloromethyl ketone. FVIIai was radiolabeled with N-succinimidyl 4-(18)F-fluorobenzoate and purified. The corresponding product, (18)F-FVIIai, was injected into nude mice with subcutaneous human pancreatic xenograft tumors (BxPC-3) and investigated using small-animal PET/CT imaging 1, 2, and 4 h after injection. Ex vivo biodistribution was performed after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by enzyme-linked immunosorbent assay and correlated with the uptake of (18)F-FVIIai in the tumors measured in vivo by PET imaging. The PET images showed high uptake of (18)F-FVIIai in the tumor regions, with a mean uptake of 2.5 ± 0.3 percentage injected dose per gram (%ID/g) (mean ± SEM) 4 h after injection of 7.3-9.3 MBq of (18)F-FVIIai and with an average maximum uptake in the tumors of 7.1 ± 0.7 %ID/g at 4 h. In comparison, the muscle uptake was 0.2 ± 0.01 %ID/g at 4 h. At 4 h, the tumors had the highest uptake of any organ. Blocking with FVIIai significantly reduced the uptake of (18)F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P < 0.001). The uptake of (18)F-FVIIai measured in vivo by PET imaging correlated (r = 0.72, P < 0.02) with TF protein level measured ex vivo. (18)F-FVIIai is a promising PET tracer for specific and noninvasive imaging of tumor TF expression. The tracer merits further development and clinical translation, with potential to become a companion diagnostics for emerging TF-targeted therapies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analyses of the NAC transcription factor gene family in Gossypium raimondii Ulbr.: chromosomal location, structure, phylogeny, and expression patterns.

PubMed

Shang, Haihong; Li, Wei; Zou, Changsong; Yuan, Youlu

2013-07-01

NAC domain proteins are plant-specific transcription factors known to play diverse roles in various plant developmental processes. In the present study, we performed the first comprehensive study of the NAC gene family in Gossypium raimondii Ulbr., incorporating phylogenetic, chromosomal location, gene structure, conserved motif, and expression profiling analyses. We identified 145 NAC transcription factor (NAC-TF) genes that were phylogenetically clustered into 18 distinct subfamilies. Of these, 127 NAC-TF genes were distributed across the 13 chromosomes, 80 (55%) were preferentially retained duplicates located in both duplicated regions and six were located in triplicated chromosomal regions. The majority of NAC-TF genes showed temporal-, spatial-, and tissue-specific expression patterns based on transcriptomic and qRT-PCR analyses. However, the expression patterns of several duplicate genes were partially redundant, suggesting the occurrence of sub-functionalization during their evolution. Based on their genomic organization, we concluded that genomic duplications contributed significantly to the expansion of the NAC-TF gene family in G. raimondii. Comprehensive analysis of their expression profiles could provide novel insights into the functional divergence among members of the NAC gene family in G. raimondii. © 2013 Institute of Botany, Chinese Academy of Sciences.

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1

PubMed Central

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-01-01

AIM: To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes. PMID:17009409

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1.

PubMed

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-10-07

To study the expression of HBV enhancer II by transcription factor COUP-TF1. In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

FANCD2 functions as a critical factor downstream of MiTF to maintain the proliferation and survival of melanoma cells.

PubMed

Bourseguin, Julie; Bonet, Caroline; Renaud, Emilie; Pandiani, Charlotte; Boncompagni, Marina; Giuliano, Sandy; Pawlikowska, Patrycja; Karmous-Benailly, Houda; Ballotti, Robert; Rosselli, Filippo; Bertolotto, Corine

2016-11-09

Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment.

FANCD2 functions as a critical factor downstream of MiTF to maintain the proliferation and survival of melanoma cells

PubMed Central

Bourseguin, Julie; Bonet, Caroline; Renaud, Emilie; Pandiani, Charlotte; Boncompagni, Marina; Giuliano, Sandy; Pawlikowska, Patrycja; Karmous-Benailly, Houda; Ballotti, Robert; Rosselli, Filippo; Bertolotto, Corine

2016-01-01

Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment. PMID:27827420

[siRNA-mediated tissue factor knockdown in porcine neonatal islet cell clusters in vitro].

PubMed

Ji, Ming; Yi, Shounan; Yu, Deling; Wang, Wei

2011-12-01

To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro. Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively. Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS. The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.

Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

PubMed

Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

2004-02-15

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

Stochastic model of transcription factor-regulated gene expression

NASA Astrophysics Data System (ADS)

Karmakar, Rajesh; Bose, Indrani

2006-09-01

We consider a stochastic model of transcription factor (TF)-regulated gene expression. The model describes two genes, gene A and gene B, which synthesize the TFs and the target gene proteins, respectively. We show through analytic calculations that the TF fluctuations have a significant effect on the distribution of the target gene protein levels when the mean TF level falls in the highest sensitive region of the dose-response curve. We further study the effect of reducing the copy number of gene A from two to one. The enhanced TF fluctuations yield results different from those in the deterministic case. The probability that the target gene protein level exceeds a threshold value is calculated with the knowledge of the probability density functions associated with the TF and target gene protein levels. Numerical simulation results for a more detailed stochastic model are shown to be in agreement with those obtained through analytic calculations. The relevance of these results in the context of the genetic disorder haploinsufficiency is pointed out. Some experimental observations on the haploinsufficiency of the tumour suppressor gene, Nkx 3.1, are explained with the help of the stochastic model of TF-regulated gene expression.

Toll-like receptor (TLR)-4 mediates anti-β2GPI/β2GPI-induced tissue factor expression in THP-1 cells

PubMed Central

Zhou, H; Yan, Y; Xu, G; Zhou, B; Wen, H; Guo, D; Zhou, F; Wang, H

2011-01-01

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-β2-glycoprotein I/β2-glycoprotein I complex (anti-β2GPI/β2GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an ‘adaptor’ for intracellular signal transduction in anti-β2GPI/β2GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-β2GPI/β2GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-β2GPI (10 µg/ml)/β2GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-β2GPI/β2GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to β2GPI that had been conjugated to a column (β2GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-β2GPI/β2GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-β2GPI/β2GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative. PMID:21091668

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

PubMed

Coppola, S; Narciso, L; Feccia, T; Bonci, D; Calabrò, L; Morsilli, O; Gabbianelli, M; De Maria, R; Testa, U; Peschle, C

2006-01-01

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

PubMed

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

2015-07-01

To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Graft Product for Autologous Peripheral Blood Stem Cell Transplantation Enhances Thrombin Generation and Expresses Procoagulant Microparticles and Tissue Factor.

PubMed

Sidibe, Fatoumata; Spanoudaki, Anastasia; Vanneaux, Valerie; Mbemba, Elisabeth; Larghero, Jerome; Van Dreden, Patrick; Lotz, Jean-Pierre; Elalamy, Ismail; Larsen, Annette K; Gerotziafas, Grigoris T

2018-05-01

The beneficial effect of autologous peripheral blood stem cell transplantation (APBSCT) may be compromised by acute vascular complications related to hypercoagulability. We studied the impact of graft product on thrombin generation of normal plasma and the expression of tissue factor (TF) and procoagulant platelet-derived procoagulant microparticles (Pd-MPs) in samples of graft products. Graft products from 10 patients eligible for APBSCT were mixed with platelet-poor plasma (PPP) or platelet-rich plasma (PRP) from healthy volunteers and assessed for in vitro thrombin generation. In control experiments, thrombin generation was assessed in (1) PPP and PRP without any exogenous TF and/or procoagulant phospholipids, (2) PPP with the addition of TF (5 pM) and procoagulant phospholipids (4 μM), (3) in PRP with the addition of TF (5 pM). Graft products were assessed with Western blot assay for TF expression, with a specific clotting assay for TF activity and with flow cytometry assay for Pd-MPs. The graft product enhanced thrombin generation and its procoagulant activity was related to the presence of Pd-MPs and TF. The concentration of Pd-MPs in the graft product was characterized by a significant interindividual variability. The present study reveals the need for a thorough quality control of the graft products regarding their procoagulant potential.

Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer.

PubMed

Eriksson, Oskar; Thulin, Åsa; Asplund, Anna; Hegde, Geeta; Navani, Sanjay; Siegbahn, Agneta

2016-05-31

Tissue Factor (TF) forms a proteolytically active complex together with coagulation factor VIIa (FVIIa) and functions as the trigger of blood coagulation or alternatively activates cell signaling. We recently described that EphA2 of the Eph tyrosine kinase receptor family is cleaved directly by the TF/FVIIa complex. The aim of the present study was to further characterize the cross-talk between TF/FVIIa and EphA2 using in vitro model systems and human cancer specimens. Cleavage and phosphorylation of EphA2 was studied by Western blot. Subcellular localization of TF and EphA2 was investigated by a proximity ligation assay and confocal microscopy. Phalloidin staining of the actin cytoskeleton was used to study cell rounding and retraction fiber formation. Expression of TF and EphA2 in human colorectal cancer specimens was examined by immunohistochemistry. TF and EphA2 co-localized constitutively in MDA-MB-231 cells, and addition of FVIIa resulted in cleavage of EphA2 by a PAR2-independent mechanism. Overexpression of TF in U251 glioblastoma cells lead to co-localization with EphA2 at the leading edge and FVIIa-dependent cleavage of EphA2. FVIIa potentiated ephrin-A1-induced cell rounding and retraction fiber formation in MDA-MB-231 cells through a RhoA/ROCK-dependent pathway that did not require PAR2-activation. TF and EphA2 were expressed in colorectal cancer specimens, and were significantly correlated. These results suggest that TF/FVIIa-EphA2 cross-talk might potentiate ligand-dependent EphA2 signaling in human cancers, and provide initial evidence that it is possible for this interaction to occur in vivo.

Identification of transcription factors potential related to brown planthopper resistance in rice via microarray expression profiling

PubMed Central

2012-01-01

Background Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most destructive insect pests of rice. The molecular responses of plants to sucking insects resemble responses to pathogen infection. However, the molecular mechanism of BPH-resistance in rice remains unclear. Transcription factors (TF) are up-stream regulators of various genes that bind to specific DNA sequences, thereby controlling the transcription from DNA to mRNA. They are key regulators for transcriptional expression in biological processes, and are probably involved in the BPH-induced pathways in resistant rice varieties. Results We conducted a microarray experiment to analyze TF genes related to BPH resistance in a Sri Lankan rice cultivar, Rathu Heenati (RHT). We compared the expression profiles of TF genes in RHT with those of the susceptible rice cultivar Taichun Native 1 (TN1). We detected 2038 TF genes showing differential expression signals between the two rice varieties. Of these, 442 TF genes were probably related to BPH-induced resistance in RHT and TN1, and 229 may be related to constitutive resistance only in RHT. These genes showed a fold change (FC) of more than 2.0 (P<0.05). Among the 442 TF genes related to BPH-induced resistance, most of them were readily induced in TN1 than in RHT by BPH feeding, for instance, 154 TF genes were up-regulated in TN1, but only 31 TF genes were up-regulated in RHT at 24 hours after BPH infestation; 2–4 times more TF genes were induced in TN1 than in RHT by BPH. At an FC threshold of >10, there were 37 induced TF genes and 26 constitutive resistance TF genes. Of these, 13 were probably involved in BPH-induced resistance, and 8 in constitutive resistance to BPH in RHT. Conclusions We explored the molecular mechanism of resistance to BPH in rice by comparing expressions of TF genes between RHT and TN1. We speculate that the level of gene repression, especially for early TF genes, plays an important role in the defense response. The fundamental point of the resistance strategy is that plants protect themselves by reducing their metabolic level to inhibit feeding by BPH and prevent damage from water and nutrient loss. We have selected 21 TF genes related to BPH resistance for further analyses to understand the molecular responses to BPH feeding in rice. PMID:23228240

Identification of transcription factors potential related to brown planthopper resistance in rice via microarray expression profiling.

PubMed

Wang, Yubing; Guo, Huimin; Li, Haichao; Zhang, Hao; Miao, Xuexia

2012-12-10

Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most destructive insect pests of rice. The molecular responses of plants to sucking insects resemble responses to pathogen infection. However, the molecular mechanism of BPH-resistance in rice remains unclear. Transcription factors (TF) are up-stream regulators of various genes that bind to specific DNA sequences, thereby controlling the transcription from DNA to mRNA. They are key regulators for transcriptional expression in biological processes, and are probably involved in the BPH-induced pathways in resistant rice varieties. We conducted a microarray experiment to analyze TF genes related to BPH resistance in a Sri Lankan rice cultivar, Rathu Heenati (RHT). We compared the expression profiles of TF genes in RHT with those of the susceptible rice cultivar Taichun Native 1 (TN1). We detected 2038 TF genes showing differential expression signals between the two rice varieties. Of these, 442 TF genes were probably related to BPH-induced resistance in RHT and TN1, and 229 may be related to constitutive resistance only in RHT. These genes showed a fold change (FC) of more than 2.0 (P<0.05). Among the 442 TF genes related to BPH-induced resistance, most of them were readily induced in TN1 than in RHT by BPH feeding, for instance, 154 TF genes were up-regulated in TN1, but only 31 TF genes were up-regulated in RHT at 24 hours after BPH infestation; 2-4 times more TF genes were induced in TN1 than in RHT by BPH. At an FC threshold of >10, there were 37 induced TF genes and 26 constitutive resistance TF genes. Of these, 13 were probably involved in BPH-induced resistance, and 8 in constitutive resistance to BPH in RHT. We explored the molecular mechanism of resistance to BPH in rice by comparing expressions of TF genes between RHT and TN1. We speculate that the level of gene repression, especially for early TF genes, plays an important role in the defense response. The fundamental point of the resistance strategy is that plants protect themselves by reducing their metabolic level to inhibit feeding by BPH and prevent damage from water and nutrient loss. We have selected 21 TF genes related to BPH resistance for further analyses to understand the molecular responses to BPH feeding in rice.

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Xiao; Gang, Yi; Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province

2015-02-06

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself.more » The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.« less

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

PubMed

Shoji, Mamoru; Sun, Aiming; Kisiel, Walter; Lu, Yang J; Shim, Hyunsuk; McCarey, Bernard E; Nichols, Christopher; Parker, Ernest T; Pohl, Jan; Mosley, Cara A; Alizadeh, Aaron R; Liotta, Dennis C; Snyder, James P

2008-04-01

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Indications, usage, and dosage of the transfer factor.

PubMed

Berrón-Pérez, Renato; Chávez-Sánchez, Raúl; Estrada-García, Iris; Espinosa-Padilla, Sara; Cortez-Gómez, Rudyard; Serrano-Miranda, Ernestina; Ondarza-Aguilera, Rodolfo; Pérez-Tapia, Mayra; Pineda Olvera, Benjamín; Jiménez-Martínez, María del Carmen; Portugués, Abraham; Rodríguez, Azucena; Cano, Laura; Pacheco, Pedro Urcino; Barrientos, Javier; Chacón, Rommel; Serafín, Jeannet; Mendez, Patricia; Monges, Abelardo; Cervantes, Edgar; Estrada-Parra, Sergio

2007-01-01

The transfer factor (TF) was described in 1955 by S. Lawrence. In 1992 Kirkpatrick characterized the specific TF at molecular level. The TF is constituted by a group of numerous molecules, of low molecular weight, from 1.0 to 6.0 kDa. The 5 kDa fraction corresponds to the TF specific to antigens. There are a number of publications about the clinical indications of the TF for diverse diseases, in particular those where the cellular immune response is compromised or in those where there is a deficient regulation of the immune response. In this article we present our clinical and basic experiences, especially regarding the indications, usage and dosage of the TF. Our group demonstrated that the TF increases the expression of IFN-gamma and RANTES, while decreases the expression of osteopontine. Using animal models we have worked with M. tuberculosis, and with a model of glioma with good therapeutic results. In the clinical setting we have worked with herpes zoster, herpes simplex type I, herpetic keratitis, atopic dermatitis, osteosarcoma, tuberculosis, asthma, post-herpetic neuritis, anergic coccidioidomycosis, leishmaniasis, toxoplasmosis, mucocutaneous candidiasis, pediatric infections produced by diverse pathogen germs, sinusitis, pharyngitis, and otits media. All of these diseases were studied through protocols which main goals were to study the therapeutic effects of the TF, and to establish in a systematic way diverse dosage schema and time for treatment to guide the prescription of the TF.

Statistical mechanical model of coupled transcription from multiple promoters due to transcription factor titration

PubMed Central

Rydenfelt, Mattias; Cox, Robert Sidney; Garcia, Hernan; Phillips, Rob

2014-01-01

Transcription factors (TFs) with regulatory action at multiple promoter targets is the rule rather than the exception, with examples ranging from the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes simultaneously to situations involving multiple copies of the same gene, such as plasmids, retrotransposons, or highly replicated viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in correlation (“promoter entanglement”) between transcription of different genes. We develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression for a general set of promoters and the resulting correlation in transcription rates of different genes. Our results show that the TF titration effect could be important for understanding gene expression in many regulatory settings. PMID:24580252

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy.

PubMed

Hu, Zhiwei; Cheng, Jijun; Xu, Jie; Ruf, Wolfram; Lockwood, Charles J

2017-02-01

Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836.

PubMed

Hernandez, Reinier; England, Christopher G; Yang, Yunan; Valdovinos, Hector F; Liu, Bai; Wong, Hing C; Barnhart, Todd E; Cai, Weibo

2017-10-28

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89 Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89 Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89 Zr-Df-ALT-836 for TF in vivo. 89 Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89 Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89 Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89 Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural, functional and evolutionary characterization of major drought transcription factors families in maize

NASA Astrophysics Data System (ADS)

Mittal, Shikha; Banduni, Pooja; Mallikarjuna, Mallana G.; Rao, Atmakuri R.; Jain, Prashant A.; Dash, Prasanta K.; Thirunavukkarasu, Nepolean

2018-05-01

Drought is one of the major threats to maize production. In order to improve the production and to breed tolerant hybrids, understanding the genes and regulatory mechanisms during drought stress is important. Transcription factors (TFs) play a major role in gene regulation and many TFs have been identified in response to drought stress. In our experiment, a set of 15 major TF families comprising 1436 genes was structurally and functionally characterized using in-silico tools and a gene expression assay. All 1436 genes were mapped on 10 chromosome of maize. The functional annotation indicated the involvement of these genes in ABA signaling, ROS scavenging, photosynthesis, stomatal regulation, and sucrose metabolism. Duplication was identified as the primary force in divergence and expansion of TF families. Phylogenetic relationship was developed individually for each TF family as well as combined TF families. Phylogenetic analysis grouped the TF family of genes into TF-specific and mixed groups. Phylogenetic analysis of genes belonging to various TF families suggested that the origin of TFs occurred in the lineage of maize evolution. Gene structure analysis revealed that more number of genes were intron-rich as compared to intronless genes. Drought-responsive CRE’s such as ABREA, ABREB, DRE1 and DRECRTCOREAT have been identified. Expression and interaction analyses identified leaf-specific bZIP TF, GRMZM2G140355, as a potential contributor toward drought tolerance in maize. We also analyzed protein-protein interaction network of 269 drought-responsive genes belonging to different drought-related TFs. The information generated on structural and functional characteristics, expression and interaction of the drought-related TF families will be useful to decipher the drought tolerance mechanisms and to derive drought-tolerant genotypes in maize.

Fructose induces prothrombotic phenotype in human endothelial cells : A new role for "added sugar" in cardio-metabolic risk.

PubMed

Cirillo, Plinio; Pellegrino, Grazia; Conte, Stefano; Maresca, Fabio; Pacifico, Francesco; Leonardi, Antonio; Trimarco, Bruno

2015-11-01

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5 mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.

Dynamic Transcription Factor Networks in Epithelial-Mesenchymal Transition in Breast Cancer Models

PubMed Central

Siletz, Anaar; Schnabel, Michael; Kniazeva, Ekaterina; Schumacher, Andrew J.; Shin, Seungjin; Jeruss, Jacqueline S.; Shea, Lonnie D.

2013-01-01

The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy. PMID:23593114

Dynamic transcription factor networks in epithelial-mesenchymal transition in breast cancer models.

PubMed

Siletz, Anaar; Schnabel, Michael; Kniazeva, Ekaterina; Schumacher, Andrew J; Shin, Seungjin; Jeruss, Jacqueline S; Shea, Lonnie D

2013-01-01

The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Huang-Joe; Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan; Lo, Wan-Yu

Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease inmore » TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.« less

The clinical value of tissue factor assays.

PubMed

Francis, J L; Carvalho, M; Francis, D A

1995-06-01

Tissue factor (TF) is now considered to be the primary physiologic activator of the blood coagulation system. Coupled with recent advances in our understanding of the biochemistry of TF this has heightened interest in measuring aspects of TF activity in disease states. Expression of TF by blood monocytes in various diseases is an established trigger for intravascular coagulation and there is now a considerable body of experience with its measurement. This has considerable clinical potential although more widespread application awaits a consensus on the most appropriate methodologic approach to its measurement. TF can be detected in urine and may reflect the activation state of renal macrophages. Urinary TF is increased in cancer and could have diagnostic and prognostic value in a variety of malignant diseases. Finally, it is now possible to measure soluble TF in plasma. One such assay is commercially available and is technically simple to perform. The clinical value of such assays, however, must await better understanding of the source and function of soluble TF in plasma.

COUP-TF1 Modulates Thyroid Hormone Action in an Embryonic Stem-Cell Model of Cortical Pyramidal Neuronal Differentiation.

PubMed

Teng, Xiaochun; Liu, Yan-Yun; Teng, Weiping; Brent, Gregory A

2018-05-01

Thyroid hormone is critical for normal brain development and acts in a spatial and temporal specific pattern. Thyroid hormone excess, or deficiency, can lead to irreversible impairment of brain and sensory development. Chicken ovalbumin upstream-transcription factor 1 (COUP-TF1), expressed early in neuronal development, is essential to achieve normal brain structure. Thyroid hormone stimulation of gene expression is inversely correlated with the level of COUP-TF1 expression. An in vitro method of differentiating mouse embryonic stem (mES) cells into cortical neurons was utilized to study the influence of COUP-TF1 on thyroid hormone signaling in brain development. mES cells were cultured and differentiated in specific conditioned media, and a high percentage of nestin-positive progenitor neurons in the first stage, and cortical neurons in the second stage, was obtained with characteristic neuronal firing. The number of nestin-positive progenitors, as determined by fluorescence-activated cell sorting analysis, was significantly greater with triiodothyronine (T3) treatment compared to control (p < 0.05). T3 enhanced the expression of cortical neuron marker (Tbr1 and Rc3) mRNAs. After COUP-TF1 knockdown, the number of nestin-positive progenitors was reduced compared to control (p < 0.05), but the number increased with T3 treatment. The mRNA of cortical neuronal gene markers was measured after COUP-TF1 knockdown. In the presence of T3, the peak expression of neuron markers Emx1, Tbr1, Camkiv, and Rc3 mRNA was earlier, at day 18 of differentiation, compared to control cells, at day 22. Furthermore, after COUP-TF1 knockdown, T3 induction of Rc3 and Tbr1 mRNA was significantly enhanced compared to cells expressing COUP-TF1. These results indicate that COUP-TF1 plays an important role in modulating the timing and magnitude of T3-stimulated gene expression required for normal corticogenesis.

The seed dormancy defect of Arabidopsis mutants lacking the transcript elongation factor TFIIS is caused by reduced expression of the DOG1 gene.

PubMed

Mortensen, Simon A; Grasser, Klaus D

2014-01-03

TFIIS is a transcript elongation factor that facilitates transcription by RNA polymerase II, as it assists the enzyme to bypass blocks to mRNA synthesis. Previously, we have reported that Arabidopsis plants lacking TFIIS exhibit reduced seed dormancy. Among the genes differentially expressed in tfIIs seeds, the DOG1 gene was identified that is a known QTL for seed dormancy. Here we have analysed plants that overexpress TFIIS in wild type background, or that harbour an additional copy of DOG1 in tfIIs mutant background. These experiments demonstrate that the down-regulation of DOG1 expression causes the seed dormancy phenotype of tfIIs mutants. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.

PubMed

Pawlinski, Rafal; Pedersen, Brian; Kehrle, Bettina; Aird, William C; Frank, Rolf D; Guha, Mausumee; Mackman, Nigel

2003-05-15

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Effect of dibutyryl cyclic adenosine monophosphate on the gene expression of plasminogen activator inhibitor-1 and tissue factor in adipocytes.

PubMed

Taniguchi, Makoto; Ono, Naoko; Hayashi, Akira; Yakura, Yuwna; Takeya, Hiroyuki

2011-10-01

Hypertrophic adipocytes in obese states express the elevated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF). An increase in the intracellular concentration of cyclic adenosine monophosphate (cAMP) promotes triglyceride hydrolysis and may improve dysregulation of adipocyte metabolism. Here, we investigate the effect of dibutyryl-cAMP (a phosphodiesterase-resistant analog of cAMP) on the gene expression of PAI-1 and TF in adipocytes. Differentiated 3T3-L1 adipocytes were treated with dibutyryl-cAMP and agents that would be expected to elevate intracellular cAMP, including cilostazol (a phosphodiesterase inhibitor with anti-platelet and vasodilatory properties), isoproterenol (a beta adrenergic agonist) and forskolin (an adenylyl cyclase activator). The levels of PAI-1 and TF mRNAs were measured using real-time quantitative reverse transcription-PCR. The treatment of adipocytes with dibutyryl-cAMP resulted in the inhibition of both lipid accumulation and TF gene expression. However, PAI-1 gene expression was slightly but significantly increased by dibutyryl-cAMP. On the other hand, cilostazol inhibited the expression of PAI-1 without affecting lipid accumulation. When the adipocytes were treated with cilostazol in combination with isoproterenol or forskolin, the inhibitory effect of cilostazol on PAI-1 gene expression was counteracted, thus suggesting that inhibition by cilostazol may not be the result of intracellular cAMP accumulation by phosphodiesterase inhibition. These results suggest the implication of cAMP in regulation of the gene expression of TF and PAI-1 in adipocytes. Our findings will serve as a useful basis for further research in therapy for obesity-associated thrombosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mimosa: Mixture Model of Co-expression to Detect Modulators of Regulatory Interaction

NASA Astrophysics Data System (ADS)

Hansen, Matthew; Everett, Logan; Singh, Larry; Hannenhalli, Sridhar

Functionally related genes tend to be correlated in their expression patterns across multiple conditions and/or tissue-types. Thus co-expression networks are often used to investigate functional groups of genes. In particular, when one of the genes is a transcription factor (TF), the co-expression-based interaction is interpreted, with caution, as a direct regulatory interaction. However, any particular TF, and more importantly, any particular regulatory interaction, is likely to be active only in a subset of experimental conditions. Moreover, the subset of expression samples where the regulatory interaction holds may be marked by presence or absence of a modifier gene, such as an enzyme that post-translationally modifies the TF. Such subtlety of regulatory interactions is overlooked when one computes an overall expression correlation. Here we present a novel mixture modeling approach where a TF-Gene pair is presumed to be significantly correlated (with unknown coefficient) in a (unknown) subset of expression samples. The parameters of the model are estimated using a Maximum Likelihood approach. The estimated mixture of expression samples is then mined to identify genes potentially modulating the TF-Gene interaction. We have validated our approach using synthetic data and on three biological cases in cow and in yeast. While limited in some ways, as discussed, the work represents a novel approach to mine expression data and detect potential modulators of regulatory interactions.

Peritumoral adipose tissue as a source of inflammatory and angiogenic factors in colorectal cancer.

PubMed

Amor, S; Iglesias-de la Cruz, M C; Ferrero, E; García-Villar, O; Barrios, V; Fernandez, N; Monge, L; García-Villalón, A L; Granado, M

2016-02-01

Obesity is a risk factor for the development of human colorectal cancer (CC). The aim of this work is to report the inflammatory and angiogenic scenario in lean (BMI < 25 kg/m2) and obese (BMI > 30 kg/m2) patients with and without CC and to assess the role of peritumoral adipose tissue in CC-induced inflammation. Patients were divided in four experimental groups: obese patients with CC (OB-CC), lean patients with CC (LEAN-CC), obese patients without CC (OB), and lean patients without CC (LEAN). Plasma levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-4, IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in OB-CC patients. Peritumoral adipose tissue (TF) explants and cultured mature adipocytes secreted higher amounts of nitrites and nitrates than did control and non-tumoral (NTF) adipose tissue both alone and in response to lipopolysaccharide (LPS). Nitrite and nitrate secretion was also increased in TF explants from OB-CC patients compared with that from LEAN-CC patients. Gene expression of adiponectin, tumor necrosis factor alpha (TNF-α), insulin-like growth factor type I (IGF-I), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor γ (PPAR-γ) was increased in TF explants from CC patients. LPS increased the gene expression of IL-6, IL-10, TNF-α, vascular endothelial growth factor (VEGF), and COX-2 in OB and in TF explants from OB-CC patients. COX-2 and PPAR-γ inhibition further increased LPS-induced release of nitrites and nitrates in TF explants and adipocytes from OB-CC patients. In conclusion, OB-CC patients have increased plasma levels of pro-inflammatory and angiogenic factors. TF from OB-CC patients shows an increased secretion of inflammatory markers compared with both TF from LEAN-CC and non-tumoral adipose tissue (AT) through a COX-2- and PPAR-γ-independent mechanism.

Thyroid hormone and COUP-TF1 regulate kallikrein-binding protein (KBP) gene expression.

PubMed

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen; Brent, Gregory A

2011-03-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

Thyroid Hormone and COUP-TF1 Regulate Kallikrein-Binding Protein (KBP) Gene Expression

PubMed Central

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen

2011-01-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T3 and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5′ flanking region (−53 to −29) and nTRE2, located in the first intron (104–132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T3. COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T3 repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T3. Nuclear corepressor knockdown resulted in loss of T3 repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T3 induction of positive thyroid hormone response elements, reverses T3 repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T3 and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T3. PMID:21266512

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.

Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

PubMed

Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

2015-05-15

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

ASXL gain-of-function truncation mutants: defective and dysregulated forms of a natural ribosomal frameshifting product?

PubMed

Dinan, Adam M; Atkins, John F; Firth, Andrew E

2017-10-16

Programmed ribosomal frameshifting (PRF) is a gene expression mechanism which enables the translation of two N-terminally coincident, C-terminally distinct protein products from a single mRNA. Many viruses utilize PRF to control or regulate gene expression, but very few phylogenetically conserved examples are known in vertebrate genes. Additional sex combs-like (ASXL) genes 1 and 2 encode important epigenetic and transcriptional regulatory proteins that control the expression of homeotic genes during key developmental stages. Here we describe an ~150-codon overlapping ORF (termed TF) in ASXL1 and ASXL2 that, with few exceptions, is conserved throughout vertebrates. Conservation of the TF ORF, strong suppression of synonymous site variation in the overlap region, and the completely conserved presence of an EH[N/S]Y motif (a known binding site for Host Cell Factor-1, HCF-1, an epigenetic regulatory factor), all indicate that TF is a protein-coding sequence. A highly conserved UCC_UUU_CGU sequence (identical to the known site of +1 ribosomal frameshifting for influenza virus PA-X expression) occurs at the 5' end of the region of enhanced synonymous site conservation in ASXL1. Similarly, a highly conserved RG_GUC_UCU sequence (identical to a known site of -2 ribosomal frameshifting for arterivirus nsp2TF expression) occurs at the 5' end of the region of enhanced synonymous site conservation in ASXL2. Due to a lack of appropriate splice forms, or initiation sites, the most plausible mechanism for translation of the ASXL1 and 2 TF regions is ribosomal frameshifting, resulting in a transframe fusion of the N-terminal half of ASXL1 or 2 to the TF product, termed ASXL-TF. Truncation or frameshift mutants of ASXL are linked to myeloid malignancies and genetic diseases, such as Bohring-Opitz syndrome, likely at least in part as a result of gain-of-function or dominant-negative effects. Our hypothesis now indicates that these disease-associated mutant forms represent overexpressed defective versions of ASXL-TF. This article was reviewed by Laurence Hurst and Eugene Koonin.

Expression of Tissue Factor in Epithelial Ovarian Carcinoma Is Involved in the Development of Venous Thromboembolism.

PubMed

Sakurai, Manabu; Matsumoto, Koji; Gosho, Masahiko; Sakata, Akiko; Hosokawa, Yoshihiko; Tenjimbayashi, Yuri; Katoh, Takashi; Shikama, Ayumi; Komiya, Haruna; Michikami, Hiroo; Tasaka, Nobutaka; Akiyama-Abe, Azusa; Nakao, Sari; Ochi, Hiroyuki; Onuki, Mamiko; Minaguchi, Takeo; Yoshikawa, Hiroyuki; Satoh, Toyomi

2017-01-01

Our 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor (TF) in the development of venous thromboembolism (VTE) before treatment, especially in clear cell carcinoma (CCC). This follow-up study further investigated this possibility in a larger cohort. We investigated the intensity of TF expression (ITFE) and other variables for associations with VTE using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between November 2004 and December 2010, none of whom had received neoadjuvant chemotherapy. Before starting treatment, all patients were ultrasonographically screened for VTE. The ITFE was graded based on immunostaining of surgical specimens. Histological types were serous carcinoma (n = 42), CCC (n = 12), endometrioid carcinoma (n = 15), mucinous carcinoma (n = 53), and undifferentiated carcinoma (n = 6). The prevalence of VTE was significantly higher in CCC (34%) than in non-CCC (17%, P = 0.03). As ITFE increased, the frequencies of CCC and VTE increased significantly (P < 0.001 and P = 0.014, respectively). Multivariate analysis identified TF expression and pretreatment dimerized plasmin fragment D level as significant independent risk factors for VTE development. These factors showed particularly strong impacts on advanced-stage disease (P = 0.021). The 2007 cohort was small, preventing multivariate analysis. This study of a larger cohort yielded stronger evidence that the development of VTE in epithelial ovarian cancer may involve TF expression in cancer tissues.

Genome-Wide Analysis of Androgen Receptor Targets Reveals COUP-TF1 as a Novel Player in Human Prostate Cancer

PubMed Central

Perets, Ruth; Kaplan, Tommy; Stein, Ilan; Hidas, Guy; Tayeb, Shay; Avraham, Eti; Ben-Neriah, Yinon; Simon, Itamar; Pikarsky, Eli

2012-01-01

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer. PMID:23056316

Genome-wide analysis of androgen receptor targets reveals COUP-TF1 as a novel player in human prostate cancer.

PubMed

Perets, Ruth; Kaplan, Tommy; Stein, Ilan; Hidas, Guy; Tayeb, Shay; Avraham, Eti; Ben-Neriah, Yinon; Simon, Itamar; Pikarsky, Eli

2012-01-01

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR - COUP-TF1 - which could possibly play a role in human prostate cancer.

Prostaglandin E2 Regulates Its Own Inactivating Enzyme, 15-PGDH, by EP2 Receptor-Mediated Cervical Cell-Specific Mechanisms

PubMed Central

Kishore, A. Hari; Owens, David

2014-01-01

Context: Prostaglandins play important roles in parturition and have been used to induce cervical ripening and labor. Prior to cervical ripening at term, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is highly expressed in the cervix and metabolizes cyclooxygenase-2-mediated increases in active prostaglandin E2 (PGE2) to inactive 15-keto PGE2. At term, 15-PGDH gene expression decreases and PGE2 accumulates, leading to cervical ripening and labor. Previously, we found that the cervical isoform of microphthalmia-associated transcription factor (MiTF-CX) serves as a progestational transcription factor that represses IL-8 and hypoxia-mediated increases in cyclooxygenase-2. Objective: We tested the hypothesis that PGE2 regulates its own inactivation through MiTF-CX. Design: We used human cervical stromal cells to investigate the regulation of 15-PGDH. Setting: This was a laboratory-based study using cells from clinical tissue samples. Main Outcome Measures: We evaluated the mechanisms by which PGE2 regulates 15-PGDH in human cervical stromal cells. Results: PGE2 repressed MiTF-CX and 15-PGDH, whereas ectopic overexpression of MiTF-CX induced 15-PGDH expression levels. Stabilization of HIF-1α by deferoxamine resulted in concomitant down-regulation of MiTF-CX and 15-PGDH. Ectopic overexpression of MiTF-CX abrogated PGE2- and deferoxamine-mediated loss of MiTF-CX and 15-PGDH. PGE2-induced loss of MiTF-CX and 15-PGDH was mediated through prostaglandin E2 receptor (EP2) receptors (PTGER2), but not cAMP. Conclusions: The 15-PGDH gene is a MiTF-CX target gene in cervical stromal cells and is down-regulated by PGE2 through EP2 receptors. The findings suggest that EP2 receptor-specific antagonists may be used as an adjunct to present clinical management for the prevention of preterm cervical ripening and preterm labor. PMID:24471568

Regression Analysis of Combined Gene Expression Regulation in Acute Myeloid Leukemia

PubMed Central

Li, Yue; Liang, Minggao; Zhang, Zhaolei

2014-01-01

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. PMID:25340776

SM-TF: A structural database of small molecule-transcription factor complexes.

PubMed

Xu, Xianjin; Ma, Zhiwei; Sun, Hongmin; Zou, Xiaoqin

2016-06-30

Transcription factors (TFs) are the proteins involved in the transcription process, ensuring the correct expression of specific genes. Numerous diseases arise from the dysfunction of specific TFs. In fact, over 30 TFs have been identified as therapeutic targets of about 9% of the approved drugs. In this study, we created a structural database of small molecule-transcription factor (SM-TF) complexes, available online at http://zoulab.dalton.missouri.edu/SM-TF. The 3D structures of the co-bound small molecule and the corresponding binding sites on TFs are provided in the database, serving as a valuable resource to assist structure-based drug design related to TFs. Currently, the SM-TF database contains 934 entries covering 176 TFs from a variety of species. The database is further classified into several subsets by species and organisms. The entries in the SM-TF database are linked to the UniProt database and other sequence-based TF databases. Furthermore, the druggable TFs from human and the corresponding approved drugs are linked to the DrugBank. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Air pollution upregulates endothelial cell procoagulant activity via ultrafine particle-induced oxidant signaling and tissue factor expression.

PubMed

Snow, S J; Cheng, W; Wolberg, A S; Carraway, M S

2014-07-01

Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure. Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Antiphospholipid antibodies promote tissue factor-dependent angiogenic switch and tumor progression.

PubMed

Wu, Yuan-Yuan; V Nguyen, Andrew; Wu, Xiao-Xuan; Loh, Mingyu; Vu, Michelle; Zou, Yiyu; Liu, Qiang; Guo, Peng; Wang, Yanhua; Montgomery, Leslie L; Orlofsky, Amos; Rand, Jacob H; Lin, Elaine Y

2014-12-01

Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell-derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

PubMed

Koizume, Shiro; Ito, Shin; Yoshioka, Yusuke; Kanayama, Tomohiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Yamada, Roppei; Ochiya, Takahiro; Ruf, Wolfram; Miyagi, Etsuko; Hirahara, Fumiki; Miyagi, Yohei

2016-01-01

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

PubMed

Schmidt, Lars Henning; Stucke-Ring, Janine; Brand, Caroline; Schliemann, Christoph; Harrach, Saliha; Muley, Thomas; Herpel, Esther; Kessler, Torsten; Mohr, Michael; Görlich, Dennis; Kreuter, Michael; Lenz, Georg; Wardelmann, Eva; Thomas, Michael; Berdel, Wolfgang E; Schwöppe, Christian; Hartmann, Wolfgang

2017-11-01

Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered iron metabolism, inflammation, transferrin receptors, and ferritin expression in non-small-cell lung cancer.

PubMed

Kukulj, Suzana; Jaganjac, Morana; Boranic, Milivoj; Krizanac, Simun; Santic, Zarko; Poljak-Blazi, Marija

2010-06-01

The involvement of iron and inflammation parameters on overall survival in non-small-cell lung cancer (NSCLC) patients was studied. Furthermore, transferrin receptors 1 (TfR1) and ferritin expression in tumor tissue, tumor stroma, and normal lung tissue were analyzed. Iron metabolism and inflammation parameters were determined by automated laboratory measurements at the time of diagnosis. TfR1 and ferritin expression were determined by immuno-histochemical methods. About 50% of patients survived 12 months only. At the time of diagnosis more than half of the patients had anemia and significantly elevated serum ferritin. Iron content of serum ferritin (ICF) was below the reference values in 90% of patients. Furthermore, ICF showed positive correlation with iron metabolic parameters and survival but negative correlation with serum ferritin and ESR. The expression of TfR1 and ferritin in tumor cells was observed in 88% or 62% of patients, respectively. Tumor stroma was TfR1 negative and sporadically ferritin positive. Tumor tissue ferritin expression showed negative correlation with serum iron and hematokrit (Ht), and positive correlation with ferritin, erythrocyte sedimentation rate (ESR), alpha-1 globulin, and alpha-2 globulin. Positive correlation was found between TfR1 expression in tumor tissue and alpha-globulin. The correlation between TfR1/ferritin expression in tumor tissue and ICF or survival was not observed. Therefore, we conclude that elevated serum ferritin in sera of NSCLC patients is the result of inflammation and oxidative stress rather than body iron overload. Higher expression of ferritin in tumor tissue may be the consequence of iron deficiency or local toxicity induced by environmental factors.

Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice.

PubMed

Thomas, G M; Brill, A; Mezouar, S; Crescence, L; Gallant, M; Dubois, C; Wagner, D D

2015-07-01

The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain. To investigate how pancreatic cancer MPs promote DVT in vivo. We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT. Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs. TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis. © 2015 International Society on Thrombosis and Haemostasis.

[Endothelial microparticles (EMP) in physiology and pathology].

PubMed

Sierko, Ewa; Sokół, Monika; Wojtukiewicz, Marek Z

2015-08-18

Endothelial microparticles (EMP) are released from endothelial cells (ECs) in the process of activation and/or apoptosis. They harbor adhesive molecules, enzymes, receptors and cytoplasmic structures and express a wide range of various constitutive antigens, typical for ECs, at their surface. Under physiological conditions the concentration of EMP in the blood is clinically insignificant. However, it was reported that under pathological conditions EMP concentration in the blood might slightly increase and contribute to blood coagulation, angiogenesis and inflammation. It has been shown that EMP directly and indirectly contribute to the activation of blood coagulation. Endothelial microparticles directly participate in blood coagulation through their surface tissue factor (TF) - a major initiator of blood coagulation. Furthermore, EMP exhibit procoagulant potential via expression of negatively charged phospholipids at their surface, which may promote assembly of coagulation enzymes (TF/VII, tenases and prothrombinase complexes), leading to thrombus formation. In addition, they provide a binding surface for coagulation factors: IXa, VIII, Va and IIa. Moreover, it is possible that EMP transfer TF from TF-bearing EMP to activated platelets and monocytes by binding them through adhesion molecules. Also, EMP express von Willebrand factor, which may facilitate platelet aggregation. Apart from their procoagulant properties, it was demonstrated that EMP may express adhesive molecules and metalloproteinases (MMP-2, MMP-9) at their surface and release growth factors, which may contribute to angiogenesis. Additionally, surface presence of C3 and C4 - components of the classical pathway - suggests pro-inflammatory properties of these structures. This article contains a summary of available data on the biology and pathophysiology of endothelial microparticles and their potential role in blood coagulation, angiogenesis and inflammation.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

PubMed

Richter, D U; Jeschke, U; Bergemann, C; Makovitzky, J; Lüthen, F; Karsten, U; Briese, V

2005-01-01

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Identification of active miRNA and transcription factor regulatory pathways in human obesity-related inflammation.

PubMed

Zhang, Xi-Mei; Guo, Lin; Chi, Mei-Hua; Sun, Hong-Mei; Chen, Xiao-Wen

2015-03-07

Obesity-induced chronic inflammation plays a fundamental role in the pathogenesis of metabolic syndrome (MS). Recently, a growing body of evidence supports that miRNAs are largely dysregulated in obesity and that specific miRNAs regulate obesity-associated inflammation. We applied an approach aiming to identify active miRNA-TF-gene regulatory pathways in obesity. Firstly, we detected differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) from mRNA and miRNA expression profiles, respectively. Secondly, by mapping the DEGs and DEmiRs to the curated miRNA-TF-gene regulatory network as active seed nodes and connect them with their immediate neighbors, we obtained the potential active miRNA-TF-gene regulatory subnetwork in obesity. Thirdly, using a Breadth-First-Search (BFS) algorithm, we identified potential active miRNA-TF-gene regulatory pathways in obesity. Finally, through the hypergeometric test, we identified the active miRNA-TF-gene regulatory pathways that were significantly related to obesity. The potential active pathways with FDR < 0.0005 were considered to be the active miRNA-TF regulatory pathways in obesity. The union of the active pathways is visualized and identical nodes of the active pathways were merged. We identified 23 active miRNA-TF-gene regulatory pathways that were significantly related to obesity-related inflammation.

Inhibition effects of total flavonoids from Scutellaria barbata D. Don on human breast carcinoma bone metastasis via downregulating PTHrP pathway

PubMed Central

Liu, Huihui; Guo, Shanyu

2018-01-01

It is abundantly clear that tumor-derived parathyroid hormone-related protein (PTHrP), receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) are central contributors in promoting osteolytic process of breast carcinoma bone metastasis. Forcusing on this molecular basis, the study was undertaken to explore the inhibition effects of total flavonoids from Scutellaria barbata D. Don (TF-SB) on human breast carcinoma bone metastasis. MDA-MB-231 cells and nude mouse models of breast cancer bone metastasis were given TF-SB in different concentrations. The proliferation, migration and invasion potentials of MDA-MB-231 cells were respectively tested. The effects of TF-SB on tumor weights and bone destruction were investigated. The mRNA and protein expression of PTHrP, OPG and RANKL were assessed by qPCR and western blot analysis. In vitro, TF-SB inhibited the proliferation, migration and invasion of MDA-MB-231 cells in a dose-dependent manner. In vivo, TF-SB prevented bone metastasis of breast cancer by decreasing the number of osteoclast cells per field in a dose-dependent manner, but not affecting tumor growth or mouse survival. Molecular analysis revealed that TF-SB controled the secretion of osteolysis-related factors PTHrP and its downstream RANKL/OPG. Together, by controlling the expression of PTHrP and its downstream OPG/RANKL, TF-SB has significant inhibition effects on breast cancer bone metastasis, which indicates a new therapeutic method. PMID:29512770

Plasmodium falciparum-infected erythrocytes induce Tissue Factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes

PubMed Central

Francischetti, Ivo MB; Seydel, Karl B; Monteiro, Robson Q; Whitten, Richard O; Erexson, Cindy R; Noronha, Almério LL; Ostera, Graciela R.; Kamiza, Steve B; Molyneux, Malcolm E; Ward, Jerrold M; Taylor, Terrie E

2010-01-01

Summary Background Plasmodium falciparum malaria infects 300–500 million people every year causing 1–2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. Objectives We have asked whether parasitized red blood cells (pRBC) interaction with EC induces Tissue Factor expression in vitro and in vivo. The potential of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. Results We demonstrate that mature forms of pRBC induce functional expression of tissue factor (TF) by endothelial cells (EC) in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, postmortem brain sections obtained from P. falciparum-infected children who died from Cerebral Malaria and other causes display a consistent staining for TF in the EC. Conclusions These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications. PMID:17002660

Skin extracts from 2 Italian table grapes (Italia and Palieri) inhibit tissue factor expression by human blood mononuclear cells.

PubMed

Milella, Rosa Anna; Antonacci, Donato; Crupi, Pasquale; Incampo, Francesca; Carrieri, Cosimo; Semeraro, Nicola; Colucci, Mario

2012-08-01

Grape and its products such as red wine and grape juice have well-known antithrombotic properties, which have been attributed to their high content in polyphenolic compounds. Most studies on the mechanisms underlying these beneficial effects, among which the suppression of tissue factor (TF) synthesis in blood mononuclear cells (MNC) and vascular endothelium is a prominent one, have been performed with purified polyphenols, while little is known about the effect of fresh grapes which contain a multitude of phytochemicals whose interaction may lead to different cell responses. In this study, we investigated the effect of grape skin extracts (GSEs) on TF expression in isolated blood MNC and in whole blood. Alcoholic extracts from skins of 2 grape varieties (Palieri and Italia) inhibited TF expression in lipopolysaccharide (LPS)-stimulated MNC in a concentration-dependent manner with ≥90% inhibition of TF activity and antigen at 6 μg/mL of gallic acid equivalents. Noteworthy, GSEs were also able to inhibit the appearance of TF in whole blood challenged with LPS. The 2 grape varieties displayed a fairly similar TF-inhibiting capacity despite marked differences in phenolic profile. When selected purified polyphenols were tested, their ability to inhibit TF expression was markedly lower as compared to grape extracts, whereas a mixture of some representative polyphenols was much more efficient, supporting the occurrence of a synergistic effect. Given the key role of cell TF in thrombotic diseases, the inhibition of MNC-mediated clotting activation, if confirmed by in vivo studies, might represent an important antithrombotic mechanism. Our data indicate that the combination of different polyphenols, as in grape extracts, is much more efficient than the single constituents, a finding that might be useful as starting point for the development of new antithrombotic nutraceutics. In addition, our study validated a simple, inexpensive, and physiologically relevant in vitro method on whole blood that allows the evaluation of one of the most important antithrombotic activities of food and food-derived products. The simplicity of the method makes it suitable also for screening purposes in large-scale studies. © 2012 Institute of Food Technologists®

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol.

PubMed

Kaur, Gurjeet; Roberti, Marinella; Raul, Francis; Pendurthi, Usha R

2007-01-01

Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction.

Inhibition of platelet activation prevents the P-selectin and integrin-dependent accumulation of cancer cell microparticles and reduces tumor growth and metastasis in vivo.

PubMed

Mezouar, Soraya; Darbousset, Roxane; Dignat-George, Françoise; Panicot-Dubois, Laurence; Dubois, Christophe

2015-01-15

Venous thromboembolism constitutes one of the main causes of death during the progression of a cancer. We previously demonstrated that tissue factor (TF)-bearing cancer cell-derived microparticles accumulate at the site of injury in mice developing a pancreatic cancer. The presence of these microparticles at the site of thrombosis correlates with the size of the platelet-rich thrombus. The objective of this study was to determine the involvement of TF expressed by cancer cell-derived microparticles on thrombosis associated with cancer. We observed that pancreatic cancer cell derived microparticles expressed TF, its inhibitor tissue factor pathway inhibitor (TFPI) as well as the integrins αvβ1 and αvβ3. In mice bearing a tumor under-expressing TF, a significant decrease in circulating TF activity associated with an increase bleeding time and a 100-fold diminished fibrin generation and platelet accumulation at the site of injury were observed. This was mainly due to the interaction of circulating cancer cell-derived microparticles expressing TFPI with activated platelets and fibrinogen. In an ectopic model of cancer, treatment of mice with Clopidogrel, an anti-platelet drug, decreased the size of the tumors and restored hemostasis by preventing the accumulation of cancer cell-derived microparticles at the site of thrombosis. In a syngeneic orthotopic model of pancreatic cancer Clopidogrel also significantly inhibited the development of metastases. Together, these results indicate that an anti-platelet strategy may efficiently treat thrombosis associated with cancer and reduce the progression of pancreatic cancer in mice. © 2014 UICC.

Novel Genomic and Evolutionary Insight of WRKY Transcription Factors in Plant Lineage

PubMed Central

Mohanta, Tapan Kumar; Park, Yong-Hwan; Bae, Hanhong

2016-01-01

The evolutionarily conserved WRKY transcription factor (TF) regulates different aspects of gene expression in plants, and modulates growth, development, as well as biotic and abiotic stress responses. Therefore, understanding the details regarding WRKY TFs is very important. In this study, large-scale genomic analyses of the WRKY TF gene family from 43 plant species were conducted. The results of our study revealed that WRKY TFs could be grouped and specifically classified as those belonging to the monocot or dicot plant lineage. In this study, we identified several novel WRKY TFs. To our knowledge, this is the first report on a revised grouping system of the WRKY TF gene family in plants. The different forms of novel chimeric forms of WRKY TFs in the plant genome might play a crucial role in their evolution. Tissue-specific gene expression analyses in Glycine max and Phaseolus vulgaris showed that WRKY11-1, WRKY11-2 and WRKY11-3 were ubiquitously expressed in all tissue types, and WRKY15-2 was highly expressed in the stem, root, nodule and pod tissues in G. max and P. vulgaris. PMID:27853303

Novel Genomic and Evolutionary Insight of WRKY Transcription Factors in Plant Lineage.

PubMed

Mohanta, Tapan Kumar; Park, Yong-Hwan; Bae, Hanhong

2016-11-17

The evolutionarily conserved WRKY transcription factor (TF) regulates different aspects of gene expression in plants, and modulates growth, development, as well as biotic and abiotic stress responses. Therefore, understanding the details regarding WRKY TFs is very important. In this study, large-scale genomic analyses of the WRKY TF gene family from 43 plant species were conducted. The results of our study revealed that WRKY TFs could be grouped and specifically classified as those belonging to the monocot or dicot plant lineage. In this study, we identified several novel WRKY TFs. To our knowledge, this is the first report on a revised grouping system of the WRKY TF gene family in plants. The different forms of novel chimeric forms of WRKY TFs in the plant genome might play a crucial role in their evolution. Tissue-specific gene expression analyses in Glycine max and Phaseolus vulgaris showed that WRKY11-1, WRKY11-2 and WRKY11-3 were ubiquitously expressed in all tissue types, and WRKY15-2 was highly expressed in the stem, root, nodule and pod tissues in G. max and P. vulgaris.

Activation of Coagulation by Lenalidomide-Based Regimens for the Treatment of Multiple Myeloma

PubMed Central

Isozumi, Yu; Arai, Reina; Fujimoto, Kazumi; Koyama, Takatoshi

2013-01-01

We investigated the procoagulant effects of lenalidomide (Len)-based regimens in vitro focusing on tissue factor (TF) and phosphatidylserine (PS). We examined the effects of a pharmacological concentration of Len with or without the corticosteroid dexamethasone (Dex) and the proteasome inhibitor bortezomib (Bor) using the human vascular endothelial cell line EAhy926 and the monocytic cell lines THP-1 and U937. Cell-surface procoagulant activity (PCA) was induced by Dex-containing regimens in all lines. Expression of TF antigen on the cell surface and of TF mRNA was markedly increased by Dex-containing regimens. PS exposure was increased modestly by a Len-based regimen. PS exposure was increased modestly in EAhy926 cells, and markedly increased in THP-1 and U937 cells by Bor-containing treatment. An anti-TF monoclonal antibody almost completely blocked the induced PCA. When Len is given in combination with Dex, PCA may be induced on endothelial cells and monocytes through TF expression and PS exposure. PMID:23696885

Activation of coagulation by lenalidomide-based regimens for the treatment of multiple myeloma.

PubMed

Isozumi, Yu; Arai, Reina; Fujimoto, Kazumi; Koyama, Takatoshi

2013-01-01

We investigated the procoagulant effects of lenalidomide (Len)-based regimens in vitro focusing on tissue factor (TF) and phosphatidylserine (PS). We examined the effects of a pharmacological concentration of Len with or without the corticosteroid dexamethasone (Dex) and the proteasome inhibitor bortezomib (Bor) using the human vascular endothelial cell line EAhy926 and the monocytic cell lines THP-1 and U937. Cell-surface procoagulant activity (PCA) was induced by Dex-containing regimens in all lines. Expression of TF antigen on the cell surface and of TF mRNA was markedly increased by Dex-containing regimens. PS exposure was increased modestly by a Len-based regimen. PS exposure was increased modestly in EAhy926 cells, and markedly increased in THP-1 and U937 cells by Bor-containing treatment. An anti-TF monoclonal antibody almost completely blocked the induced PCA. When Len is given in combination with Dex, PCA may be induced on endothelial cells and monocytes through TF expression and PS exposure.

Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

PubMed

Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

2016-06-23

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

A Transgenic Transcription Factor (TaDREB3) in Barley Affects the Expression of MicroRNAs and Other Small Non-Coding RNAs

PubMed Central

Hackenberg, Michael; Shi, Bu-Jun; Gustafson, Perry; Langridge, Peter

2012-01-01

Transcription factors (TFs), microRNAs (miRNAs), small interfering RNAs (siRNAs) and other functional non-coding small RNAs (sRNAs) are important gene regulators. Comparison of sRNA expression profiles between transgenic barley over-expressing a drought tolerant TF (TaDREB3) and non-transgenic control barley revealed many group-specific sRNAs. In addition, 42% of the shared sRNAs were differentially expressed between the two groups (|log2| >1). Furthermore, TaDREB3-derived sRNAs were only detected in transgenic barley despite the existence of homologous genes in non-transgenic barley. These results demonstrate that the TF strongly affects the expression of sRNAs and siRNAs could in turn affect the TF stability. The TF also affects size distribution and abundance of sRNAs including miRNAs. About half of the sRNAs in each group were derived from chloroplast. A sRNA derived from tRNA-His(GUG) encoded by the chloroplast genome is the most abundant sRNA, accounting for 42.2% of the total sRNAs in transgenic barley and 28.9% in non-transgenic barley. This sRNA, which targets a gene (TC245676) involved in biological processes, was only present in barley leaves but not roots. 124 and 136 miRNAs were detected in transgenic and non-transgenic barley, respectively. miR156 was the most abundant miRNA and up-regulated in transgenic barley, while miR168 was the most abundant miRNA and up-regulated in non-transgenic barley. Eight out of 20 predicted novel miRNAs were differentially expressed between the two groups. All the predicted novel miRNA targets were validated using a degradome library. Our data provide an insight into the effect of TF on the expression of sRNAs in barley. PMID:22870277

Sp8 and COUP-TF1 reciprocally regulate patterning and Fgf signaling in cortical progenitors.

PubMed

Borello, Ugo; Madhavan, Mayur; Vilinsky, Ilya; Faedo, Andrea; Pierani, Alessandra; Rubenstein, John; Campbell, Kenneth

2014-06-01

To gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors.

Sp8 and COUP-TF1 Reciprocally Regulate Patterning and Fgf Signaling in Cortical Progenitors

PubMed Central

Borello, Ugo; Madhavan, Mayur; Vilinsky, Ilya; Faedo, Andrea; Pierani, Alessandra; Rubenstein, John; Campbell, Kenneth

2014-01-01

To gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors. PMID:23307639

Systematic Analysis of Zn2Cys6 Transcription Factors Required for Development and Pathogenicity by High-Throughput Gene Knockout in the Rice Blast Fungus

PubMed Central

Huang, Pengyun; Lin, Fucheng

2014-01-01

Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn2Cys6 transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn2Cys6TF genes. A large variation in biological functions of Zn2Cys6TF genes was observed under the conditions tested. Sixty-one of 104 Zn2Cys6 TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae. PMID:25299517

Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice

PubMed Central

Thomas, G. M.; Brill, A.; Mezouar, S.; Crescence, L.; Gallant, M.; Dubois, C.; Wagner, D. D.

2015-01-01

Background The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain. Objective To address how pancreatic cancer MPs promote DVT in vivo. Methods We combined a DVT mouse model where thrombosis is induced by flow restriction of the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high and low TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT. Results Both tumor-bearing mice and mice infused with tumor MPs submitted to 3 hours of partial flow restriction developed an occlusive thrombus; fewer than a third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), functionally important players during DVT, whereas neither P-selectin nor GPIb were required for the MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT as mice infused with low TF tumor MPs had less thrombosis than mice infused with high TF tumor MPs. Conclusions TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis. PMID:25955268

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

Transcription factor evolution in eukaryotes and the assembly of the regulatory toolkit in multicellular lineages

PubMed Central

de Mendoza, Alex; Sebé-Pedrós, Arnau; Šestak, Martin Sebastijan; Matejčić, Marija; Torruella, Guifré; Domazet-Lošo, Tomislav; Ruiz-Trillo, Iñaki

2013-01-01

Transcription factors (TFs) are the main players in transcriptional regulation in eukaryotes. However, it remains unclear what role TFs played in the origin of all of the different eukaryotic multicellular lineages. In this paper, we explore how the origin of TF repertoires shaped eukaryotic evolution and, in particular, their role into the emergence of multicellular lineages. We traced the origin and expansion of all known TFs through the eukaryotic tree of life, using the broadest possible taxon sampling and an updated phylogenetic background. Our results show that the most complex multicellular lineages (i.e., those with embryonic development, Metazoa and Embryophyta) have the most complex TF repertoires, and that these repertoires were assembled in a stepwise manner. We also show that a significant part of the metazoan and embryophyte TF toolkits evolved earlier, in their respective unicellular ancestors. To gain insights into the role of TFs in the development of both embryophytes and metazoans, we analyzed TF expression patterns throughout their ontogeny. The expression patterns observed in both groups recapitulate those of the whole transcriptome, but reveal some important differences. Our comparative genomics and expression data reshape our view on how TFs contributed to eukaryotic evolution and reveal the importance of TFs to the origins of multicellularity and embryonic development. PMID:24277850

Identification of Human Lineage-Specific Transcriptional Coregulators Enabled by a Glossary of Binding Modules and Tunable Genomic Backgrounds.

PubMed

Mariani, Luca; Weinand, Kathryn; Vedenko, Anastasia; Barrera, Luis A; Bulyk, Martha L

2017-09-27

Transcription factors (TFs) control cellular processes by binding specific DNA motifs to modulate gene expression. Motif enrichment analysis of regulatory regions can identify direct and indirect TF binding sites. Here, we created a glossary of 108 non-redundant TF-8mer "modules" of shared specificity for 671 metazoan TFs from publicly available and new universal protein binding microarray data. Analysis of 239 ENCODE TF chromatin immunoprecipitation sequencing datasets and associated RNA sequencing profiles suggest the 8mer modules are more precise than position weight matrices in identifying indirect binding motifs and their associated tethering TFs. We also developed GENRE (genomically equivalent negative regions), a tunable tool for construction of matched genomic background sequences for analysis of regulatory regions. GENRE outperformed four state-of-the-art approaches to background sequence construction. We used our TF-8mer glossary and GENRE in the analysis of the indirect binding motifs for the co-occurrence of tethering factors, suggesting novel TF-TF interactions. We anticipate that these tools will aid in elucidating tissue-specific gene-regulatory programs. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcription Factor Map Alignment of Promoter Regions

PubMed Central

Blanco, Enrique; Messeguer, Xavier; Smith, Temple F; Guigó, Roderic

2006-01-01

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments. PMID:16733547

Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

PubMed Central

Fang, Xin; Sastry, Anand; Mih, Nathan; Kim, Donghyuk; Tan, Justin; Lloyd, Colton J.; Gao, Ye; Yang, Laurence; Palsson, Bernhard O.

2017-01-01

Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN—probably the best characterized TRN—several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict gene expression using this TRN; and (iii) how robust is our understanding of the TRN? First, we reconstructed a high-confidence TRN (hiTRN) consisting of 147 transcription factors (TFs) regulating 1,538 transcription units (TUs) encoding 1,764 genes. The 3,797 high-confidence regulatory interactions were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning algorithms to predict the expression of 1,364 TUs given TF activities using 441 samples. The algorithms accurately predicted condition-specific expression for 86% (1,174 of 1,364) of the TUs, while 193 TUs (14%) were predicted better than random TRNs. Third, we identified 10 regulatory modules whose definitions were robust against changes to the TRN or expression compendium. Using surrogate variable analysis, we also identified three unmodeled factors that systematically influenced gene expression. Our computational workflow comprehensively characterizes the predictive capabilities and systems-level functions of an organism’s TRN from disparate data types. PMID:28874552

A tissue factor-cascade-targeted strategy to tumor vasculature: a combination of EGFP-EGF1 conjugation nanoparticles with photodynamic therapy.

PubMed

Shi, Wei; Yin, Yanxue; Wang, Yao; Zhang, Bo; Tan, Pei; Jiang, Ting; Mei, Heng; Deng, Jun; Wang, Huafang; Guo, Tao; Pang, Zhiqing; Hu, Yu

2017-05-09

Tumor requires tumor vasculature to supply oxygen and nutrients so as to support its continued growth, as well as provide a main route for metastatic spread. In this study, a TF-cascade-targeted strategy aiming to disrupt tumor blood vessels was developed by combination of TF-targeted HMME-loaded drug delivery system and PDT. PDT is a promising new modality in the treatment of cancers, which employs the interaction between a tumor-localizing photosensitizer and light of an appropriate wavelength to bring about ROS-induced cell death. In vitro results showed that protein EGFP-EGF1modification could significantly contribute to the uptake of nanoparticles by TF over-expressed BCECs. In vivo multispectral fluorescent imaging, the EGFP-EGF1 conjugated nanoparticles showed significantly higher accumulation in tumor tissues than non-conjugated ones. Tumor tissue slides further presented that EGFP-EGF1 conjugated nanoparticles showed significantly higher accumulation in tumor vasculature than non-conjugated ones. In vitro study demonstrated that PDT increased TF expression of BCECs. In vivo imaging, ex vivo imaging and tumor tissue slides showed that PDT further contribute EGFP-EGF1-NP accumulation in tumor. These promising results indicated that PDT enhanced EGFP-EGF1modified PEG-PLGA nanoparticle accumulation in tumor vaculature. Considering that EGFP-EGF1 conjugation enhanced nanoparticles uptake by TF over-expressed endothelium and PDT increased endothelium TF expression. We conclude that PDT triggered a TF cascade targeted effect. A combination of both EGFP-EGF1 modification and PDT provided a positive feed-back target effect to tumor vessels and might have a great potential for tumor therapy.

Specificity Protein (Sp) Transcription Factors and Metformin Regulate Expression of the Long Non-coding RNA HULC

EPA Science Inventory

There is evidence that specificity protein 1 (Sp1) transcription factor (TF) regulates expression of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) cells. RNA interference (RNAi) studies showed that among several lncRNAs expressed in HepG2, SNU-449 and SK-Hep-1...

Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance.

PubMed

Bang, Seung Woon; Lee, Dong-Keun; Jung, Harin; Chung, Pil Joong; Kim, Youn Shic; Choi, Yang Do; Suh, Joo-Won; Kim, Ju-Kon

2018-05-21

Drought stress seriously impacts on plant development and productivity. Improvement of drought tolerance without yield penalty is a great challenge in crop biotechnology. Here, we report that the rice (Oryza sativa) homeodomain-leucine zipper transcription factor gene, OsTF1L (Oryza sativa transcription factor 1-like), is a key regulator of drought tolerance mechanisms. Overexpression of the OsTF1L in rice significantly increased drought tolerance at the vegetative stages of growth and promoted both effective photosynthesis and a reduction in the water loss rate under drought conditions. Importantly, the OsTF1L overexpressing plants showed a higher drought tolerance at the reproductive stage of growth with a higher grain yield than non-transgenic controls under field-drought conditions. Genome-wide analysis of OsTF1L overexpression plants revealed up-regulation of drought-inducible, stomatal movement and lignin biosynthetic genes. Overexpression of OsTF1L promoted accumulation of lignin in shoots, whereas the RNAi lines showed opposite patterns of lignin accumulation. OsTF1L is mainly expressed in outer cell layers including the epidermis, and the vasculature of the shoots, which coincides with areas of lignification. In addition, OsTF1L overexpression enhances stomatal closure under drought conditions resulted in drought tolerance. More importantly, OsTF1L directly bound to the promoters of lignin biosynthesis and drought-related genes involving poxN/PRX38, Nodulin protein, DHHC4, CASPL5B1 and AAA-type ATPase. Collectively, our results provide a new insight into the role of OsTF1L in enhancing drought tolerance through lignin biosynthesis and stomatal closure in rice. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Differential co-expression analysis of rheumatoid arthritis with microarray data.

PubMed

Wang, Kunpeng; Zhao, Liqiang; Liu, Xuefeng; Hao, Zhenyong; Zhou, Yong; Yang, Chuandong; Li, Hongqiang

2014-11-01

The aim of the present study was to investigate the underlying molecular mechanisms of rheumatoid arthritis (RA) using microarray expression profiles from osteoarthritis and RA patients, to improve diagnosis and treatment strategies for the condition. The gene expression profile of GSE27390 was downloaded from Gene Expression Omnibus, including 19 samples from patients with RA (n=9) or osteoarthritis (n=10). Firstly, the differentially expressed genes (DEGs) were obtained with the thresholds of |logFC|>1.0 and P<0.05, using the t‑test method in LIMMA package. Then, differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) were screened with q<0.25 by the differential coexpression analysis and differential regulation analysis of gene expression microarray data package. Secondly, pathway enrichment analysis for DCGs was performed by the Database for Annotation, Visualization and Integrated Discovery and the DCLs associated with RA were selected by comparing the obtained DCLs with known transcription factor (TF)-targets in the TRANSFAC database. Finally, the obtained TFs were mapped to the known TF-targets to construct the network using cytoscape software. A total of 1755 DEGs, 457 DCGs and 101988 DCLs were achieved and there were 20 TFs in the obtained six TF-target relations (STAT3-TNF, PBX1‑PLAU, SOCS3-STAT3, GATA1-ETS2, ETS1-ICAM4 and CEBPE‑GATA1) and 457 DCGs. A number of TF-target relations in the constructed network were not within DCLs when the TF and target gene were DCGs. The identified TFs may have an important role in the pathogenesis of RA and have the potential to be used as biomarkers for the development of novel diagnostic and therapeutic strategies for RA.

Detection of eQTL modules mediated by activity levels of transcription factors.

PubMed

Sun, Wei; Yu, Tianwei; Li, Ker-Chau

2007-09-01

Studies of gene expression quantitative trait loci (eQTL) in different organisms have shown the existence of eQTL hot spots: each being a small segment of DNA sequence that harbors the eQTL of a large number of genes. Two questions of great interest about eQTL hot spots arise: (1) which gene within the hot spot is responsible for the linkages, i.e. which gene is the quantitative trait gene (QTG)? (2) How does a QTG affect the expression levels of many genes linked to it? Answers to the first question can be offered by available biological evidence or by statistical methods. The second question is harder to address. One simple situation is that the QTG encodes a transcription factor (TF), which regulates the expression of genes linked to it. However, previous results have shown that TFs are not overrepresented in the eQTL hot spots. In this article, we consider the scenario that the propagation of genetic perturbation from a QTG to other linked genes is mediated by the TF activity. We develop a procedure to detect the eQTL modules (eQTL hot spots together with linked genes) that are compatible with this scenario. We first detect 27 eQTL modules from a yeast eQTL data, and estimate TF activity profiles using the method of Yu and Li (2005). Then likelihood ratio tests (LRTs) are conducted to find 760 relationships supporting the scenario of TF activity mediation: (DNA polymorphism --> cis-linked gene --> TF activity --> downstream linked gene). They are organized into 4 eQTL modules: an amino acid synthesis module featuring a cis-linked gene LEU2 and the mediating TF Leu3; a pheromone response module featuring a cis-linked gene GPA1 and the mediating TF Ste12; an energy-source control module featuring two cis-linked genes, GSY2 and HAP1, and the mediating TF Hap1; a mitotic exit module featuring four cis-linked genes, AMN1, CSH1, DEM1 and TOS1, and the mediating TF complex Ace2/Swi5. Gene Ontology is utilized to reveal interesting functional groups of the downstream genes in each module. Our methods are implemented in an R package: eqtl.TF, which includes source codes and relevant data. It can be freely downloaded at http://www.stat.ucla.edu/~sunwei/software.htm. http://www.stat.ucla.edu/~sunwei/yeast_eQTL_TF/supplementary.pdf.

Mechanisms and Evolution of Control Logic in Prokaryotic Transcriptional Regulation

PubMed Central

van Hijum, Sacha A. F. T.; Medema, Marnix H.; Kuipers, Oscar P.

2009-01-01

Summary: A major part of organismal complexity and versatility of prokaryotes resides in their ability to fine-tune gene expression to adequately respond to internal and external stimuli. Evolution has been very innovative in creating intricate mechanisms by which different regulatory signals operate and interact at promoters to drive gene expression. The regulation of target gene expression by transcription factors (TFs) is governed by control logic brought about by the interaction of regulators with TF binding sites (TFBSs) in cis-regulatory regions. A factor that in large part determines the strength of the response of a target to a given TF is motif stringency, the extent to which the TFBS fits the optimal TFBS sequence for a given TF. Advances in high-throughput technologies and computational genomics allow reconstruction of transcriptional regulatory networks in silico. To optimize the prediction of transcriptional regulatory networks, i.e., to separate direct regulation from indirect regulation, a thorough understanding of the control logic underlying the regulation of gene expression is required. This review summarizes the state of the art of the elements that determine the functionality of TFBSs by focusing on the molecular biological mechanisms and evolutionary origins of cis-regulatory regions. PMID:19721087

Diurnal Cycling Transcription Factors of Pineapple Revealed by Genome-Wide Annotation and Global Transcriptomic Analysis

PubMed Central

Sharma, Anupma; Wai, Ching Man; Ming, Ray

2017-01-01

Abstract Circadian clock provides fitness advantage by coordinating internal metabolic and physiological processes to external cyclic environments. Core clock components exhibit daily rhythmic changes in gene expression, and the majority of them are transcription factors (TFs) and transcription coregulators (TCs). We annotated 1,398 TFs from 67 TF families and 80 TCs from 20 TC families in pineapple, and analyzed their tissue-specific and diurnal expression patterns. Approximately 42% of TFs and 45% of TCs displayed diel rhythmic expression, including 177 TF/TCs cycling only in the nonphotosynthetic leaf tissue, 247 cycling only in the photosynthetic leaf tissue, and 201 cycling in both. We identified 68 TF/TCs whose cycling expression was tightly coupled between the photosynthetic and nonphotosynthetic leaf tissues. These TF/TCs likely coordinate key biological processes in pineapple as we demonstrated that this group is enriched in homologous genes that form the core circadian clock in Arabidopsis and includes a STOP1 homolog. Two lines of evidence support the important role of the STOP1 homolog in regulating CAM photosynthesis in pineapple. First, STOP1 responds to acidic pH and regulates a malate channel in multiple plant species. Second, the cycling expression pattern of the pineapple STOP1 and the diurnal pattern of malate accumulation in pineapple leaf are correlated. We further examined duplicate-gene retention and loss in major known circadian genes and refined their evolutionary relationships between pineapple and other plants. Significant variations in duplicate-gene retention and loss were observed for most clock genes in both monocots and dicots. PMID:28922793

The pig CYP2E1 promoter is activated by COUP-TF1 and HNF-1 and is inhibited by androstenone.

PubMed

Tambyrajah, Winston S; Doran, Elena; Wood, Jeffrey D; McGivan, John D

2004-11-15

Functional analysis of the pig cytochrome P4502E1 (CYP2E1) promoter identified two major activating elements. One corresponded to the hepatic nuclear factor 1 (HNF-1) consensus binding sequence at nucleotides -128/-98 and the other was located in the region -292/-266. The binding of proteins in pig liver nuclear extracts to a synthetic double-stranded oligonucleotide corresponding to this more distal activating sequence was studied by electrophoretic mobility shift assay. The minimum protein binding sequence was identified as TGTTCTGACCTCTGGG. Gel super-shift assays identified the protein binding to this site as chick ovalbumin upstream promoter transcription factor 1 (COUP-TF1). Androstenone inhibited promoter activity in transfection experiments only with constructs which included the COUP-TF1 binding site. Androstenone inhibited COUP-TF1 binding to synthetic oligonucleotides but did not affect HNF-1 binding. The results offer an explanation for the inhibition of CYP2E1 protein expression by androstenone in isolated pig hepatocytes and may be relevant to the low expression of hepatic CYP2E1 in those pigs which accumulate high levels of androstenone in vivo.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Rearing Mozambique tilapia in tidally-changing salinities: Effects on growth and the growth hormone/insulin-like growth factor I axis.

PubMed

Moorman, Benjamin P; Yamaguchi, Yoko; Lerner, Darren T; Grau, E Gordon; Seale, Andre P

2016-08-01

The growth hormone (GH)/insulin-like growth factor (IGF) axis plays a central role in the regulation of growth in teleosts and has been shown to be affected by acclimation salinity. This study was aimed at characterizing the effects of rearing tilapia, Oreochromis mossambicus, in a tidally-changing salinity on the GH/IGF axis and growth. Tilapia were raised in fresh water (FW), seawater (SW), or in a tidally-changing environment, in which salinity is switched between FW (TF) and SW (TS) every 6h, for 4months. Growth was measured over all time points recorded and fish reared in a tidally-changing environment grew significantly faster than other groups. The levels of circulating growth hormone (GH), insulin-like growth factor I (IGF-I), pituitary GH mRNA, gene expression of IGF-I, IGF-II, and growth hormone receptor 2 (GHR) in the muscle and liver were also determined. Plasma IGF-I was higher in FW and TS than in SW and TF tilapia. Pituitary GH mRNA was higher in TF and TS than in FW and SW tilapia. Gene expression of IGF-I in the liver and of GHR in both the muscle and liver changed between TF and TS fish. Fish growth was positively correlated with GH mRNA expression in the pituitary, and GHR mRNA expression in muscle and liver tissues. Our study indicates that rearing fish under tidally-changing salinities elicits a distinct pattern of endocrine regulation from that observed in fish reared in steady-state conditions, and may provide a new approach to increase tilapia growth rate and study the regulation of growth in euryhaline fish. Copyright © 2016 Elsevier Inc. All rights reserved.

Valsartan independent of AT₁ receptor inhibits tissue factor, TLR-2 and -4 expression by regulation of Egr-1 through activation of AMPK in diabetic conditions.

PubMed

Ha, Yu Mi; Park, Eun Jung; Kang, Young Jin; Park, Sang Won; Kim, Hye Jung; Chang, Ki Churl

2014-10-01

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)-1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT1R) can reduce tissue factor (TF) and toll-like receptor (TLR)-2 and -4 by regulating Egr-1 in THP-1 cells and aorta in streptozotocin-induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr-1, TF, TLR-2 and -4 which were significantly reduced by valsartan. HG increased Egr-1 expression by activation of PKC and ERK1/2 in THP-1 cells. Valsartan increased AMPK phosphorylation in a concentration and time-dependent manner via activation of LKB1. Valsartan inhibited Egr-1 without activation of PKC or ERK1/2. The reduced expression of Egr-1 by valsartan was reversed by either silencing Egr-1, or compound C, or DN-AMPK-transfected cells. Valsartan inhibited binding of NF-κB and Egr-1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF-α, IL-6 and IL-1β) production and NF-κB activity in HG-activated THP-1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT1R in THP-1 cells or CHO cells, which were devoid of AT1R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr-1, TLR-2, -4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin-induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr-1 regulation. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Valsartan independent of AT1 receptor inhibits tissue factor, TLR-2 and-4 expression by regulation of Egr-1 through activation of AMPK in diabetic conditions

PubMed Central

Ha, Yu Mi; Park, Eun Jung; Kang, Young Jin; Park, Sang Won; Kim, Hye Jung; Chang, Ki Churl

2014-01-01

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)-1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT1R) can reduce tissue factor (TF) and toll-like receptor (TLR)-2 and-4 by regulating Egr-1 in THP-1 cells and aorta in streptozotocin-induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr-1, TF, TLR-2 and-4 which were significantly reduced by valsartan. HG increased Egr-1 expression by activation of PKC and ERK1/2 in THP-1 cells. Valsartan increased AMPK phosphorylation in a concentration and time-dependent manner via activation of LKB1. Valsartan inhibited Egr-1 without activation of PKC or ERK1/2. The reduced expression of Egr-1 by valsartan was reversed by either silencing Egr-1, or compound C, or DN-AMPK-transfected cells. Valsartan inhibited binding of NF-κB and Egr-1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF-α, IL-6 and IL-1β) production and NF-κB activity in HG-activated THP-1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT1R in THP-1 cells or CHO cells, which were devoid of AT1R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr-1, TLR-2,-4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin-induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr-1 regulation. PMID:25109475

The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katayama, Seiichi; Ashizawa, Koji; Gohma, Hiroshi

2006-12-15

The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17{alpha}-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ER{alpha}-selective agonist), diarylpropionitrile (DPN, an ER{beta}-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE inmore » the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ER{alpha}-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ER{beta}-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.« less

Computational Identification of Diverse Mechanisms Underlying Transcription Factor-DNA Occupancy

PubMed Central

Cheng, Qiong; Kazemian, Majid; Pham, Hannah; Blatti, Charles; Celniker, Susan E.; Wolfe, Scot A.; Brodsky, Michael H.; Sinha, Saurabh

2013-01-01

ChIP-based genome-wide assays of transcription factor (TF) occupancy have emerged as a powerful, high-throughput method to understand transcriptional regulation, especially on a global scale. This has led to great interest in the underlying biochemical mechanisms that direct TF-DNA binding, with the ultimate goal of computationally predicting a TF's occupancy profile in any cellular condition. In this study, we examined the influence of various potential determinants of TF-DNA binding on a much larger scale than previously undertaken. We used a thermodynamics-based model of TF-DNA binding, called “STAP,” to analyze 45 TF-ChIP data sets from Drosophila embryonic development. We built a cross-validation framework that compares a baseline model, based on the ChIP'ed (“primary”) TF's motif, to more complex models where binding by secondary TFs is hypothesized to influence the primary TF's occupancy. Candidates interacting TFs were chosen based on RNA-SEQ expression data from the time point of the ChIP experiment. We found widespread evidence of both cooperative and antagonistic effects by secondary TFs, and explicitly quantified these effects. We were able to identify multiple classes of interactions, including (1) long-range interactions between primary and secondary motifs (separated by ≤150 bp), suggestive of indirect effects such as chromatin remodeling, (2) short-range interactions with specific inter-site spacing biases, suggestive of direct physical interactions, and (3) overlapping binding sites suggesting competitive binding. Furthermore, by factoring out the previously reported strong correlation between TF occupancy and DNA accessibility, we were able to categorize the effects into those that are likely to be mediated by the secondary TF's effect on local accessibility and those that utilize accessibility-independent mechanisms. Finally, we conducted in vitro pull-down assays to test model-based predictions of short-range cooperative interactions, and found that seven of the eight TF pairs tested physically interact and that some of these interactions mediate cooperative binding to DNA. PMID:23935523

Overexpression of COUP-TF1 in murine embryonic stem cells reduces retinoic acid-associated growth arrest and increases extraembryonic endoderm gene expression.

PubMed

Zhuang, Yong; Gudas, Lorraine J

2008-09-01

Vitamin A (retinol [Rol]) and its metabolites are essential for embryonic development. The Rol metabolite all-trans retinoic acid (RA) is a biologically active form of Rol. The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription-factors (COUP-TF) proteins have been implicated in the regulation of several important biological processes, such as embryonic development and neuronal cell differentiation. Because there is evidence that COUP-TFs function in the retinoid signaling network during development and differentiation, we generated murine embryonic stem (ES) cell lines which stably and constitutively overexpress COUP-TF1 (NR2F1) and we analyzed RA-induced differentiation. COUP-TF1 overexpression resulted in reduced RA-associated growth arrest. A 2.4+/-0.17-fold higher Nanog mRNA level was seen in COUP-TF1 overexpressing lines, as compared with wild-type (WT) ES cells, after a 72 hr RA treatment. We also showed that COUP-TF1 overexpression enhanced RA-induced extraembryonic endoderm gene expression. Specifically, COUP-TF1 overexpression increased mRNA levels of GATA6 by 3.3+/-0.3-fold, GATA4 by 3.6+/-0.1-fold, laminin B1 (LAMB1) by 3.4+/-0.1-fold, LAMC1 by 3.4+/-0.2-fold, Dab2 by 2.4+0.1-fold, and SOX17 by 2.5-fold at 72 hr after RA treatment plus LIF, as compared with the increases seen in WT ES cells. However, RA-induced neurogenesis was unaffected by COUP-TF1 overexpression, as shown by the equivalent levels of expression of NeuroD1, nestin, GAP43 and other neuronal markers. Our results revealed for the first time that COUP-TF1 is an important signaling molecule during vitamin A (Rol)-mediated very early stage of embryonic development.

Transferrin receptor 1 upregulation in primary tumor and downregulation in benign kidney is associated with progression and mortality in renal cell carcinoma patients

PubMed Central

Greene, Christopher J.; Attwood, Kristopher; Sharma, Nitika J.; Gross, Kenneth W.; Smith, Gary J.; Xu, Bo; Kauffman, Eric C.

2017-01-01

The central dysregulated pathway of clear cell (cc) renal cell carcinoma (RCC), the von Hippel Lindau/hypoxia inducible factor-α axis, is a key regulator of intracellular iron levels, however the role of iron uptake in human RCC tumorigenesis and progression remains unknown. We conducted a thorough, large-scale investigation of the expression and prognostic significance of the primary iron uptake protein, transferrin receptor 1 (TfR1/CD71/TFRC), in RCC patients. TfR1 immunohistochemistry was performed in over 1500 cores from 574 renal cell tumor patient tissues (primary tumors, matched benign kidneys, metastases) and non-neoplastic tissues from 36 different body sites. TfR1 levels in RCC tumors, particularly ccRCC, were significantly associated with adverse clinical prognostic features (anemia, lower body mass index, smoking), worse tumor pathology (size, stage, grade, multifocality, sarcomatoid dedifferentiation) and worse survival outcomes, including after adjustments for tumor pathology. Highest TfR1 tissue levels in the non-gravid body were detected in benign renal tubule epithelium. Opposite to TfR1 changes in the primary tumor, TfR1 levels in benign kidney dropped during tumor progression and were inversely associated with worse survival outcomes, independent of tumor pathology. Quantitative measurement of TfR1 subcellular localization in cell lines demonstrated mixed cytoplasmic and membranous expression with increased TfR1 in clusters in ccRCC versus benign renal cell lines. Results of this study support an important role for TfR1 in RCC progression and identify TfR1 as a novel RCC biomarker and therapeutic target. PMID:29291011

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

PubMed

Orellana, Renan; Kato, Sumie; Erices, Rafaela; Bravo, María Loreto; Gonzalez, Pamela; Oliva, Bárbara; Cubillos, Sofía; Valdivia, Andrés; Ibañez, Carolina; Brañes, Jorge; Barriga, María Isabel; Bravo, Erasmo; Alonso, Catalina; Bustamente, Eva; Castellon, Enrique; Hidalgo, Patricia; Trigo, Cesar; Panes, Olga; Pereira, Jaime; Mezzano, Diego; Cuello, Mauricio A; Owen, Gareth I

2015-04-15

An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Mechanism of the protective effects of the combined treatment with rhynchophylla total alkaloids and sinapine thiocyanate against a prothrombotic state caused by vascular endothelial cell inflammatory damage

PubMed Central

Li, Yunlun; Zhang, Xinya; Yang, Wenqing; Li, Chao; Chu, Yanjun; Jiang, Haiqiang; Shen, Zhenzhen

2017-01-01

The aim of the present study was to investigate the effect and the underlying mechanism of the combined treatment of rhynchophylla total alkaloids (RTA) and sinapine thiocyanate for protection against a prothrombotic state (PTS) associated with the tumor necrosis factor-alpha (TNF-α)-induced inflammatory injury of vascular endothelial cells (VECs). A TNF-α-induced VEC inflammatory injury model was established, and cell morphology of VECs was evaluated using scanning electron microscopy. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to examine the mRNA and protein expression of coagulation-related factors, including nuclear factor-κB (NF-κB), transforming growth factor-β1 (TGF-β1), tissue factor (TF), plasminogen activator inhibitor (PAI-1), protease-activation receptors (PAR-1) and protein kinase C (PKC-α) in VECs. Combined treatment with RTA and sinapine thiocyanate was demonstrated to reduce, to a varying extent, the mRNA and protein expression of NF-κB, TGF-β1, TF, PAR-1, PKC-α and PAI-1. Furthermore, combined treatment with RTA and sinapine thiocyanate was able to downregulate the expression of coagulation-related factors in injured VECs, thereby inhibiting the PTS induced by vascular endothelial injury. The underlying mechanism is partially associated with the TF-mediated activation of the thrombin-receptor signaling pathway that suppresses coagulation during inflammation and balances fibrinolysis in order to inhibit fibrin generation and deposition. PMID:28587383

Mechanism of the protective effects of the combined treatment with rhynchophylla total alkaloids and sinapine thiocyanate against a prothrombotic state caused by vascular endothelial cell inflammatory damage.

PubMed

Li, Yunlun; Zhang, Xinya; Yang, Wenqing; Li, Chao; Chu, Yanjun; Jiang, Haiqiang; Shen, Zhenzhen

2017-06-01

The aim of the present study was to investigate the effect and the underlying mechanism of the combined treatment of rhynchophylla total alkaloids (RTA) and sinapine thiocyanate for protection against a prothrombotic state (PTS) associated with the tumor necrosis factor-alpha (TNF-α)-induced inflammatory injury of vascular endothelial cells (VECs). A TNF-α-induced VEC inflammatory injury model was established, and cell morphology of VECs was evaluated using scanning electron microscopy. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to examine the mRNA and protein expression of coagulation-related factors, including nuclear factor-κB (NF-κB), transforming growth factor-β1 (TGF-β1), tissue factor (TF), plasminogen activator inhibitor (PAI-1), protease-activation receptors (PAR-1) and protein kinase C (PKC-α) in VECs. Combined treatment with RTA and sinapine thiocyanate was demonstrated to reduce, to a varying extent, the mRNA and protein expression of NF-κB, TGF-β1, TF, PAR-1, PKC-α and PAI-1. Furthermore, combined treatment with RTA and sinapine thiocyanate was able to downregulate the expression of coagulation-related factors in injured VECs, thereby inhibiting the PTS induced by vascular endothelial injury. The underlying mechanism is partially associated with the TF-mediated activation of the thrombin-receptor signaling pathway that suppresses coagulation during inflammation and balances fibrinolysis in order to inhibit fibrin generation and deposition.

Quantitative analysis of collagens and fibronectin expression in human right ventricular hypertrophy.

PubMed

Peters, T H; Sharma, H S; Yilmaz, E; Bogers, A J

1999-06-30

One of the main features in human tetralogy of Fallot (TF) is right ventricular hypertrophy (RVH) due to pressure (sub-pulmonary stenosis) and volume overload (ventricular septal defect). Currently, primary correction at a young age is the treatment of choice. To unravel the role of extracellular matrix in RVH, we examined myocardial expression of collagens and fibronectin in TF patients with primary correction (TF1, age 0.7 +/- 0.2 yr.), secondary surgery (TF2, age 36.9 +/- 4.6 yr), and in age-matched control patients. Sirius red staining quantified by video imaging showed significantly increased interstitial staining for collagens in both TF1 and TF2 groups as compared to respective controls. Fibronectin was expressed in extracellular spaces, perivascular regions, and in some cardiomyocytes. Quantitative analysis of fibronectin revealed increased expression in only TF1 group as compared to respective control. Our results indicate an increased amount of myocardial extracellular matrix deposition as a sign of fibrosis during RVH in patients with TF.

Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

PubMed

Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

1998-04-15

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes.

PubMed

Jacobs, Jelle; Atkins, Mardelle; Davie, Kristofer; Imrichova, Hana; Romanelli, Lucia; Christiaens, Valerie; Hulselmans, Gert; Potier, Delphine; Wouters, Jasper; Taskiran, Ibrahim I; Paciello, Giulia; González-Blas, Carmen B; Koldere, Duygu; Aibar, Sara; Halder, Georg; Aerts, Stein

2018-06-04

Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

Diurnal Cycling Transcription Factors of Pineapple Revealed by Genome-Wide Annotation and Global Transcriptomic Analysis.

PubMed

Sharma, Anupma; Wai, Ching Man; Ming, Ray; Yu, Qingyi

2017-09-01

Circadian clock provides fitness advantage by coordinating internal metabolic and physiological processes to external cyclic environments. Core clock components exhibit daily rhythmic changes in gene expression, and the majority of them are transcription factors (TFs) and transcription coregulators (TCs). We annotated 1,398 TFs from 67 TF families and 80 TCs from 20 TC families in pineapple, and analyzed their tissue-specific and diurnal expression patterns. Approximately 42% of TFs and 45% of TCs displayed diel rhythmic expression, including 177 TF/TCs cycling only in the nonphotosynthetic leaf tissue, 247 cycling only in the photosynthetic leaf tissue, and 201 cycling in both. We identified 68 TF/TCs whose cycling expression was tightly coupled between the photosynthetic and nonphotosynthetic leaf tissues. These TF/TCs likely coordinate key biological processes in pineapple as we demonstrated that this group is enriched in homologous genes that form the core circadian clock in Arabidopsis and includes a STOP1 homolog. Two lines of evidence support the important role of the STOP1 homolog in regulating CAM photosynthesis in pineapple. First, STOP1 responds to acidic pH and regulates a malate channel in multiple plant species. Second, the cycling expression pattern of the pineapple STOP1 and the diurnal pattern of malate accumulation in pineapple leaf are correlated. We further examined duplicate-gene retention and loss in major known circadian genes and refined their evolutionary relationships between pineapple and other plants. Significant variations in duplicate-gene retention and loss were observed for most clock genes in both monocots and dicots. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Transferrin receptor regulates pancreatic cancer growth by modulating mitochondrial respiration and ROS generation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Seung Min, E-mail: smjeong@catholic.ac.kr; Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea, Seoul 137-701; Hwang, Sunsook

2016-03-11

The transferrin receptor (TfR1) is upregulated in malignant cells and its expression is associated with cancer progression. Because of its pre-eminent role in cell proliferation, TfR1 has been an important target for the development of cancer therapy. Although TfR1 is highly expressed in pancreatic cancers, what it carries out in these refractory cancers remains poorly understood. Here we report that TfR1 supports mitochondrial respiration and ROS production in human pancreatic ductal adenocarcinoma (PDAC) cells, which is required for their tumorigenic growth. Elevated TfR1 expression in PDAC cells contributes to oxidative phosphorylation, which allows for the generation of ROS. Importantly, mitochondrial-derivedmore » ROS are essential for PDAC growth. However, exogenous iron supplement cannot rescue the defects caused by TfR1 knockdown. Moreover, we found that TfR1 expression determines PDAC cells sensitivity to oxidative stress. Together, our findings reveal that TfR1 can contribute to the mitochondrial respiration and ROS production, which have essential roles in growth and survival of pancreatic cancer. - Highlights: • Pancreatic ductal adenocarcinoma (PDAC) exhibits an elevated transferrin receptor (TfR1) expression in comparison with non-transformed pancreatic cells. • TfR1 is required for PDAC growth by regulating mitochondrial respiration and ROS production. • TfR1 functions as a determinant of cell viability to oxidative stress in PDAC cells.« less

The Role of Genome Accessibility in Transcription Factor Binding in Bacteria.

PubMed

Gomes, Antonio L C; Wang, Harris H

2016-04-01

ChIP-seq enables genome-scale identification of regulatory regions that govern gene expression. However, the biological insights generated from ChIP-seq analysis have been limited to predictions of binding sites and cooperative interactions. Furthermore, ChIP-seq data often poorly correlate with in vitro measurements or predicted motifs, highlighting that binding affinity alone is insufficient to explain transcription factor (TF)-binding in vivo. One possibility is that binding sites are not equally accessible across the genome. A more comprehensive biophysical representation of TF-binding is required to improve our ability to understand, predict, and alter gene expression. Here, we show that genome accessibility is a key parameter that impacts TF-binding in bacteria. We developed a thermodynamic model that parameterizes ChIP-seq coverage in terms of genome accessibility and binding affinity. The role of genome accessibility is validated using a large-scale ChIP-seq dataset of the M. tuberculosis regulatory network. We find that accounting for genome accessibility led to a model that explains 63% of the ChIP-seq profile variance, while a model based in motif score alone explains only 35% of the variance. Moreover, our framework enables de novo ChIP-seq peak prediction and is useful for inferring TF-binding peaks in new experimental conditions by reducing the need for additional experiments. We observe that the genome is more accessible in intergenic regions, and that increased accessibility is positively correlated with gene expression and anti-correlated with distance to the origin of replication. Our biophysically motivated model provides a more comprehensive description of TF-binding in vivo from first principles towards a better representation of gene regulation in silico, with promising applications in systems biology.

Clinical Significance of Tissue Factor and CD13 Double-Positive Microparticles in Sirs Patients with Trauma and Severe Sepsis.

PubMed

Matsumoto, Hisatake; Yamakawa, Kazuma; Ogura, Hiroshi; Koh, Taichin; Matsumoto, Naoya; Shimazu, Takeshi

2017-04-01

Activated immune cells such as monocytes are key factors in systemic inflammatory response syndrome (SIRS) following trauma and sepsis. Activated monocytes induce almost all tissue factor (TF) expression contributing to inflammation and coagulation. TF and CD13 double-positive microparticles (TF/CD13MPs) are predominantly released from these activated monocytes. This study aimed to evaluate TF/CD13MPs and assess their usefulness as a biomarker of pathogenesis in early SIRS following trauma and sepsis. This prospective study comprising 24 trauma patients, 25 severe sepsis patients, and 23 healthy controls was conducted from November 2012 to February 2015. Blood samples were collected from patients within 24 h after injury and diagnosis of severe sepsis and from healthy controls. Numbers of TF/CD13MPs were measured by flow cytometry immediately thereafter. Injury Severity Score (ISS) and Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores were calculated at patient enrollment. APACHE II and SOFA scores and International Society of Thrombosis and Haemostasis (ISTH) overt disseminated intravascular coagulation (DIC) diagnostic criteria algorithm were calculated at the time of enrollment of severe sepsis patients. Numbers of TF/CD13MPs were significantly increased in both trauma and severe sepsis patients versus controls and correlated significantly with ISS and APACHE II score in trauma patients and with APACHE II and ISTH DIC scores in severe sepsis patients. Increased numbers of TF/CD13MPs correlated significantly with severities in the acute phase in trauma and severe sepsis patients, suggesting that TF/CD13MPs are important in the pathogenesis of early SIRS following trauma and sepsis.

An efficient method to transcription factor binding sites imputation via simultaneous completion of multiple matrices with positional consistency.

PubMed

Guo, Wei-Li; Huang, De-Shuang

2017-08-22

Transcription factors (TFs) are DNA-binding proteins that have a central role in regulating gene expression. Identification of DNA-binding sites of TFs is a key task in understanding transcriptional regulation, cellular processes and disease. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) enables genome-wide identification of in vivo TF binding sites. However, it is still difficult to map every TF in every cell line owing to cost and biological material availability, which poses an enormous obstacle for integrated analysis of gene regulation. To address this problem, we propose a novel computational approach, TFBSImpute, for predicting additional TF binding profiles by leveraging information from available ChIP-seq TF binding data. TFBSImpute fuses the dataset to a 3-mode tensor and imputes missing TF binding signals via simultaneous completion of multiple TF binding matrices with positional consistency. We show that signals predicted by our method achieve overall similarity with experimental data and that TFBSImpute significantly outperforms baseline approaches, by assessing the performance of imputation methods against observed ChIP-seq TF binding profiles. Besides, motif analysis shows that TFBSImpute preforms better in capturing binding motifs enriched in observed data compared with baselines, indicating that the higher performance of TFBSImpute is not simply due to averaging related samples. We anticipate that our approach will constitute a useful complement to experimental mapping of TF binding, which is beneficial for further study of regulation mechanisms and disease.

Diversification of transcription factor-DNA interactions and the evolution of gene regulatory networks.

PubMed

Rogers, Julia M; Bulyk, Martha L

2018-04-25

Sequence-specific transcription factors (TFs) bind short DNA sequences in the genome to regulate the expression of target genes. In the last decade, numerous technical advances have enabled the determination of the DNA-binding specificities of many of these factors. Large-scale screens of many TFs enabled the creation of databases of TF DNA-binding specificities, typically represented as position weight matrices (PWMs). Although great progress has been made in determining and predicting binding specificities systematically, there are still many surprises to be found when studying a particular TF's interactions with DNA in detail. Paralogous TFs' binding specificities can differ in subtle ways, in a manner that is not immediately apparent from looking at their PWMs. These differences affect gene regulatory outputs and enable TFs to rewire transcriptional networks over evolutionary time. This review discusses recent observations made in the study of TF-DNA interactions that highlight the importance of continued in-depth analysis of TF-DNA interactions and their inherent complexity. This article is categorized under: Biological Mechanisms > Regulatory Biology. © 2018 Wiley Periodicals, Inc.

BloodChIP: a database of comparative genome-wide transcription factor binding profiles in human blood cells.

PubMed

Chacon, Diego; Beck, Dominik; Perera, Dilmi; Wong, Jason W H; Pimanda, John E

2014-01-01

The BloodChIP database (http://www.med.unsw.edu.au/CRCWeb.nsf/page/BloodChIP) supports exploration and visualization of combinatorial transcription factor (TF) binding at a particular locus in human CD34-positive and other normal and leukaemic cells or retrieval of target gene sets for user-defined combinations of TFs across one or more cell types. Increasing numbers of genome-wide TF binding profiles are being added to public repositories, and this trend is likely to continue. For the power of these data sets to be fully harnessed by experimental scientists, there is a need for these data to be placed in context and easily accessible for downstream applications. To this end, we have built a user-friendly database that has at its core the genome-wide binding profiles of seven key haematopoietic TFs in human stem/progenitor cells. These binding profiles are compared with binding profiles in normal differentiated and leukaemic cells. We have integrated these TF binding profiles with chromatin marks and expression data in normal and leukaemic cell fractions. All queries can be exported into external sites to construct TF-gene and protein-protein networks and to evaluate the association of genes with cellular processes and tissue expression.

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

PubMed Central

Ul-Haq, R; Chitambar, C R

1993-01-01

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8379943

Effect of cigarette smoke on monocyte procoagulant activity: Focus on platelet-derived brain-derived neurotrophic factor (BDNF).

PubMed

Amadio, Patrizia; Baldassarre, Damiano; Sandrini, Leonardo; Weksler, Babette B; Tremoli, Elena; Barbieri, Silvia S

2017-01-01

Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor

PubMed Central

Tang, Haiwang; Ivanciu, Lacramioara; Popescu, Narcis; Peer, Glenn; Hack, Erik; Lupu, Cristina; Taylor, Fletcher B.; Lupu, Florea

2007-01-01

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis. PMID:17640967

Synthesis of EF24-tripeptide chloromethyl ketone: a novel curcumin-related anticancer drug delivery system.

PubMed

Sun, Aiming; Shoji, Mamoru; Lu, Yang J; Liotta, Dennis C; Snyder, James P

2006-06-01

The blood coagulation cascade includes a step in which the soluble protein, factor VIIa (fVIIa), complexes with its transmembrane receptor, tissue factor (TF). The fVIIa/TF protein-protein complex is subsequently drawn into the cell by endocytosis. The observation that TF is aberrantly and abundantly expressed on many cancer cells offers an opportunity to specifically target those cells with an effective anticancer drug. Thus, we propose a new drug delivery system, drug-linker-Phe-Phe-Arg-mk-fVIIa, which can associate with TF on the surface of cancer cells, but release the cytotoxic agent in the cytoplasm. Synthetic procedures have been developed for the preparation of phenylalanine-phenylalanine-arginine chloromethyl ketone, (FFRck) followed by coupling with the cytotoxin EF24 and subsequently fVIIa to give EF-24-FFRmk-fVIIa. When breast cancer cells (MDA-MB-231) and human melanoma cells (RPMI-7951) are treated with the complex, the cells are arrested to a greater extent than EF24 alone by comparison with controls.

Mechanisms of Normal and Abnormal Endometrial Bleeding

PubMed Central

Lockwood, Charles J.

2011-01-01

Expression of tissue factor (TF), the primary initiator of coagulation, is enhanced in decidualized human endometrial stromal cells (HESC) during the progesterone-dominated luteal phase. Progesterone also augments a second HESC hemostatic factor, plasminogen activator inhibitor-1 (PAI-1). In contrast, progestins inhibit HESC matrix metalloproteinase (MMP)-1, 3 and 9 expression to stabilize endometrial stromal and vascular extracellular matrix. Through these mechanisms decidualized endometrium is rendered both hemostatic and resistant to excess trophoblast invasion in the mid-luteal phase and throughout gestation to prevent hemorrhage and accreta. In non-fertile cycles, progesterone withdrawal results in decreased HESC TF and PAI-expression and increased MMP activity and inflammatory cytokine production promoting the controlled hemorrhage of menstruation and related tissue sloughing. In contrast to these well ordered biochemical processes, unpredictable endometrial bleeding associated with anovulation reflects absence of progestational effects on TF, PAI-1 and MMP activity as well as unrestrained angiogenesis rendering the endometrium non-hemostatic, proteolytic and highly vascular. Abnormal bleeding associated with long-term progestin-only contraceptives results not from impaired hemostasis but from unrestrained angiogenesis leading to large fragile endometrial vessels. This abnormal angiogenesis reflects progestational inhibition of endometrial blood flow promoting local hypoxia and generation of reactive oxygen species that increase production of angiogenic factors such as vascular endothelial growth factor (VEGF) in HESCs and Angiopoietin-2 (Ang-2) in endometrial endothelial cells while decreasing HESC expression of angiostatic, Ang-1. The resulting vessel fragility promotes bleeding. Aberrant angiogenesis also underlies abnormal bleeding associated with myomas and endometrial polyps however there are gaps in our understanding of this pathology. PMID:21499503

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding deltaEF1.

PubMed

Miller, Myrna M; Jarosinski, Keith W; Schat, Karel A

2008-12-01

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (deltaEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and deltaEF1.

PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants.

PubMed

Chow, Chi-Nga; Zheng, Han-Qin; Wu, Nai-Yun; Chien, Chia-Hung; Huang, Hsien-Da; Lee, Tzong-Yi; Chiang-Hsieh, Yi-Fan; Hou, Ping-Fu; Yang, Tien-Yi; Chang, Wen-Chi

2016-01-04

Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Decreasing TfR1 expression reverses anemia and hepcidin suppression in β-thalassemic mice

PubMed Central

Li, Huihui; Choesang, Tenzin; Bao, Weili; Chen, Huiyong; Feola, Maria; Garcia-Santos, Daniel; Li, Jie; Sun, Shuming; Follenzi, Antonia; Pham, Petra; Liu, Jing; Zhang, Jinghua; Ponka, Prem; An, Xiuli; Mohandas, Narla; Fleming, Robert E.; Rivella, Stefano; Li, Guiyuan

2017-01-01

Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin’s effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/− mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1+/− haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice. PMID:28151426

Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network

PubMed Central

Gubelmann, Carine; Schwalie, Petra C; Raghav, Sunil K; Röder, Eva; Delessa, Tenagne; Kiehlmann, Elke; Waszak, Sebastian M; Corsinotti, Andrea; Udin, Gilles; Holcombe, Wiebke; Rudofsky, Gottfried; Trono, Didier; Wolfrum, Christian; Deplancke, Bart

2014-01-01

Adipose tissue is a key determinant of whole body metabolism and energy homeostasis. Unraveling the regulatory mechanisms underlying adipogenesis is therefore highly relevant from a biomedical perspective. Our current understanding of fat cell differentiation is centered on the transcriptional cascades driven by the C/EBP protein family and the master regulator PPARγ. To elucidate further components of the adipogenic gene regulatory network, we performed a large-scale transcription factor (TF) screen overexpressing 734 TFs in mouse pre-adipocytes and probed their effect on differentiation. We identified 22 novel pro-adipogenic TFs and characterized the top ranking TF, ZEB1, as being essential for adipogenesis both in vitro and in vivo. Moreover, its expression levels correlate with fat cell differentiation potential in humans. Genomic profiling further revealed that this TF directly targets and controls the expression of most early and late adipogenic regulators, identifying ZEB1 as a central transcriptional component of fat cell differentiation. DOI: http://dx.doi.org/10.7554/eLife.03346.001 PMID:25163748

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

Identifying Stress Transcription Factors Using Gene Expression and TF-Gene Association Data

PubMed Central

Wu, Wei-Sheng; Chen, Bor-Sen

2007-01-01

Unicellular organisms such as yeasts have evolved to survive environmental stresses by rapidly reorganizing the genomic expression program to meet the challenges of harsh environments. The complex adaptation mechanisms to stress remain to be elucidated. In this study, we developed Stress Transcription Factor Identification Algorithm (STFIA), which integrates gene expression and TF-gene association data to identify the stress transcription factors (TFs) of six kinds of stresses. We identified some general stress TFs that are in response to various stresses, and some specific stress TFs that are in response to one specific stress. The biological significance of our findings is validated by the literature. We found that a small number of TFs may be sufficient to control a wide variety of expression patterns in yeast under different stresses. Two implications can be inferred from this observation. First, the adaptation mechanisms to different stresses may have a bow-tie structure. Second, there may exist extensive regulatory cross-talk among different stress responses. In conclusion, this study proposes a network of the regulators of stress responses and their mechanism of action. PMID:20066130

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia

PubMed Central

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-01-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre-processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)-gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein-DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid-repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF-pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF-gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA-box binding protein associated factor 1 and CCCTC-binding factor, which may be potential therapeutic targets of AML. PMID:28498449

Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

PubMed Central

Ohkawara, Hiroshi; Ishibashi, Toshiyuki; Sugimoto, Koichi; Ikeda, Kazuhiko; Ogawa, Kazuei; Takeishi, Yasuchika

2014-01-01

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs. PMID:25162582

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver.

PubMed

Adrian, G S; Rivera, E V; Adrian, E K; Lu, Y; Buchanan, J; Herbert, D C; Weaker, F J; Walter, C A; Bowman, B H

1993-01-01

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

PubMed Central

Schütte, Judith; Wang, Huange; Antoniou, Stella; Jarratt, Andrew; Wilson, Nicola K; Riepsaame, Joey; Calero-Nieto, Fernando J; Moignard, Victoria; Basilico, Silvia; Kinston, Sarah J; Hannah, Rebecca L; Chan, Mun Chiang; Nürnberg, Sylvia T; Ouwehand, Willem H; Bonzanni, Nicola; de Bruijn, Marella FTR; Göttgens, Berthold

2016-01-01

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. DOI: http://dx.doi.org/10.7554/eLife.11469.001 PMID:26901438

Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

PubMed Central

Fang, Bin; Everett, Logan J.; Jager, Jennifer; Briggs, Erika; Armour, Sean M.; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A.

2014-01-01

SUMMARY Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ. PMID:25416951

Circadian enhancers coordinate multiple phases of rhythmic gene transcription in vivo.

PubMed

Fang, Bin; Everett, Logan J; Jager, Jennifer; Briggs, Erika; Armour, Sean M; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A

2014-11-20

Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of enhancer RNAs (eRNAs) that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ.

MAGIA2: from miRNA and genes expression data integrative analysis to microRNA–transcription factor mixed regulatory circuits (2012 update)

PubMed Central

Bisognin, Andrea; Sales, Gabriele; Coppe, Alessandro; Bortoluzzi, Stefania; Romualdi, Chiara

2012-01-01

MAGIA2 (http://gencomp.bio.unipd.it/magia2) is an update, extension and evolution of the MAGIA web tool. It is dedicated to the integrated analysis of in silico target prediction, microRNA (miRNA) and gene expression data for the reconstruction of post-transcriptional regulatory networks. miRNAs are fundamental post-transcriptional regulators of several key biological and pathological processes. As miRNAs act prevalently through target degradation, their expression profiles are expected to be inversely correlated to those of the target genes. Low specificity of target prediction algorithms makes integration approaches an interesting solution for target prediction refinement. MAGIA2 performs this integrative approach supporting different association measures, multiple organisms and almost all target predictions algorithms. Nevertheless, miRNAs activity should be viewed as part of a more complex scenario where regulatory elements and their interactors generate a highly connected network and where gene expression profiles are the result of different levels of regulation. The updated MAGIA2 tries to dissect this complexity by reconstructing mixed regulatory circuits involving either miRNA or transcription factor (TF) as regulators. Two types of circuits are identified: (i) a TF that regulates both a miRNA and its target and (ii) a miRNA that regulates both a TF and its target. PMID:22618880

Genome-wide patterns of promoter sharing and co-expression in bovine skeletal muscle.

PubMed

Gu, Quan; Nagaraj, Shivashankar H; Hudson, Nicholas J; Dalrymple, Brian P; Reverter, Antonio

2011-01-12

Gene regulation by transcription factors (TF) is species, tissue and time specific. To better understand how the genetic code controls gene expression in bovine muscle we associated gene expression data from developing Longissimus thoracis et lumborum skeletal muscle with bovine promoter sequence information. We created a highly conserved genome-wide promoter landscape comprising 87,408 interactions relating 333 TFs with their 9,242 predicted target genes (TGs). We discovered that the complete set of predicted TGs share an average of 2.75 predicted TF binding sites (TFBSs) and that the average co-expression between a TF and its predicted TGs is higher than the average co-expression between the same TF and all genes. Conversely, pairs of TFs sharing predicted TGs showed a co-expression correlation higher that pairs of TFs not sharing TGs. Finally, we exploited the co-occurrence of predicted TFBS in the context of muscle-derived functionally-coherent modules including cell cycle, mitochondria, immune system, fat metabolism, muscle/glycolysis, and ribosome. Our findings enabled us to reverse engineer a regulatory network of core processes, and correctly identified the involvement of E2F1, GATA2 and NFKB1 in the regulation of cell cycle, fat, and muscle/glycolysis, respectively. The pivotal implication of our research is two-fold: (1) there exists a robust genome-wide expression signal between TFs and their predicted TGs in cattle muscle consistent with the extent of promoter sharing; and (2) this signal can be exploited to recover the cellular mechanisms underpinning transcription regulation of muscle structure and development in bovine. Our study represents the first genome-wide report linking tissue specific co-expression to co-regulation in a non-model vertebrate.

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid

PubMed Central

Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

2015-01-01

Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5′-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5′-TGTAAGCCCTAACA-3′) upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network. PMID:26581248

Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry.

PubMed

Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H; Petersen, Lars C; Kristensen, Jesper B; Behrens, Carsten; Madsen, Jacob; Kjaer, Andreas

2018-01-17

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64 Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64 Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64 Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Ovine uterine space restriction alters placental transferrin receptor and fetal iron status during late pregnancy

PubMed Central

Sun, Mary Y.; Habeck, Jason M.; Meyer, Katie M.; Koch, Jill M.; Ramadoss, Jayanth; Blohowiak, Sharon E.; Magness, Ronald R.; Kling, Pamela J.

2013-01-01

Background Fetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans, but is unstudied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial NOS (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron. Methods An ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non-space restricted (NSR) singletons were compared to USR fetuses at 120 or 130 days of gestation (GD). Results When expressed proportionate to fetal weight, liver iron content did not differ while renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport. Conclusion In conclusion, fetal iron was regulated in an organ-specific fashion. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130. PMID:23202722

Seasonal Abscisic Acid Signal and a Basic Leucine Zipper Transcription Factor, DkbZIP5, Regulate Proanthocyanidin Biosynthesis in Persimmon Fruit1[C][W][OA

PubMed Central

Akagi, Takashi; Katayama-Ikegami, Ayako; Kobayashi, Shozo; Sato, Akihiko; Kono, Atsushi; Yonemori, Keizo

2012-01-01

Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5. PMID:22190340

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

PubMed

Holy, Erik W; Besler, Christian; Reiner, Martin F; Camici, Giovanni G; Manz, Jasmin; Beer, Jürg H; Lüscher, Thomas F; Landmesser, Ulf; Tanner, Felix C

2014-11-01

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

PubMed

Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

2013-03-01

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of bax protein in sphingosine-induced apoptosis in the human leukemic cell line TF1 and its bcl-2 transfectants.

PubMed

Isogai, C; Murate, T; Tamiya-Koizumi, K; Yoshida, S; Ito, T; Nagai, H; Kinoshita, T; Kagami, Y; Hotta, T; Hamaguchi, M; Saito, H

1998-11-01

Sphingosine, a sphingolipid breakdown product, has been proposed as an apoptosis-inducing agent. In this study, we examined the effect of sphingosine in bcl-2-overexpressing cells compared with cells that do not express the bcl-2 gene. The human erythroleukemic cell line TF1, which lacks bcl-2 expression, was easily induced to undergo apoptotic cell death by a variety of stimuli, including depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) or exposure to methylmethane sulfonate (MMS) (100 microg/mL), ultraviolet light (15 J/m2), X-ray irradiation (20 Gy), or sphingosine, a sphingolipid breakdown product (5 microM). In contrast, bcl-2 transfectants of TF1 (TF1-bcl2), which we established, were resistant to most of these treatments but remained sensitive to sphingosine. Neither C2- nor C6-ceramide (short-chain ceramide) induced apoptosis in TF1-mock and TF1-bcl2 cells. Sphingosine-induced apoptosis could not be inhibited by fumonisin B1, which can prevent conversion of sphingosine to ceramide, suggesting that sphingosine itself, not ceramide, possesses apoptosis-inducing capability. Western blotting, which revealed a 21-kDa bax protein in untreated cells, revealed the presence of an additional 18-kDa protein in GM-CSF-depleted and MMS- or sphingosine-treated TF1-mock cells. In TF1-bcl2 cells, this protein was not detected after GM-CSF depletion or MMS treatment, but was observed after sphingosine treatment. Immunoprecipitation with anti-bcl2 antibody, followed by immunoblotting with anti-bax antibody, showed that both the 21-kDa bax protein and the 18-kDa protein heterodimerized with bcl-2 protein. These results suggest that sphingosine is a unique reagent for apoptosis and that it can overcome bcl-2 gene expression. Furthermore, induction of 18-kDa bax-related protein may play an important role in apoptosis. Sphingosine, but not ceramide, may prove applicable as a reagent for future cytotoxic drugs used to treat intractable tumors overexpressing bcl-2.

Predicting the binding preference of transcription factors to individual DNA k-mers.

PubMed

Alleyne, Trevis M; Peña-Castillo, Lourdes; Badis, Gwenael; Talukder, Shaheynoor; Berger, Michael F; Gehrke, Andrew R; Philippakis, Anthony A; Bulyk, Martha L; Morris, Quaid D; Hughes, Timothy R

2009-04-15

Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA-protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF-DNA recognition, and suggest a rational approach for future analyses of TF families.

Gallium Maltolate Disrupts Tumor Iron Metabolism and Retards the Growth of Glioblastoma by Inhibiting Mitochondrial Function and Ribonucleotide Reductase.

PubMed

Chitambar, Christopher R; Al-Gizawiy, Mona M; Alhajala, Hisham S; Pechman, Kimberly R; Wereley, Janine P; Wujek, Robert; Clark, Paul A; Kuo, John S; Antholine, William E; Schmainda, Kathleen M

2018-06-01

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) in vitro After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control ( P = 0.0159) and reduced tumor mitotic figures ( P = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells in vitro We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma in vivo, thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. Mol Cancer Ther; 17(6); 1240-50. ©2018 AACR . ©2018 American Association for Cancer Research.

A transcription factor hierarchy defines an environmental stress response network.

PubMed

Song, Liang; Huang, Shao-Shan Carol; Wise, Aaron; Castanon, Rosa; Nery, Joseph R; Chen, Huaming; Watanabe, Marina; Thomas, Jerushah; Bar-Joseph, Ziv; Ecker, Joseph R

2016-11-04

Environmental stresses are universally encountered by microbes, plants, and animals. Yet systematic studies of stress-responsive transcription factor (TF) networks in multicellular organisms have been limited. The phytohormone abscisic acid (ABA) influences the expression of thousands of genes, allowing us to characterize complex stress-responsive regulatory networks. Using chromatin immunoprecipitation sequencing, we identified genome-wide targets of 21 ABA-related TFs to construct a comprehensive regulatory network in Arabidopsis thaliana Determinants of dynamic TF binding and a hierarchy among TFs were defined, illuminating the relationship between differential gene expression patterns and ABA pathway feedback regulation. By extrapolating regulatory characteristics of observed canonical ABA pathway components, we identified a new family of transcriptional regulators modulating ABA and salt responsiveness and demonstrated their utility to modulate plant resilience to osmotic stress. Copyright © 2016, American Association for the Advancement of Science.

Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice: involvement of tissue factor, inducible NO synthase and nuclear factor (NF)-κB.

PubMed

Sun, Qi; Chen, Ling; Gao, Mengyu; Jiang, Wenwen; Shao, Fangxian; Li, Jingjing; Wang, Jun; Kou, Junping; Yu, Boyang

2012-01-01

Acute lung injury is still a significant clinical problem with a high mortality rate and there are few effective therapies in clinic. Here, we studied the inhibitory effect of ruscogenin, an anti-inflammatory and anti-thrombotic natural product, on lipopolysaccharide (LPS)-induced acute lung injury in mice basing on our previous studies. The results showed that a single oral administration of ruscogenin significantly decreased lung wet to dry weight (W/D) ratio at doses of 0.3, 1.0 and 3.0 mg/kg 1 h prior to LPS challenge (30 mg/kg, intravenous injection). Histopathological changes such as pulmonary edema, coagulation and infiltration of inflammatory cells were also attenuated by ruscogenin. In addition, ruscogenin markedly decreased LPS-induced myeloperoxidase (MPO) activity and nitrate/nitrite content, and also downregulated expression of tissue factor (TF), inducible NO synthase (iNOS) and nuclear factor (NF)-κB p-p65 (Ser 536) in the lung tissue at three doses. Furthermore, ruscogenin reduced plasma TF procoagulant activity and nitrate/nitrite content in LPS-induced ALI mice. These findings confirmed that ruscogenin significantly attenuate LPS-induced acute lung injury via inhibiting expressions of TF and iNOS and NF-κB p65 activation, indicating it as a potential therapeutic agent for ALI or sepsis. Copyright © 2011 Elsevier B.V. All rights reserved.

The transcription factor titration effect dictates level of gene expression.

PubMed

Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

2014-03-13

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

On the Design of VLSI Circuits for the Winograd Fourier Transform Algorithm

DTIC Science & Technology

1991-12-01

3-:3 T’able 8: Twiddle factors in TF1 (real side) ... .. .. .. .. .... ... .. .. ...- 4 T;l1)a1e 9: Twiddle factors I n... TF1 (imaginary side) .. .. .. .. ... ... .... .. 13-5 ’fable 10: Twiddle factors in TF2 (r’eal side) ... .. .. .. .. .... ... .... .. 13-6 ’Table 11...reads its twiddle factor from Y. The four other possibilities (TFO, TF1 , TF2, and TF3) correspond to the fixed values that are necessary for computing 20

Curated collection of yeast transcription factor DNA binding specificity data reveals novel structural and gene regulatory insights

PubMed Central

2011-01-01

Background Transcription factors (TFs) play a central role in regulating gene expression by interacting with cis-regulatory DNA elements associated with their target genes. Recent surveys have examined the DNA binding specificities of most Saccharomyces cerevisiae TFs, but a comprehensive evaluation of their data has been lacking. Results We analyzed in vitro and in vivo TF-DNA binding data reported in previous large-scale studies to generate a comprehensive, curated resource of DNA binding specificity data for all characterized S. cerevisiae TFs. Our collection comprises DNA binding site motifs and comprehensive in vitro DNA binding specificity data for all possible 8-bp sequences. Investigation of the DNA binding specificities within the basic leucine zipper (bZIP) and VHT1 regulator (VHR) TF families revealed unexpected plasticity in TF-DNA recognition: intriguingly, the VHR TFs, newly characterized by protein binding microarrays in this study, recognize bZIP-like DNA motifs, while the bZIP TF Hac1 recognizes a motif highly similar to the canonical E-box motif of basic helix-loop-helix (bHLH) TFs. We identified several TFs with distinct primary and secondary motifs, which might be associated with different regulatory functions. Finally, integrated analysis of in vivo TF binding data with protein binding microarray data lends further support for indirect DNA binding in vivo by sequence-specific TFs. Conclusions The comprehensive data in this curated collection allow for more accurate analyses of regulatory TF-DNA interactions, in-depth structural studies of TF-DNA specificity determinants, and future experimental investigations of the TFs' predicted target genes and regulatory roles. PMID:22189060

The significance of measuring monocyte tissue factor activity in patients with breast and colorectal cancer

PubMed Central

Lwaleed, B A; Chisholm, M; Francis, J L

1999-01-01

Monocytes express tissue factor (mTF) in several conditions including cancer where levels may be valuable in assessing tumour presence and progression. Using a two-stage kinetic chromogenic assay (KCA), mTF levels were measured in controls [normal subjects (n = 60) and patients undergoing hernia repair or cholecystectomy (n = 60)], in patients with benign and malignant disease of the breast (n = 83) and of the large bowel (n = 62). This was performed under fresh (resting) conditions and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin. The malignant groups showed higher mTF levels than each of the three controls for resting (P < 0.05 breast, P < 0.05 colorectal) unstimulated (P < 0.05 breast, P < 0.05 colorectal) and stimulated cells (P < 0.001 breast, P < 0.01 colorectal). Similarly, the benign inflammatory groups had higher mTF levels than controls for resting (P < 0.05 colorectal), unstimulated (P < 0.05 colorectal) and stimulated cells (P < 0.01 breast, P < 0.01 colorectal). There was no significant difference between malignant and benign inflammatory groups in each organ. mTF levels showed an increase corresponding to that of histological tumour progression and were higher in non-surviving patients. In conclusion, mTF levels are raised in malignant and inflammatory disease compared to controls and patients with non-inflammatory conditions. Stimulated cells give better discrimination between the groups and may be of value in identifying high risk individuals. mTF levels showed an association with tumour grade or stage and the patients' survival time. © 1999 Cancer Research Campaign PMID:10390009

PlantTFDB: a comprehensive plant transcription factor database

PubMed Central

Guo, An-Yuan; Chen, Xin; Gao, Ge; Zhang, He; Zhu, Qi-Hui; Liu, Xiao-Chuan; Zhong, Ying-Fu; Gu, Xiaocheng; He, Kun; Luo, Jingchu

2008-01-01

Transcription factors (TFs) play key roles in controlling gene expression. Systematic identification and annotation of TFs, followed by construction of TF databases may serve as useful resources for studying the function and evolution of transcription factors. We developed a comprehensive plant transcription factor database PlantTFDB (http://planttfdb.cbi.pku.edu.cn), which contains 26 402 TFs predicted from 22 species, including five model organisms with available whole genome sequence and 17 plants with available EST sequences. To provide comprehensive information for those putative TFs, we made extensive annotation at both family and gene levels. A brief introduction and key references were presented for each family. Functional domain information and cross-references to various well-known public databases were available for each identified TF. In addition, we predicted putative orthologs of those TFs among the 22 species. PlantTFDB has a simple interface to allow users to search the database by IDs or free texts, to make sequence similarity search against TFs of all or individual species, and to download TF sequences for local analysis. PMID:17933783

Dynamic CRM occupancy reflects a temporal map of developmental progression.

PubMed

Wilczyński, Bartek; Furlong, Eileen E M

2010-06-22

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.

Innate lymphoid cells in normal and disease: An introductory overview.

PubMed

Moretta, Lorenzo; Locatelli, Franco

2016-11-01

Innate lymphoid cells (ILC) represent a novel group of lymphocytes that, different from T and B-lymphocytes lack recombinant activating genes (RAG-1 or RAG-2) and thus do not express rearranged antigen-specific receptors. Members of this family, i.e. NK cells, have been known since long time, while the other ILCs have been discovered only in recent years, possibly because of their predominant localization in tissues, primarily in mucosal tissues, skin and mucosa-associated lymphoid organs. ILC have been grouped in three major subsets on the basis of their phenotypic and functional features as well as of their dependency on given transcription factors (TF). Briefly, ILC-1 are dependent on T-bet TF and produce interferon (IFN)-γ. Group 2 ILC (ILC2) express GATA-3 TF and produce IL-5, IL-4 and IL-13 (Type 2) cytokines while group 3 ILC (ILC3) express RORγt TF and produce IL-17 and IL-22. ILC provide early defenses against pathogens and intervene in the repair of damaged tissues. ILC activation is mediated by cytokines (specifically acting on different ILC groups) and/or by activating receptors that are, at least in part, the same that had been previously identified in NK cells [1]. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

MiRNA and TF co-regulatory network analysis for the pathology and recurrence of myocardial infarction.

PubMed

Lin, Ying; Sibanda, Vusumuzi Leroy; Zhang, Hong-Mei; Hu, Hui; Liu, Hui; Guo, An-Yuan

2015-04-13

Myocardial infarction (MI) is a leading cause of death in the world and many genes are involved in it. Transcription factor (TFs) and microRNAs (miRNAs) are key regulators of gene expression. We hypothesized that miRNAs and TFs might play combinatory regulatory roles in MI. After collecting MI candidate genes and miRNAs from various resources, we constructed a comprehensive MI-specific miRNA-TF co-regulatory network by integrating predicted and experimentally validated TF and miRNA targets. We found some hub nodes (e.g. miR-16 and miR-26) in this network are important regulators, and the network can be severed as a bridge to interpret the associations of previous results, which is shown by the case of miR-29 in this study. We also constructed a regulatory network for MI recurrence and found several important genes (e.g. DAB2, BMP6, miR-320 and miR-103), the abnormal expressions of which may be potential regulatory mechanisms and markers of MI recurrence. At last we proposed a cellular model to discuss major TF and miRNA regulators with signaling pathways in MI. This study provides more details on gene expression regulation and regulators involved in MI progression and recurrence. It also linked up and interpreted many previous results.

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

PubMed

Witsø, Ingun Lund; Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

2016-01-01

Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli.

Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling

PubMed Central

Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

2016-01-01

Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli. PMID:27309855

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

PubMed

Rae, C; MacEwan, D J

2004-12-01

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

microRNA as a Potential Vector for the Propagation of Robustness in Protein Expression and Oscillatory Dynamics within a ceRNA Network

PubMed Central

Gérard, Claude; Novák, Béla

2013-01-01

microRNAs (miRNAs) are small noncoding RNAs that are important post-transcriptional regulators of gene expression. miRNAs can induce thresholds in protein synthesis. Such thresholds in protein output can be also achieved by oligomerization of transcription factors (TF) for the control of gene expression. First, we propose a minimal model for protein expression regulated by miRNA and by oligomerization of TF. We show that miRNA and oligomerization of TF generate a buffer, which increases the robustness of protein output towards molecular noise as well as towards random variation of kinetics parameters. Next, we extend the model by considering that the same miRNA can bind to multiple messenger RNAs, which accounts for the dynamics of a minimal competing endogenous RNAs (ceRNAs) network. The model shows that, through common miRNA regulation, TF can control the expression of all proteins formed by the ceRNA network, even if it drives the expression of only one gene in the network. The model further suggests that the threshold in protein synthesis mediated by the oligomerization of TF can be propagated to the other genes, which can increase the robustness of the expression of all genes in such ceRNA network. Furthermore, we show that a miRNA could increase the time delay of a “Goodwin-like” oscillator model, which may favor the occurrence of oscillations of large amplitude. This result predicts important roles of miRNAs in the control of the molecular mechanisms leading to the emergence of biological rhythms. Moreover, a model for the latter oscillator embedded in a ceRNA network indicates that the oscillatory behavior can be propagated, via the shared miRNA, to all proteins formed by such ceRNA network. Thus, by means of computational models, we show that miRNAs could act as vectors allowing the propagation of robustness in protein synthesis as well as oscillatory behaviors within ceRNA networks. PMID:24376695

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Yiming; Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Sullenbarger, Brent

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis;more » however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.« less

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-specific repression mechanism that so far has not been characterised for this class of mesodermal CRMs. PMID:23144600

Exploring the bZIP transcription factor regulatory network in Neurospora crassa

PubMed Central

Tian, Chaoguang; Li, Jingyi; Glass, N. Louise

2011-01-01

Transcription factors (TFs) are key nodes of regulatory networks in eukaryotic organisms, including filamentous fungi such as Neurospora crassa. The 178 predicted DNA-binding TFs in N. crassa are distributed primarily among six gene families, which represent an ancient expansion in filamentous ascomycete genomes; 98 TF genes show detectable expression levels during vegetative growth of N. crassa, including 35 that show a significant difference in expression level between hyphae at the periphery versus hyphae in the interior of a colony. Regulatory networks within a species genome include paralogous TFs and their respective target genes (TF regulon). To investigate TF network evolution in N. crassa, we focused on the basic leucine zipper (bZIP) TF family, which contains nine members. We performed baseline transcriptional profiling during vegetative growth of the wild-type and seven isogenic, viable bZIP deletion mutants. We further characterized the regulatory network of one member of the bZIP family, NCU03905. NCU03905 encodes an Ap1-like protein (NcAp-1), which is involved in resistance to multiple stress responses, including oxidative and heavy metal stress. Relocalization of NcAp-1 from the cytoplasm to the nucleus was associated with exposure to stress. A comparison of the NcAp-1 regulon with Ap1-like regulons in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus showed both conservation and divergence. These data indicate how N. crassa responds to stress and provide information on pathway evolution. PMID:21081763

Exploring the bZIP transcription factor regulatory network in Neurospora crassa.

PubMed

Tian, Chaoguang; Li, Jingyi; Glass, N Louise

2011-03-01

Transcription factors (TFs) are key nodes of regulatory networks in eukaryotic organisms, including filamentous fungi such as Neurospora crassa. The 178 predicted DNA-binding TFs in N. crassa are distributed primarily among six gene families, which represent an ancient expansion in filamentous ascomycete genomes; 98 TF genes show detectable expression levels during vegetative growth of N. crassa, including 35 that show a significant difference in expression level between hyphae at the periphery versus hyphae in the interior of a colony. Regulatory networks within a species genome include paralogous TFs and their respective target genes (TF regulon). To investigate TF network evolution in N. crassa, we focused on the basic leucine zipper (bZIP) TF family, which contains nine members. We performed baseline transcriptional profiling during vegetative growth of the wild-type and seven isogenic, viable bZIP deletion mutants. We further characterized the regulatory network of one member of the bZIP family, NCU03905. NCU03905 encodes an Ap1-like protein (NcAp-1), which is involved in resistance to multiple stress responses, including oxidative and heavy metal stress. Relocalization of NcAp-1 from the cytoplasm to the nucleus was associated with exposure to stress. A comparison of the NcAp-1 regulon with Ap1-like regulons in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus showed both conservation and divergence. These data indicate how N. crassa responds to stress and provide information on pathway evolution.

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation.

PubMed

Sasaki, Katsutomo; Yamaguchi, Hiroyasu; Aida, Ryutaro; Shikata, Masahito; Abe, Tomoko; Ohtsubo, Norihiro

2012-09-01

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Ethylene-responsive element-binding factor 5, ERF5, is involved in chitin-induced innate immunity response.

PubMed

Son, Geon Hui; Wan, Jinrong; Kim, Hye Jin; Nguyen, Xuan Canh; Chung, Woo Sik; Hong, Jong Chan; Stacey, Gary

2012-01-01

Our recent work demonstrated that chitin treatment modulated the expression of 118 transcription factor (TF) genes in Arabidopsis. To investigate the potential roles of these TF in chitin signaling and plant defense, we initiated an interaction study among these TF proteins, as well as two chitin-activated mitogen-activated protein kinases (MPK3 and MPK6), using a yeast two-hybrid system. This study revealed interactions among the following proteins: three ethylene-responsive element-binding factors (ERF), five WRKY transcription factors, one scarecrow-like (SCL), and the two MPK, in addition to many other interactions, reflecting a complex TF interaction network. Most of these interactions were subsequently validated by other methods, such as pull-down and in planta bimolecular fluorescence complementation assays. The key node ERF5 was shown to interact with multiple proteins in the network, such as ERF6, ERF8, and SCL13, as well as MPK3 and MPK6. Interestingly, ERF5 appeared to negatively regulate chitin signaling and plant defense against the fungal pathogen Alternaria brassicicola and positively regulate salicylic acid signaling and plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Therefore, ERF5 may play an important role in plant innate immunity, likely through coordinating chitin and other defense pathways in plants in response to different pathogens.

Transcription factor trapping by RNA in gene regulatory elements.

PubMed

Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Carbohydrates and activity of natural and recombinant tissue factor.

PubMed

Krudysz-Amblo, Jolanta; Jennings, Mark E; Mann, Kenneth G; Butenas, Saulius

2010-01-29

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF.factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (V(max)) of the complex increased in the order rTF(1-243) (Escherichia coli) < rTF(1-263) (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K(m) of FX for rTF(1-263)-FVIIa remained unchanged after deglycosylation, whereas the k(cat) decreased slightly. A pronounced decrease, 4-fold, in k(cat) was observed for pTF.FVIIa upon deglycosylation, whereas the K(m) was minimally altered. The parameters of FX activation by both rTF(1-263D)-FVIIa and pTF(D)-FVIIa were identical and similar to those for rTF(1-243)-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF(1-263) and pTF. The carbohydrates of rTF(1-263) contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.

Lung fibrosis-associated soluble mediators and bronchoalveolar lavage from idiopathic pulmonary fibrosis patients promote the expression of fibrogenic factors in subepithelial lung myofibroblasts.

PubMed

Bouros, Evangelos; Filidou, Eirini; Arvanitidis, Konstantinos; Mikroulis, Dimitrios; Steiropoulos, Paschalis; Bamias, George; Bouros, Demosthenes; Kolios, George

2017-10-01

Idiopathic pulmonary fibrosis (IPF) is characterized by infiltration of inflammatory cells, excessive collagen production and accumulation of myofibroblasts. We explored the possible role of subepithelial lung myofibroblasts (SELMs) in the development of fibrosis in IPF. SELMs, isolated from surgical specimens of healthy lung tissue, were cultured with pro-inflammatory factors or bronchoalveolar lavage fluid (BALF) from patients with IPF or idiopathic non-specific interstitial pneumonia (iNSIP) and their fibrotic activity was assessed. Stimulation of SELMs with pro-inflammatory factors induced a significant increase of Tissue Factor (TF) and Tumor necrosis factor-Like cytokine 1 A (TL1A) expression and collagen production in culture supernatants. Stimulation with BALF from IPF patients with mild to moderate, but not severe disease, and from iNSIP patients induced a significant increase of TF expression. BALF from all IPF patients induced a significant increase of TL1A expression and collagen production, while BALF from iNSIP patients induced a significant increase of TL1A, but not of collagen production. Interestingly, TGF-β1 and BALF from all IPF, but not iNSIP patients, induced a significant increase in SELMs migration. In conclusion, BALF from IPF patients induces fibrotic activity in lung myofibroblasts, similar to mediators associated with lung fibrosis, indicating a key role of SELMs in IPF. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gypenoside XLIX, a naturally occurring gynosaponin, PPAR-alpha dependently inhibits LPS-induced tissue factor expression and activity in human THP-1 monocytic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Tom Hsun-Wei; Van Hoan Tran; Roufogalis, Basil D.

2007-01-01

Tissue factor (TF) is involved not only in the progression of atherosclerosis and other cardiovascular diseases, but is also associated with tumor growth, metastasis, and angiogenesis and hence may be an attractive target for directed cancer therapeutics. Gynostemma pentaphyllum (GP) is widely used in the treatment of various cardiovascular diseases including atherosclerosis, as well as cancers. Gypenoside (Gyp) XLIX, a dammarane-type glycoside, is one of the prominent components in GP. We have recently reported Gyp XLIX to be a potent peroxisome proliferator-activated receptor (PPAR)-alpha activator. Here we demonstrate that Gyp XLIX (0-300 {mu}M) concentration dependently inhibited TF promoter activity aftermore » induction by the inflammatory stimulus lipopolysaccharide (LPS) in human monocytic THP-1 cells transfected with promoter reporter constructs pTF-LUC. Furthermore, Gyp XLIX inhibited LPS-induced TF mRNA and protein overexpression in THP-1 monocyte cells. Its inhibition of LPS-induced TF hyperactivity was further confirmed by chromogenic enzyme activity assay. The activities of Gyp XLIX reported in this study were similar to those of Wy-14643, a potent synthetic PPAR-alpha activator. Furthermore, the Gyp XLIX-induced inhibitory effect on TF luciferase activity was completely abolished in the presence of the PPAR-alpha selective antagonist MK-886. The present findings suggest that Gyp XLIX inhibits LPS-induced TF overexpression and enhancement of its activity in human THP-1 monocytic cells via PPAR-alpha-dependent pathways. The data provide new insights into the basis of the use of the traditional Chinese herbal medicine G. pentaphyllum for the treatment of cardiovascular and inflammatory diseases, as well as cancers.« less

Identification of Transcription Factor Genes and Their Correlation with the High Diversity of Stramenopiles

PubMed Central

Buitrago-Flórez, Francisco Javier; Restrepo, Silvia; Riaño-Pachón, Diego Mauricio

2014-01-01

The biological diversity among Stramenopiles is striking; they range from large multicellular seaweeds to tiny unicellular species, they embrace many ecologically important autothrophic (e.g., diatoms, brown algae), and heterotrophic (e.g., oomycetes) groups. Transcription factors (TFs) and other transcription regulators (TRs) regulate spatial and temporal gene expression. A plethora of transcriptional regulatory proteins have been identified and classified into families on the basis of sequence similarity. The purpose of this work is to identify the TF and TR complement in diverse species belonging to Stramenopiles in order to understand how these regulators may contribute to their observed diversity. We identified and classified 63 TF and TR families in 11 species of Stramenopiles. In some species we found gene families with high relative importance. Taking into account the 63 TF and TR families identified, 28 TF and TR families were established to be positively correlated with specific traits like number of predicted proteins, number of flagella and number of cell types during the life cycle. Additionally, we found gains and losses in TF and TR families specific to some species and clades, as well as, two families with high abundance specific to the autotrophic species and three families with high abundance specific to the heterotropic species. For the first time, there is a systematic search of TF and TR families in Stramenopiles. The attempts to uncover relationships between these families and the complexity of this group may be of great impact, considering that there are several important pathogens of plants and animals, as well as, important species involved in carbon cycling. Specific TF and TR families identified in this work appear to be correlated with particular traits in the Stramenopiles group and may be correlated with the high complexity and diversity in Stramenopiles. PMID:25375671

Identification of transcription factor genes and their correlation with the high diversity of stramenopiles.

PubMed

Buitrago-Flórez, Francisco Javier; Restrepo, Silvia; Riaño-Pachón, Diego Mauricio

2014-01-01

The biological diversity among Stramenopiles is striking; they range from large multicellular seaweeds to tiny unicellular species, they embrace many ecologically important autothrophic (e.g., diatoms, brown algae), and heterotrophic (e.g., oomycetes) groups. Transcription factors (TFs) and other transcription regulators (TRs) regulate spatial and temporal gene expression. A plethora of transcriptional regulatory proteins have been identified and classified into families on the basis of sequence similarity. The purpose of this work is to identify the TF and TR complement in diverse species belonging to Stramenopiles in order to understand how these regulators may contribute to their observed diversity. We identified and classified 63 TF and TR families in 11 species of Stramenopiles. In some species we found gene families with high relative importance. Taking into account the 63 TF and TR families identified, 28 TF and TR families were established to be positively correlated with specific traits like number of predicted proteins, number of flagella and number of cell types during the life cycle. Additionally, we found gains and losses in TF and TR families specific to some species and clades, as well as, two families with high abundance specific to the autotrophic species and three families with high abundance specific to the heterotropic species. For the first time, there is a systematic search of TF and TR families in Stramenopiles. The attempts to uncover relationships between these families and the complexity of this group may be of great impact, considering that there are several important pathogens of plants and animals, as well as, important species involved in carbon cycling. Specific TF and TR families identified in this work appear to be correlated with particular traits in the Stramenopiles group and may be correlated with the high complexity and diversity in Stramenopiles.

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

PubMed

Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

2016-05-01

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Predictive regulatory models in Drosophila melanogaster by integrative inference of transcriptional networks

PubMed Central

Marbach, Daniel; Roy, Sushmita; Ay, Ferhat; Meyer, Patrick E.; Candeias, Rogerio; Kahveci, Tamer; Bristow, Christopher A.; Kellis, Manolis

2012-01-01

Gaining insights on gene regulation from large-scale functional data sets is a grand challenge in systems biology. In this article, we develop and apply methods for transcriptional regulatory network inference from diverse functional genomics data sets and demonstrate their value for gene function and gene expression prediction. We formulate the network inference problem in a machine-learning framework and use both supervised and unsupervised methods to predict regulatory edges by integrating transcription factor (TF) binding, evolutionarily conserved sequence motifs, gene expression, and chromatin modification data sets as input features. Applying these methods to Drosophila melanogaster, we predict ∼300,000 regulatory edges in a network of ∼600 TFs and 12,000 target genes. We validate our predictions using known regulatory interactions, gene functional annotations, tissue-specific expression, protein–protein interactions, and three-dimensional maps of chromosome conformation. We use the inferred network to identify putative functions for hundreds of previously uncharacterized genes, including many in nervous system development, which are independently confirmed based on their tissue-specific expression patterns. Last, we use the regulatory network to predict target gene expression levels as a function of TF expression, and find significantly higher predictive power for integrative networks than for motif or ChIP-based networks. Our work reveals the complementarity between physical evidence of regulatory interactions (TF binding, motif conservation) and functional evidence (coordinated expression or chromatin patterns) and demonstrates the power of data integration for network inference and studies of gene regulation at the systems level. PMID:22456606

Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis.

PubMed

Chang, Yao-Ming; Liu, Wen-Yu; Shih, Arthur Chun-Chieh; Shen, Meng-Ni; Lu, Chen-Hua; Lu, Mei-Yeh Jade; Yang, Hui-Wen; Wang, Tzi-Yuan; Chen, Sean C-C; Chen, Stella Maris; Li, Wen-Hsiung; Ku, Maurice S B

2012-09-01

To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C(4) metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.

Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

NASA Astrophysics Data System (ADS)

Tormoen, Garth W.; Recht, Olivia; Gruber, András; Levine, Ross L.; McCarty, Owen J. T.

2013-10-01

Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF.

PubMed

Prasad, Ramesh; Sen, Prosenjit

2018-02-01

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF 1-263 -FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF 1-263 -FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.

Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection.

PubMed

Leal de Azeredo, Elzinandes; Solórzano, Victor Edgar Fiestas; de Oliveira, Débora Batista; Marinho, Cintia Ferreira; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Kubelka, Claire Fernandes; de-Oliveira-Pinto, Luzia Maria

2017-01-01

Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors

PubMed Central

Jin, Jinpu; Zhang, He; Kong, Lei; Gao, Ge; Luo, Jingchu

2014-01-01

With the aim to provide a resource for functional and evolutionary study of plant transcription factors (TFs), we updated the plant TF database PlantTFDB to version 3.0 (http://planttfdb.cbi.pku.edu.cn). After refining the TF classification pipeline, we systematically identified 129 288 TFs from 83 species, of which 67 species have genome sequences, covering main lineages of green plants. Besides the abundant annotation provided in the previous version, we generated more annotations for identified TFs, including expression, regulation, interaction, conserved elements, phenotype information, expert-curated descriptions derived from UniProt, TAIR and NCBI GeneRIF, as well as references to provide clues for functional studies of TFs. To help identify evolutionary relationship among identified TFs, we assigned 69 450 TFs into 3924 orthologous groups, and constructed 9217 phylogenetic trees for TFs within the same families or same orthologous groups, respectively. In addition, we set up a TF prediction server in this version for users to identify TFs from their own sequences. PMID:24174544

Effect of increased HoxB4 on human megakaryocytic development

PubMed Central

Zhong, Yiming; Sullenbarger, Brent; Lasky, Larry C.

2010-01-01

In order to ex vivo produce clinically useful quantity of platelets, we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data indicate that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating Tpo R and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine. PMID:20599537

Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines.

PubMed

Yamamizu, Kohei; Sharov, Alexei A; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B; Schlessinger, David; Ko, Minoru S H

2016-05-06

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs.

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

PubMed Central

Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

2008-01-01

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

APG: an Active Protein-Gene network model to quantify regulatory signals in complex biological systems.

PubMed

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information.

APG: an Active Protein-Gene Network Model to Quantify Regulatory Signals in Complex Biological Systems

PubMed Central

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information. PMID:23346354

Allelic variants of OsSUB1A cause differential expression of transcription factor genes in response to submergence in rice.

PubMed

Sharma, Niharika; Dang, Trang Minh; Singh, Namrata; Ruzicic, Slobodan; Mueller-Roeber, Bernd; Baumann, Ute; Heuer, Sigrid

2018-01-08

Flooding during seasonal monsoons affects millions of hectares of rice-cultivated areas across Asia. Submerged rice plants die within a week due to lack of oxygen, light and excessive elongation growth to escape the water. Submergence tolerance was first reported in an aus-type rice landrace, FR13A, and the ethylene-responsive transcription factor (TF) gene SUB1A-1 was identified as the major tolerance gene. Intolerant rice varieties generally lack the SUB1A gene but some intermediate tolerant varieties, such as IR64, carry the allelic variant SUB1A-2. Differential effects of the two alleles have so far not been addressed. As a first step, we have therefore quantified and compared the expression of nearly 2500 rice TF genes between IR64 and its derived tolerant near isogenic line IR64-Sub1, which carries the SUB1A-1 allele. Gene expression was studied in internodes, where the main difference in expression between the two alleles was previously shown. Nineteen and twenty-six TF genes were identified that responded to submergence in IR64 and IR64-Sub1, respectively. Only one gene was found to be submergence-responsive in both, suggesting different regulatory pathways under submergence in the two genotypes. These differentially expressed genes (DEGs) mainly included MYB, NAC, TIFY and Zn-finger TFs, and most genes were downregulated upon submergence. In IR64, but not in IR64-Sub1, SUB1B and SUB1C, which are also present in the Sub1 locus, were identified as submergence responsive. Four TFs were not submergence responsive but exhibited constitutive, genotype-specific differential expression. Most of the identified submergence responsive DEGs are associated with regulatory hormonal pathways, i.e. gibberellins (GA), abscisic acid (ABA), and jasmonic acid (JA), apart from ethylene. An in-silico promoter analysis of the two genotypes revealed the presence of allele-specific single nucleotide polymorphisms, giving rise to ABRE, DRE/CRT, CARE and Site II cis-elements, which can partly explain the observed differential TF gene expression. This study identified new gene targets with the potential to further enhance submergence tolerance in rice and provides insights into novel aspects of SUB1A-mediated tolerance.

Identification and characterization of TF1(phox), a DNA-binding protein that increases expression of gp91(phox) in PLB985 myeloid leukemia cells.

PubMed

Eklund, E A; Kakar, R

1997-04-04

The CYBB gene encodes gp91(phox), the heavy chain of the phagocyte-specific NADPH oxidase. CYBB is transcriptionally inactive until the promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bind to one of these cis-elements (-214 to -262 base pairs) and identified TF1(phox), a protein with sequence-specific binding to the CYBB promoter. Electrophoretic mobility shift assay with nuclear proteins from a variety of cell lines demonstrated binding of a protein to the CYBB promoter that was cross-immunoreactive with TF1(phox). DNA binding of this protein was increased by IFN-gamma treatment in the myeloid cell line PLB985, but not in the non-myeloid cell line HeLa. Overexpression of recombinant TF1(phox) in PLB985 cells increased endogenous gp91(phox) message abundance, but did not lead to cellular differentiation. Overexpression of TF1(phox) in myeloid leukemia cell lines increased reporter gene expression from artificial promoter constructs containing CYBB promoter sequence. These data suggested that TF1(phox) increased expression of gp91(phox).

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei.

PubMed

Salmon, D; Geuskens, M; Hanocq, F; Hanocq-Quertier, J; Nolan, D; Ruben, L; Pays, E

1994-07-15

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

An RpoS-dependent sRNA regulates the expression of a chaperone involved in protein folding

PubMed Central

Silva, Inês Jesus; Ortega, Álvaro Darío; Viegas, Sandra Cristina; García-del Portillo, Francisco; Arraiano, Cecília Maria

2013-01-01

Small noncoding RNAs (sRNAs) are usually expressed in the cell to face a variety of stresses. In this report we disclose the first target for SraL (also known as RyjA), a sRNA present in many bacteria, which is highly induced in stationary phase. We also demonstrate that this sRNA is directly transcribed by the major stress σ factor σS (RpoS) in Salmonella enterica serovar Typhimurium. We show that SraL sRNA down-regulates the expression of the chaperone Trigger Factor (TF), encoded by the tig gene. TF is one of the three major chaperones that cooperate in the folding of the newly synthesized cytosolic proteins and is the only ribosome-associated chaperone known in bacteria. By use of bioinformatic tools and mutagenesis experiments, SraL was shown to directly interact with the 5′ UTR of the tig mRNA a few nucleotides upstream of the Shine-Dalgarno region. Namely, point mutations in the sRNA (SraL*) abolished the repression of tig mRNA and could only down-regulate a tig transcript target with the respective compensatory mutations. We have also validated in vitro that SraL forms a stable duplex with the tig mRNA. This work constitutes the first report of a small RNA affecting protein folding. Taking into account that both SraL and TF are very well conserved in enterobacteria, this work will have important repercussions in the field. PMID:23893734

An RpoS-dependent sRNA regulates the expression of a chaperone involved in protein folding.

PubMed

Silva, Inês Jesus; Ortega, Alvaro Darío; Viegas, Sandra Cristina; García-Del Portillo, Francisco; Arraiano, Cecília Maria

2013-09-01

Small noncoding RNAs (sRNAs) are usually expressed in the cell to face a variety of stresses. In this report we disclose the first target for SraL (also known as RyjA), a sRNA present in many bacteria, which is highly induced in stationary phase. We also demonstrate that this sRNA is directly transcribed by the major stress σ factor σ(S) (RpoS) in Salmonella enterica serovar Typhimurium. We show that SraL sRNA down-regulates the expression of the chaperone Trigger Factor (TF), encoded by the tig gene. TF is one of the three major chaperones that cooperate in the folding of the newly synthesized cytosolic proteins and is the only ribosome-associated chaperone known in bacteria. By use of bioinformatic tools and mutagenesis experiments, SraL was shown to directly interact with the 5' UTR of the tig mRNA a few nucleotides upstream of the Shine-Dalgarno region. Namely, point mutations in the sRNA (SraL*) abolished the repression of tig mRNA and could only down-regulate a tig transcript target with the respective compensatory mutations. We have also validated in vitro that SraL forms a stable duplex with the tig mRNA. This work constitutes the first report of a small RNA affecting protein folding. Taking into account that both SraL and TF are very well conserved in enterobacteria, this work will have important repercussions in the field.

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

PubMed

Maekawa, T; Sudo, T; Kurimoto, M; Ishii, S

1991-09-11

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

PubMed

Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

2015-04-03

Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

PubMed

Calonge, María Julia; Seoane, Joan; Massagué, Joan

2004-05-28

A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

Activation of blood coagulation in cancer: implications for tumour progression

PubMed Central

Lima, Luize G.; Monteiro, Robson Q.

2013-01-01

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies. PMID:23889169

Reciprocal patterns of allergen-induced GATA-3 expression in peripheral blood mononuclear cells from atopics vs. non-atopics.

PubMed

Macaubas, C; Lee, P T; Smallacombe, T B; Holt, B J; Wee, C; Sly, P D; Holt, P G

2002-01-01

T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes. To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization. PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation. These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.

Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation.

PubMed

Xiao, M; Luo, Z; Mantel, C; Broxmeyer, H E; Lu, L

2000-02-01

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

PubMed

Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2015-12-01

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

SALT-RESPONSIVE ERF1 is a negative regulator of grain filling and gibberellin-mediated seedling establishment in rice.

PubMed

Schmidt, Romy; Schippers, Jos H M; Mieulet, Delphine; Watanabe, Mutsumi; Hoefgen, Rainer; Guiderdoni, Emmanuel; Mueller-Roeber, Bernd

2014-02-01

Grain quality is an important agricultural trait that is mainly determined by grain size and composition. Here, we characterize the role of the rice transcription factor (TF) SALT-RESPONSIVE ERF1 (SERF1) during grain development. Through genome-wide expression profiling and chromatin immunoprecipitation, we found that SERF1 directly regulates RICE PROLAMIN-BOX BINDING FACTOR (RPBF), a TF that functions as a positive regulator of grain filling. Loss of SERF1 enhances RPBF expression resulting in larger grains with increased starch content, while SERF1 overexpression represses RPBF resulting in smaller grains. Consistently, during grain filling, starch biosynthesis genes such as GRANULE-BOUND STARCH SYNTHASEI (GBSSI), STARCH SYNTHASEI (SSI), SSIIIa, and ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2 (AGPL2) are up-regulated in SERF1 knockout grains. Moreover, SERF1 is a direct upstream regulator of GBSSI. In addition, SERF1 negatively regulates germination by controlling RPBF expression, which mediates the gibberellic acid (GA)-induced expression of RICE AMYLASE1A (RAmy1A). Loss of SERF1 results in more rapid seedling establishment, while SERF1 overexpression has the opposite effect. Our study reveals that SERF1 represents a negative regulator of grain filling and seedling establishment by timing the expression of RPBF.

DNA breathing dynamics distinguish binding from nonbinding consensus sites for transcription factor YY1 in cells.

PubMed

Alexandrov, Boian S; Fukuyo, Yayoi; Lange, Martin; Horikoshi, Nobuo; Gelev, Vladimir; Rasmussen, Kim Ø; Bishop, Alan R; Usheva, Anny

2012-11-01

The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.

Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

He Yingbo; Chang Guodong; Zhan Shunli

2008-06-06

The level of circulating tissue factor (TF) is up-regulated in human angiogenesis-related malignancies. However, whether circulating TF has angiogenic activities has not been determined. Soluble TF (sTF) is the main domain of circulating TF. Here, using cell migration, wound healing, and tubule formation assays, human recombinant sTF was found to significantly promote the migration and differentiation of endothelial cells. The stress fiber formation and rearrangement induced by sTF observed through immunofluorescence microscope may be responsible for the stimulatory migration effect of sTF. Nevertheless, sTF had no effects on endothelial cell proliferation. Interestingly, sTF can be internalized by endothelial cells, whichmore » implies a novel mechanism for sTF in angiogenesis. These results suggest that sTF has unique angiogenic activities and may serve as a potential therapeutic target to treat diseases associated with angiogenesis such as cancer and rheumatoid arthritis.« less

Chronic sleep deprivation markedly reduces coagulation factor VII expression

PubMed Central

Pinotti, Mirko; Bertolucci, Cristiano; Frigato, Elena; Branchini, Alessio; Cavallari, Nicola; Baba, Kenkichi; Contreras-Alcantara, Susana; Ehlen, J. Christopher; Bernardi, Francesco; Paul, Ketema N.; Tosini, Gianluca

2010-01-01

Chronic sleep loss, a common feature of human life in industrialized countries, is associated to cardiovascular disorders. Variations in functional parameters of coagulation might contribute to explain this relationship. By exploiting the mouse model and a specifically designed protocol, we demonstrated that seven days of partial sleep deprivation significantly decreases (−30.5%) the thrombin generation potential in plasma evaluated upon extrinsic (TF/FVIIa pathway) but not intrinsic activation of coagulation. This variation was consistent with a decrease (−49.8%) in the plasma activity levels of factor VII (FVII), the crucial physiologicalal trigger of coagulation, which was even more pronounced at the liver mRNA level (−85.7%). The recovery in normal sleep conditions for three days completely restored thrombin generation and FVII activity in plasma. For the first time, we demonstrate that chronic sleep deprivation on its own reduces, in a reversible manner, the FVII expression levels, thus influencing the TF/FVIIa activation pathway efficiency. PMID:20418241

Environmental contaminants and microRNA regulation: Transcription factors as regulators of toxicant-altered microRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sollome, James; Martin, Elizabeth

MicroRNAs (miRNAs) regulate gene expression by binding mRNA and inhibiting translation and/or inducing degradation of the associated transcripts. Expression levels of miRNAs have been shown to be altered in response to environmental toxicants, thus impacting cellular function and influencing disease risk. Transcription factors (TFs) are known to be altered in response to environmental toxicants and play a critical role in the regulation of miRNA expression. To date, environmentally-responsive TFs that are important for regulating miRNAs remain understudied. In a state-of-the-art analysis, we utilized an in silico bioinformatic approach to characterize potential transcriptional regulators of environmentally-responsive miRNAs. Using the miRStart database,more » genomic sequences of promoter regions for all available human miRNAs (n = 847) were identified and promoter regions were defined as − 1000/+500 base pairs from the transcription start site. Subsequently, the promoter region sequences of environmentally-responsive miRNAs (n = 128) were analyzed using enrichment analysis to determine overrepresented TF binding sites (TFBS). While most (56/73) TFs differed across environmental contaminants, a set of 17 TFs was enriched for promoter binding among miRNAs responsive to numerous environmental contaminants. Of these, one TF was common to miRNAs altered by the majority of environmental contaminants, namely SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 3 (SMARCA3). These identified TFs represent candidate common transcriptional regulators of miRNAs perturbed by environmental toxicants. - Highlights: • Transcription factors that regulate environmentally-modulated miRNA expression are understudied • Transcription factor binding sites (TFBS) located within DNA promoter regions of miRNAs were identified. • Specific transcription factors may serve as master regulators of environmentally-mediated microRNA expression.« less

Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules

PubMed Central

Faure, Andre J.; Schmidt, Dominic; Watt, Stephen; Schwalie, Petra C.; Wilson, Michael D.; Xu, Huiling; Ramsay, Robert G.; Odom, Duncan T.; Flicek, Paul

2012-01-01

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein–DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels. PMID:22780989

The genetic regulatory network centered on Pto-Wuschela and its targets involved in wood formation revealed by association studies.

PubMed

Yang, Xiaohui; Wei, Zunzheng; Du, Qingzhang; Chen, Jinhui; Wang, Qingshi; Quan, Mingyang; Song, Yuepeng; Xie, Jianbo; Zhang, Deqiang

2015-11-09

Transcription factors (TFs) regulate gene expression and can strongly affect phenotypes. However, few studies have examined TF variants and TF interactions with their targets in plants. Here, we used genetic association in 435 unrelated individuals of Populus tomentosa to explore the variants in Pto-Wuschela and its targets to decipher the genetic regulatory network of Pto-Wuschela. Our bioinformatics and co-expression analysis identified 53 genes with the motif TCACGTGA as putative targets of Pto-Wuschela. Single-marker association analysis showed that Pto-Wuschela was associated with wood properties, which is in agreement with the observation that it has higher expression in stem vascular tissues in Populus. Also, SNPs in the 53 targets were associated with growth or wood properties under additive or dominance effects, suggesting these genes and Pto-Wuschela may act in the same genetic pathways that affect variation in these quantitative traits. Epistasis analysis indicated that 75.5% of these genes directly or indirectly interacted Pto-Wuschela, revealing the coordinated genetic regulatory network formed by Pto-Wuschela and its targets. Thus, our study provides an alternative method for dissection of the interactions between a TF and its targets, which will strength our understanding of the regulatory roles of TFs in complex traits in plants.

Opposing Control by Transcription Factors MYB61 and MYB3 Increases Freezing Tolerance by Relieving C-Repeat Binding Factor Suppression1[OPEN

PubMed Central

Zhang, Yunqin; Miao, Zhenyan; Xie, Can; Meng, Xiangzhao; Deng, Jie; Mysore, Kirankumar S.; Frugier, Florian; Wang, Tao

2016-01-01

Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula. In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula. PMID:27578551

cGRNB: a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets.

PubMed

Xu, Huayong; Yu, Hui; Tu, Kang; Shi, Qianqian; Wei, Chaochun; Li, Yuan-Yuan; Li, Yi-Xue

2013-01-01

We are witnessing rapid progress in the development of methodologies for building the combinatorial gene regulatory networks involving both TFs (Transcription Factors) and miRNAs (microRNAs). There are a few tools available to do these jobs but most of them are not easy to use and not accessible online. A web server is especially needed in order to allow users to upload experimental expression datasets and build combinatorial regulatory networks corresponding to their particular contexts. In this work, we compiled putative TF-gene, miRNA-gene and TF-miRNA regulatory relationships from forward-engineering pipelines and curated them as built-in data libraries. We streamlined the R codes of our two separate forward-and-reverse engineering algorithms for combinatorial gene regulatory network construction and formalized them as two major functional modules. As a result, we released the cGRNB (combinatorial Gene Regulatory Networks Builder): a web server for constructing combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. The cGRNB enables two major network-building modules, one for MPGE (miRNA-perturbed gene expression) datasets and the other for parallel miRNA/mRNA expression datasets. A miRNA-centered two-layer combinatorial regulatory cascade is the output of the first module and a comprehensive genome-wide network involving all three types of combinatorial regulations (TF-gene, TF-miRNA, and miRNA-gene) are the output of the second module. In this article we propose cGRNB, a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. Since parallel miRNA/mRNA expression datasets are rapidly accumulated by the advance of next-generation sequencing techniques, cGRNB will be very useful tool for researchers to build combinatorial gene regulatory networks based on expression datasets. The cGRNB web-server is free and available online at http://www.scbit.org/cgrnb.

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi

PubMed Central

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-01-01

The PKS-NRPS-derived tetramic acid equisetin and its N-desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus. The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum, a species distantly related to the notorious rice pathogen Fusarium fujikuroi. Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi. Bioinformatic analysis revealed that this cluster does not contain the equisetin N-methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi. Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22, led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23, encoding a second Zn(II)2Cys6 TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T. TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus. PMID:28379186

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi.

PubMed

Janevska, Slavica; Arndt, Birgit; Baumann, Leonie; Apken, Lisa Helene; Mauriz Marques, Lucas Maciel; Humpf, Hans-Ulrich; Tudzynski, Bettina

2017-04-05

The PKS-NRPS-derived tetramic acid equisetin and its N -desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus . The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum , a species distantly related to the notorious rice pathogen Fusarium fujikuroi . Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi . Bioinformatic analysis revealed that this cluster does not contain the equisetin N -methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi . Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22 , led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23 , encoding a second Zn(II)₂Cys₆ TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T . TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus.

Positive Feedback Loops for Factor V and Factor VII Activation Supply Sensitivity to Local Surface Tissue Factor Density During Blood Coagulation

PubMed Central

Balandina, A.N.; Shibeko, A.M.; Kireev, D.A.; Novikova, A.A.; Shmirev, I.I.; Panteleev, M.A.; Ataullakhanov, F.I.

2011-01-01

Blood coagulation is triggered not only by surface tissue factor (TF) density but also by surface TF distribution. We investigated recognition of surface TF distribution patterns during blood coagulation and identified the underlying molecular mechanisms. For these investigations, we employed 1), an in vitro reaction-diffusion experimental model of coagulation; and 2), numerical simulations using a mathematical model of coagulation in a three-dimensional space. When TF was uniformly immobilized over the activating surface, the clotting initiation time in normal plasma increased from 4 min to >120 min, with a decrease in TF density from 100 to 0.7 pmol/m2. In contrast, surface-immobilized fibroblasts initiated clotting within 3–7 min, independently of fibroblast quantity and despite a change in average surface TF density from 0.5 to 130 pmol/m2. Experiments using factor V-, VII-, and VIII-deficient plasma and computer simulations demonstrated that different responses to these two TF distributions are caused by two positive feedback loops in the blood coagulation network: activation of the TF–VII complex by factor Xa, and activation of factor V by thrombin. This finding suggests a new role for these reactions: to supply sensitivity to local TF density during blood coagulation. PMID:22004734

The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation.

PubMed Central

Lazennec, G; Kern, L; Valotaire, Y; Salbert, G

1997-01-01

The rainbow trout estrogen receptor (rtER) is a positively autoregulated gene in liver cells. In a previous report, we showed that upregulation is mediated by an estrogen response element (ERE) located in the proximal promoter of the gene and that a half binding site for nuclear receptors (5'-TGACCT-3') located 15 bp upstream of the ERE is involved in the magnitude of the estrogen response. We now report that the human orphan receptor COUP-TF and a COUP-TF-like protein from trout liver are able to bind to the consensus half-site. When cotransfected with the rtER gene proximal promoter, COUP-TF had no regulatory functions on its own. Interestingly, COUP-TF enhanced rtER transactivation properties in the presence of estradiol in a dose-dependent manner when cotransfected with the rtER gene promoter. Unliganded retinoid receptor heterodimers had the same helper function as COUP-TF in the presence of estradiol but were switched to repressors when the ligand all-trans-retinoic acid was added. Mutation of the consensus half-site only slightly reduced COUP-TF helper function, suggesting that it actually results from a complex mechanism that probably involves both DNA binding of COUP-TF to the promoter and protein-protein interaction with another transcription factor bound to the promoter. Nevertheless, a DNA-binding-defective mutant of COUP-TF was also defective in ER helper function. Competition footprinting analysis suggested that COUP-TF actually establishes contacts with the consensus upstream half-site and the downstream ERE half-site that would form a DR-24-like response element. Interaction of COUP-TF with the DR-24 element was confirmed in footprinting assays by using nuclear extracts from Saccharomyces cerevisiae expressing COUP-TF. Finally, interaction of COUP-TF with mutants of the rtER gene promoter showed that COUP-TF recognizes the ERE when the upstream half-site is mutated. These data show that COUP-TF may activate transcription through interaction with other nuclear receptors. This cross-talk between liganded nuclear receptors and orphan receptors is likely to modulate the spectrum of action of a particular ligand-receptor complex and may participate in the cell-type specificity of the ligand effect. PMID:9271383

A gene expression signature that correlates with CD8+T cell expansion in acute Epstein Barr virus infection1

PubMed Central

Greenough, Thomas C.; Straubhaar, Juerg R.; Kamga, Larisa; Weiss, Eric R.; Brody, Robin M.; McManus, Margaret M.; Lambrecht, Linda K.; Somasundaran, Mohan; Luzuriaga, Katherine F.

2015-01-01

Virus specific CD8+ T cells expand dramatically during acute Epstein Barr virus (EBV) infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of ten individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and Eomesodermin (Eomes), were upregulated and maintained at similar levels in both AIM and CONV; by contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Altogether, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus-specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and Eomes may help to maintain effector mechanisms in activated cells, and to enable proliferation and transition to earlier differentiation states in CONV. PMID:26416268

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

PubMed Central

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-01-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

PubMed

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-04-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy.

PubMed

Duanmu, J; Cheng, J; Xu, J; Booth, C J; Hu, Z

2011-04-26

The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR. The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume. To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model. We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

CORE_TF: a user-friendly interface to identify evolutionary conserved transcription factor binding sites in sets of co-regulated genes

PubMed Central

Hestand, Matthew S; van Galen, Michiel; Villerius, Michel P; van Ommen, Gert-Jan B; den Dunnen, Johan T; 't Hoen, Peter AC

2008-01-01

Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites) that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at . It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conserved transcription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites. PMID:19036135

PRISM offers a comprehensive genomic approach to transcription factor function prediction

PubMed Central

Wenger, Aaron M.; Clarke, Shoa L.; Guturu, Harendra; Chen, Jenny; Schaar, Bruce T.; McLean, Cory Y.; Bejerano, Gill

2013-01-01

The human genome encodes 1500–2000 different transcription factors (TFs). ChIP-seq is revealing the global binding profiles of a fraction of TFs in a fraction of their biological contexts. These data show that the majority of TFs bind directly next to a large number of context-relevant target genes, that most binding is distal, and that binding is context specific. Because of the effort and cost involved, ChIP-seq is seldom used in search of novel TF function. Such exploration is instead done using expression perturbation and genetic screens. Here we propose a comprehensive computational framework for transcription factor function prediction. We curate 332 high-quality nonredundant TF binding motifs that represent all major DNA binding domains, and improve cross-species conserved binding site prediction to obtain 3.3 million conserved, mostly distal, binding site predictions. We combine these with 2.4 million facts about all human and mouse gene functions, in a novel statistical framework, in search of enrichments of particular motifs next to groups of target genes of particular functions. Rigorous parameter tuning and a harsh null are used to minimize false positives. Our novel PRISM (predicting regulatory information from single motifs) approach obtains 2543 TF function predictions in a large variety of contexts, at a false discovery rate of 16%. The predictions are highly enriched for validated TF roles, and 45 of 67 (67%) tested binding site regions in five different contexts act as enhancers in functionally matched cells. PMID:23382538

PLGA-encapsulated tea polyphenols enhance the chemotherapeutic efficacy of cisplatin against human cancer cells and mice bearing Ehrlich ascites carcinoma

PubMed Central

Singh, Madhulika; Bhatnagar, Priyanka; Mishra, Sanjay; Kumar, Pradeep; Shukla, Yogeshwer; Gupta, Kailash Chand

2015-01-01

The clinical success of the applicability of tea polyphenols awaits efficient systemic delivery and bioavailability. Herein, following the concept of nanochemoprevention, which uses nanotechnology for enhancing the efficacy of chemotherapeutic drugs, we employed tea polyphenols, namely theaflavin (TF) and epigallocatechin-3-gallate (EGCG) encapsulated in a biodegradable nanoparticulate formulation based on poly(lactide-co-glycolide) (PLGA) with approximately 26% and 18% encapsulation efficiency, respectively. It was observed that TF/EGCG encapsulated PLGA nanoparticles (NPs) offered an up to ~7-fold dose advantage when compared with bulk TF/EGCG in terms of exerting its antiproliferative effects and also enhanced the anticancer potential of cisplatin (CDDP) in A549 (lung carcinoma), HeLa (cervical carcinoma), and THP-1 (acute monocytic leukemia) cells. Cell cycle analysis revealed that TF/EGCG-NPs were more efficient than bulk TF/EGCG in sensitizing A549 cells to CDDP-induced apoptosis, with a dose advantage of up to 20-fold. Further, TF/EGCG-NPs, alone or in combination with CDDP, were more effective in inhibiting NF-κB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase-9, and vascular endothelial growth factor, involved in cell proliferation, metastasis, and angiogenesis, respectively. EGCG and TF-NPs were also found to be more effective than bulk TF/EGCG in inducing the cleavage of caspase-3 and caspase-9 and Bax/Bcl2 ratio in favor of apoptosis. Further, in vivo evaluation of these NPs in combination with CDDP showed an increase in life span (P<0.05) in mice bearing Ehrlich’s ascites carcinoma cells, with apparent regression of tumor volume in comparison with mice treated with bulk doses with CDDP. These results indicate that EGCG and TF-NPs have superior cancer chemosensitization activity when compared with bulk TF/EGCG. PMID:26586942

Incremental Drag due to Grooves and Threads for KE (Kinetic Energy) Projectiles

DTIC Science & Technology

1989-03-01

RFI • CTsB TF * - MF (3a) q L where TF1 is the Thread Factor defined as: TF 0.84 + 0.117 P - o (3b) where p is the groove pitch in inches, MF1 is...g2) MF RF CD (4) where TF11 and TF1 2 are the thread factors for the threads of pitch p, and P2, respectively. 5 One can notice the large

Identification of Tf1 integration events in S. pombe under nonselective conditions

PubMed Central

Cherry, Kristina E.; Hearn, Willis E.; Seshie, Osborne Y.; Singleton, Teresa L.; Singleton, Teresa L.

2014-01-01

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the S. pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418S/neo+ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418S/neo+ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1’s ability to insert within silent regions of S. pombe’s genome. PMID:24680781

Identification of Tf1 integration events in S. pombe under nonselective conditions.

PubMed

Cherry, Kristina E; Hearn, Willis E; Seshie, Osborne Y K; Singleton, Teresa L

2014-06-01

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418(S)/neo(+) Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418(S)/neo(+) clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression and characterization of a novel reverse transcriptase of the LTR retrotransposon Tf1.

PubMed

Kirshenboim, Noa; Hayouka, Zvi; Friedler, Assaf; Hizi, Amnon

2007-09-30

The LTR retrotransposon of Schizosacharomyces pombe, Tf1, has several distinctive properties that can be related to the unique properties of its reverse transcriptase (RT). Consequently, we expressed, purified and studied the recombinant Tf1 RT. This monomeric protein possesses all activities typical to RTs: DNA and RNA-dependent DNA polymerase as well as an inherent ribonuclease H. The DNA polymerase activity shows preference to Mn(+)(2) or Mg(+)(2), depending on the substrate used, whereas the ribonuclease H strongly prefers Mn(+)(2). The most outstanding feature of Tf1 RT is its capacity to add non-templated nucleotides to the 3'-ends of the nascent DNA. This is mainly apparent in the presence of Mn(+)(2), as is the noticeable low fidelity of DNA synthesis. In all, Tf1 RT has a marked infidelity in synthesizing DNA at template ends, a phenomenon that can explain, as discussed herein, some of the features of Tf1 replication in the host cells.

Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

NASA Astrophysics Data System (ADS)

Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

2013-06-01

Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

ESF-EMBO Symposium "Molecular Biology and Innovative Therapies in Sarcomas of Childhood and Adolescence" Sept 29-Oct 4, Polonia Castle Pultusk, Poland.

PubMed

Schäfer, Beat W; Koscielniak, Ewa; Kovar, Heinrich; Fulda, Simone

2013-01-01

Rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) are among the most common pediatric sarcomas (Arndt et al., 2012). Despite sarcomas representing a highly heterogeneous group of tumors, ES and alveolar RMS (ARMS) typically share one common genetic characteristic, namely a specific chromosomal translocation (Helman and Meltzer, 2003; Lessnick and Ladanyi, 2012). These translocations generate fusion proteins, which are composed of two transcription factors (TF). Typically, one TF is a developmentally regulated factor that is essential for proper specification of a given lineage and provides the DNA-binding domain, while the partner TF contributes a transactivation domain that drives aberrant expression of target genes. Based on these common genetic characteristics, the first ESF-EMBO research conference entitled "Molecular Biology and Innovative Therapies in Sarcomas of Childhood and Adolescence" with special focus on RMS and ES was held at the Polonia Castle in Pultusk, Poland. The conference gathered 70 participants from more than 15 countries and several continents representing most research groups that are active in this field.

Transfer of radionuclides to plants of natural ecosystems at the Semipalatinsk Test Site.

PubMed

Larionova, N V; Lukashenko, S N; Kabdyrakova, A M; Kunduzbayeva, A Ye; Panitskiy, A V; Ivanova, A R

2018-06-01

A systematic study devoted to 137 Cs, 90 Sr, 241 Am, 239+240 Pu radionuclides in vegetation cover from several spots of the Semipalatinsk test site (STS) is summarised in this paper, highlighting the main findings obtained. The analysed spots are characterized by various types of radioactive contamination. Transfer factors (Tf) required for the quantitative description of the radionuclides transition from the soil to aboveground plant parts were determined, being found that, on average, the minimum Tf for all the radionuclides concerned were determined on the "Experimental Field" ground, followed by the determined ones in the "plumes" of radioactive fallout and in the conditionally "background" territories analysed. The highest transfer factors were characteristic of zones of radioactive streamflows and places of warfare radioactive agent (WRA) tests. On the other hand, ordering the radionuclide transferring factors in descending order, the following sequence was obtained: 90 Sr Tf > Cs Tf >  239+240 Pu Tf >  241 Am Tf, with the 90 Sr Tf, on the average, exceeding the 137 Cs Tf by 8 times and exceeding the 239+240 Pu Tf by up 16 times. 239+240 Pu Tf values were up to 3 times higher than the 241 Am Tf. The exception to the indicated radionuclide Tf descending order corresponded to places of WRA tests where Tf of radionuclides of interest by plants follows the sequence 90 Sr >  239+240 Pu >  137 Cs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Constitutive expression of the promyelocytic leukemia-associated oncogene PML-RARalpha in TF1 cells: isoform-specific and retinoic acid-dependent effects on growth, bcl-2 expression, and apoptosis.

PubMed

Slack, J L; Yu, M

1998-05-01

Two major isoforms of PML-RARalpha are associated with (15;17)-positive acute promyelocytic leukemia (APL); however, functional differences between these isoforms have been difficult to define, and the molecular mechanism by which each isoform contributes to the pathogenesis of APL is not fully understood. To address these issues, the 'short' (S) and 'long' (L) isoforms of PML-RARalpha were constitutively expressed in the factor-dependent human erythroleukemia cell line, TF1. Expression of the L, but not the S, isoform inhibited growth of these cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the absence of GM-CSF, the S isoform partially protected against apoptosis, while the L isoform accelerated cell death. Treatment with all-trans retinoic acid (ATRA) inhibited cell growth and caused apoptosis only in PML-RARalpha-expressing cells, and these effects of ATRA were more marked in cells expressing the L isoform. ATRA treatment also led to downregulation of bcl-2 and endogenous RARalpha in PML-RARalpha-expressing cells, but had little effect on the level of exogenously expressed PML-RARalpha. We conclude that (1) subtle differences exist in the biologic activities of the L and S isoforms of PML-RARalpha, and (2) both isoforms are capable of transducing an ATRA-mediated signal that leads to downregulation of bcl-2 and induction of programmed cell death.

Nifedipine Increases Iron Content in WKPT-0293 Cl.2 Cells via Up-Regulating Iron Influx Proteins

PubMed Central

Yu, Shuang-Shuang; Jiang, Li-Rong; Ling, Yan; Qian, Zhong-Ming; Zhou, Yu-Fu; Li, Juan; Ke, Ya

2017-01-01

Nifedipine was reported to enhance urinary iron excretion in iron overloaded mice. However, it remains unknown how nifedipine stimulates urinary iron excretion in the kidney. We speculated that nifedipine might inhibit the TfR1/ DMT1 (transferrin receptor 1/divalent metal transporter1)-mediated iron uptake by proximal tubule cells in addition to blocking L-type Ca2+ channels, leading to an increase in iron in lumen-fluid and then urinary iron excretion. To test this hypothesis, we investigated the effects of nifedipine on iron content and expression of TfR1, DMT1 and ferroportin1 (Fpn1) in WKPT-0293 Cl.2 cells of the S1 segment of the proximal tubule in rats, using a graphite furnace atomic absorption spectrophotometer and Western blot analysis, respectively. We demonstrated for the first time that nifedipine significantly enhanced iron content as well as TfR1 and DMT1 expression and had no effect on Fpn1 levels in the cells. We also found that ferric ammonium citrate decreased TfR1 levels, increased Fpn1 expression and had no effect on DMT1 content, while co-treatment with nifedipine and FAC increase TfR1 and DMT1 expression and also had no effect on Fpn1 levels. These findings suggest that the nifedipine-induced increase in cell iron may mainly be due to the corresponding increase in TfR1 and DMT1 expression and also imply that the effects of nifedipine on iron transport in proximal tubule cells can not explain the increase in urinary iron excretion. PMID:28243203

Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis).

PubMed

Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Gao, Wu-Jun

2017-08-22

Garden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood. De novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development. Our study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L.

PubMed

Kakeshpour, Tayebeh; Nayebi, Shadi; Rashidi Monfared, Sajad; Moieni, Ahmad; Karimzadeh, Ghasem

2015-10-01

Papaver somniferum L. is an herbaceous, annual and diploid plant that is important from pharmacological and strategic point of view. The cDNA clones of two putative MYB and WRKY genes were isolated (GeneBank accession numbers KP411870 and KP203854, respectively) from this plant, via the nested-PCR method, and characterized. The MYB transcription factor (TF) comprises 342 amino acids, and exhibits the structural features of the R2R3MYB protein family. The WRKY TF, a 326 amino acid-long polypeptide, falls structurally into the group II of WRKY protein family. Quantitative real-time PCR (qRT-PCR) analyses indicate the presence of these TFs in all organs of P. somniferum L. and Papaver bracteatum L. Highest expression levels of these two TFs were observed in the leaf tissues of P. somniferum L. while in P. bracteatum L. the espression levels were highest in the root tissues. Promoter analysis of the 10 co-expressed gene clustered involved in noscapine biosynthesis pathway in P. somniferum L. suggested that not only these 10 genes are co-expressed, but also share common regulatory motifs and TFs including MYB and WRKY TFs, and that may explain their common regulation.

Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

PubMed

Zhang, Peter G Y; Yeung, Joanna; Gupta, Ishita; Ramirez, Miguel; Ha, Thomas; Swanson, Douglas J; Nagao-Sato, Sayaka; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; de Hoon, Michiel; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Goldowitz, Dan

2018-06-01

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

PubMed

Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G

2017-01-05

Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2 -/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2 -/- mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2 -/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2 -/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2 -/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

A proximity-based graph clustering method for the identification and application of transcription factor clusters.

PubMed

Spadafore, Maxwell; Najarian, Kayvan; Boyle, Alan P

2017-11-29

Transcription factors (TFs) form a complex regulatory network within the cell that is crucial to cell functioning and human health. While methods to establish where a TF binds to DNA are well established, these methods provide no information describing how TFs interact with one another when they do bind. TFs tend to bind the genome in clusters, and current methods to identify these clusters are either limited in scope, unable to detect relationships beyond motif similarity, or not applied to TF-TF interactions. Here, we present a proximity-based graph clustering approach to identify TF clusters using either ChIP-seq or motif search data. We use TF co-occurrence to construct a filtered, normalized adjacency matrix and use the Markov Clustering Algorithm to partition the graph while maintaining TF-cluster and cluster-cluster interactions. We then apply our graph structure beyond clustering, using it to increase the accuracy of motif-based TFBS searching for an example TF. We show that our method produces small, manageable clusters that encapsulate many known, experimentally validated transcription factor interactions and that our method is capable of capturing interactions that motif similarity methods might miss. Our graph structure is able to significantly increase the accuracy of motif TFBS searching, demonstrating that the TF-TF connections within the graph correlate with biological TF-TF interactions. The interactions identified by our method correspond to biological reality and allow for fast exploration of TF clustering and regulatory dynamics.

Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

PubMed

Sakuma, Shinji; Yu, James Y H; Quang, Timothy; Hiwatari, Ken-Ichiro; Kumagai, Hironori; Kao, Stephanie; Holt, Alex; Erskind, Jalysa; McClure, Richard; Siuta, Michael; Kitamura, Tokio; Tobita, Etsuo; Koike, Seiji; Wilson, Kevin; Richards-Kortum, Rebecca; Liu, Eric; Washington, Kay; Omary, Reed; Gore, John C; Pham, Wellington

2015-03-01

Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level. © 2014 UICC.

Network-based integration of GWAS and gene expression identifies a HOX-centric network associated with serous ovarian cancer risk

PubMed Central

Kar, Siddhartha P.; Tyrer, Jonathan P.; Li, Qiyuan; Lawrenson, Kate; Aben, Katja K.H.; Anton-Culver, Hoda; Antonenkova, Natalia; Chenevix-Trench, Georgia; Baker, Helen; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Berchuck, Andrew; Bisogna, Maria; Bjørge, Line; Bogdanova, Natalia; Brinton, Louise; Brooks-Wilson, Angela; Butzow, Ralf; Campbell, Ian; Carty, Karen; Chang-Claude, Jenny; Chen, Yian Ann; Chen, Zhihua; Cook, Linda S.; Cramer, Daniel; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; Dennis, Joe; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; du Bois, Andreas; Dürst, Matthias; Eccles, Diana; Easton, Douglas F.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goode, Ellen L.; Goodman, Marc T.; Grownwald, Jacek; Harrington, Patricia; Harter, Philipp; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A.T.; Hillemanns, Peter; Hogdall, Estrid; Hogdall, Claus K.; Hosono, Satoyo; Iversen, Edwin S.; Jakubowska, Anna; Paul, James; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y; Kjaer, Susanne K.; Kelemen, Linda E.; Kellar, Melissa; Kelley, Joseph; Kiemeney, Lambertus A.; Krakstad, Camilla; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Iain A.; Menon, Usha; Modugno, Francesmary; Moysich, Kirsten B.; Narod, Steven A.; Nedergaard, Lotte; Ness, Roberta B.; Nevanlinna, Heli; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Pearce, Celeste Leigh; Pejovic, Tanja; Pelttari, Liisa M.; Permuth-Wey, Jennifer; Phelan, Catherine M.; Pike, Malcolm C.; Poole, Elizabeth M.; Ramus, Susan J.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schildkraut, Joellen M.; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Sucheston-Campbell, Lara E.; Tangen, Ingvild L.; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J; Timorek, Agnieszka; Tsai, Ya-Yu; Tworoger, Shelley S.; van Altena, Anne M.; Van Nieuwenhuysen, Els; Vergote, Ignace; Vierkant, Robert A.; Wang-Gohrke, Shan; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Woo, Yin-Ling; Wu, Xifeng; Wu, Anna; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Sellers, Thomas A.; Monteiro, Alvaro N. A.; Freedman, Matthew L.; Gayther, Simon A.; Pharoah, Paul D. P.

2015-01-01

Background Genome-wide association studies (GWAS) have so far reported 12 loci associated with serous epithelial ovarian cancer (EOC) risk. We hypothesized that some of these loci function through nearby transcription factor (TF) genes and that putative target genes of these TFs as identified by co-expression may also be enriched for additional EOC risk associations. Methods We selected TF genes within 1 Mb of the top signal at the 12 genome-wide significant risk loci. Mutual information, a form of correlation, was used to build networks of genes strongly co-expressed with each selected TF gene in the unified microarray data set of 489 serous EOC tumors from The Cancer Genome Atlas. Genes represented in this data set were subsequently ranked using a gene-level test based on results for germline SNPs from a serous EOC GWAS meta-analysis (2,196 cases/4,396 controls). Results Gene set enrichment analysis identified six networks centered on TF genes (HOXB2, HOXB5, HOXB6, HOXB7 at 17q21.32 and HOXD1, HOXD3 at 2q31) that were significantly enriched for genes from the risk-associated end of the ranked list (P<0.05 and FDR<0.05). These results were replicated (P<0.05) using an independent association study (7,035 cases/21,693 controls). Genes underlying enrichment in the six networks were pooled into a combined network. Conclusion We identified a HOX-centric network associated with serous EOC risk containing several genes with known or emerging roles in serous EOC development. Impact Network analysis integrating large, context-specific data sets has the potential to offer mechanistic insights into cancer susceptibility and prioritize genes for experimental characterization. PMID:26209509

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

PubMed Central

Merz, Frieder; Boehringer, Daniel; Schaffitzel, Christiane; Preissler, Steffen; Hoffmann, Anja; Maier, Timm; Rutkowska, Anna; Lozza, Jasmin; Ban, Nenad; Bukau, Bernd; Deuerling, Elke

2008-01-01

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome–nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors. PMID:18497744

Characterization of the interaction between diferric transferrin and transferrin receptor 2 by functional assays and atomic force microscopy*

PubMed Central

Ikuta, Katsuya; Yersin, Alexandre; Ikai, Atsushi; Aisen, Philip; Kohgo, Yutaka

2010-01-01

Transferrin receptor (TfR2), a homologue of classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α we expressed the protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary cells. The association constant for binding of diferric transferrin (Tf) to TfR2α is 5.6 × 106 M−1, which is about 50 times lower than that of TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy (AFM), a powerful tool for investigation of the interaction between ligand and receptor at the single molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by 2 energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors. PMID:20096706

The Endocrine Disrupting Chemical Tolylfluanid Alters Adipocyte Metabolism via Glucocorticoid Receptor Activation

PubMed Central

Neel, Brian A.; Brady, Matthew J.

2013-01-01

Glucocorticoid signaling plays a critical role in regulating energy metabolism. Emerging data implicate environmental endocrine-disrupting chemicals as contributors to the obesity and diabetes epidemics. Previous studies have shown that the phenylsulfamide fungicide tolylfluanid (TF) augments glucocorticoid receptor (GR)-dependent luciferase expression in 3T3-L1 preadipocytes while modulating insulin action in primary murine and human adipocytes. Studies were performed to interrogate glucocorticoid signaling in primary adipocytes exposed to TF. TF mimicked the gene transcription profile of the murine glucocorticoid corticosterone (Cort). Cellular fractionation assays demonstrated that TF treatment promoted the activating serine phosphorylation of GR, augmenting its cytoplasmic-to-nuclear translocation as well as its enrichment at glucocorticoid response elements on the glucocorticoid-induced leucine zipper gene promoter. After acute treatment, Cort or TF promoted insulin receptor substrate-1 (IRS-1) gene and protein expression. Either treatment also enriched GR binding at an identified glucocorticoid response element in the IRS-1 gene. TF or Cort each increased insulin-stimulated lipogenesis, an effect resulting from increased lipogenic gene expression and enhanced insulin-stimulated dephosphorylation of acetyl-coenzyme A carboxylase. The augmentation of insulin-stimulated lipogenesis was mediated through a specific enhancement of Akt phosphorylation at T308. These findings support modulation of IRS-1 levels as a mechanism for glucocorticoid-mediated changes in insulin action in primary adipocytes. Albeit with less affinity than Cort, in silico analysis suggests that TF can interact with the ligand binding pocket of GR. Collectively, these studies identify TF as a structurally unique environmental glucocorticoid. Glucocorticoid signaling may thus represent a novel pathway by which environmental toxicants promote the development of metabolic diseases. PMID:23340252

Activation of coagulation by a thalidomide-based regimen.

PubMed

Hoshi, Asuka; Matsumoto, Aya; Chung, Jihwa; Isozumi, Yu; Koyama, Takatoshi

2011-09-01

Combining thalidomide (Thal) with chemotherapeutic agents or steroid preparations led to improved response rates in the treatment of multiple myeloma. However, deep vein thrombosis (DVT) is one of the most serious side-effects noted with this regimen, and how a Thal-based regimen causes DVT is unclear. We investigated the procoagulant effects of Thal when combined with chemotherapeutic agents in vitro, focusing on tissue factor (TF) and phosphatidylserine. We examined the effects of the chemotherapeutic doxorubicin hydrochloride (Dox) and the steroid dexamethasone (Dex), with or without Thal. Our study used the human vascular endothelial, monocytic, and myeloma cell lines, EAhy926, THP-1, and RPMI8226, respectively. In EAhy926 and THP-1, Dex treatment increased expression of TF, which may induce procoagulant activity (PCA). Upregulation of TF mRNA correlated with activation of the Egr-1 pathway. In Thal and Dex treatments, the increase of PCA induction from phosphatidylserine exposure was modest. In contrast, Dox and Thal-Dox increased phosphatidylserine exposure in both cell types. In THP-1 cells, cell surface phosphatidylserine exposure correlated with increased PCA by Dox. Thal alone showed a modest increase in phosphatidylserine exposure in endothelial cells and monocytes. When Thal is given in combination with chemotherapies or Dex, endothelial cell and monocyte PCA may be induced through phosphatidylserine exposure, or TF expression. Induction may be protracted by Thal, which has an antiangiogenic activity. Therefore, prophylactic anticoagulant strategies should be considered in Thal-based combination regimens.

Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in Neurospora

PubMed Central

Sancar, Cigdem; Ha, Nati; Yilmaz, Rüstem; Tesorero, Rafael; Fisher, Tamas; Brunner, Michael; Sancar, Gencer

2015-01-01

Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression. PMID:25822411

A transcriptional dynamic network during Arabidopsis thaliana pollen development.

PubMed

Wang, Jigang; Qiu, Xiaojie; Li, Yuhua; Deng, Youping; Shi, Tieliu

2011-01-01

To understand transcriptional regulatory networks (TRNs), especially the coordinated dynamic regulation between transcription factors (TFs) and their corresponding target genes during development, computational approaches would represent significant advances in the genome-wide expression analysis. The major challenges for the experiments include monitoring the time-specific TFs' activities and identifying the dynamic regulatory relationships between TFs and their target genes, both of which are currently not yet available at the large scale. However, various methods have been proposed to computationally estimate those activities and regulations. During the past decade, significant progresses have been made towards understanding pollen development at each development stage under the molecular level, yet the regulatory mechanisms that control the dynamic pollen development processes remain largely unknown. Here, we adopt Networks Component Analysis (NCA) to identify TF activities over time course, and infer their regulatory relationships based on the coexpression of TFs and their target genes during pollen development. We carried out meta-analysis by integrating several sets of gene expression data related to Arabidopsis thaliana pollen development (stages range from UNM, BCP, TCP, HP to 0.5 hr pollen tube and 4 hr pollen tube). We constructed a regulatory network, including 19 TFs, 101 target genes and 319 regulatory interactions. The computationally estimated TF activities were well correlated to their coordinated genes' expressions during the development process. We clustered the expression of their target genes in the context of regulatory influences, and inferred new regulatory relationships between those TFs and their target genes, such as transcription factor WRKY34, which was identified that specifically expressed in pollen, and regulated several new target genes. Our finding facilitates the interpretation of the expression patterns with more biological relevancy, since the clusters corresponding to the activity of specific TF or the combination of TFs suggest the coordinated regulation of TFs to their target genes. Through integrating different resources, we constructed a dynamic regulatory network of Arabidopsis thaliana during pollen development with gene coexpression and NCA. The network illustrated the relationships between the TFs' activities and their target genes' expression, as well as the interactions between TFs, which provide new insight into the molecular mechanisms that control the pollen development.

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus).

PubMed

Yu, Shanshan; Yang, Hui; Chai, Yingmei; Liu, Yingying; Zhang, Qiuxia; Ding, Xinbiao; Zhu, Qian

2013-02-01

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5' untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3' UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys(61)-Cys(158), Cys(134)-Cys(150)) and a Ca(2+)/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca(2+)-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca(2+)-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Intersection of the Extrinsic Hedgehog and WNT/Wingless Signals with the Intrinsic Hox Code Underpins Branching Pattern and Tube Shape Diversity in the Drosophila Airways

PubMed Central

Matsuda, Ryo; Hosono, Chie; Saigo, Kaoru; Samakovlis, Christos

2015-01-01

The tubular networks of the Drosophila respiratory system and our vasculature show distinct branching patterns and tube shapes in different body regions. These local variations are crucial for organ function and organismal fitness. Organotypic patterns and tube geometries in branched networks are typically controlled by variations of extrinsic signaling but the impact of intrinsic factors on branch patterns and shapes is not well explored. Here, we show that the intersection of extrinsic hedgehog(hh) and WNT/wingless (wg) signaling with the tube-intrinsic Hox code of distinct segments specifies the tube pattern and shape of the Drosophila airways. In the cephalic part of the airways, hh signaling induces expression of the transcription factor (TF) knirps (kni) in the anterior dorsal trunk (DTa1). kni represses the expression of another TF spalt major (salm), making DTa1 a narrow and long tube. In DTa branches of more posterior metameres, Bithorax Complex (BX-C) Hox genes autonomously divert hh signaling from inducing kni, thereby allowing DTa branches to develop as salm-dependent thick and short tubes. Moreover, the differential expression of BX-C genes is partly responsible for the anterior-to-posterior gradual increase of the DT tube diameter through regulating the expression level of Salm, a transcriptional target of WNT/wg signaling. Thus, our results highlight how tube intrinsic differential competence can diversify tube morphology without changing availabilities of extrinsic factors. PMID:25615601

Targeted release of transcription factors for cell reprogramming by a natural micro-syringe.

PubMed

Berthoin, Lionel; Toussaint, Bertrand; Garban, Frédéric; Le Gouellec, Audrey; Caulier, Benjamin; Polack, Benoît; Laurin, David

2016-11-20

Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34 + hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34 + hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter

PubMed Central

Kim, Da-Hye; Park, Sangkyu; Lee, Jong-Yeol; Ha, Sun-Hwa; Lim, Sun-Hyung

2018-01-01

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6–43.2 fold in the PANSBP_DP plants and 2.0–124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation. PMID:29361688

Human Cytomegalovirus Protein US2 Interferes with the Expression of Human HFE, a Nonclassical Class I Major Histocompatibility Complex Molecule That Regulates Iron Homeostasis

PubMed Central

Ben-Arieh, Sayeh Vahdati; Zimerman, Baruch; Smorodinsky, Nechama I.; Yaacubovicz, Margalit; Schechter, Chana; Bacik, Igor; Gibbs, Jim; Bennink, Jack R.; Yewdell, Jon W.; Coligan, John E.; Firat, Hüseyin; Lemonnier, François; Ehrlich, Rachel

2001-01-01

HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/β2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses. PMID:11581431

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli.

PubMed

Ishihama, Akira; Kori, Ayako; Koshio, Etsuko; Yamada, Kayoko; Maeda, Hiroto; Shimada, Tomohiro; Makinoshima, Hideki; Iwata, Akira; Fujita, Nobuyuki

2014-08-01

The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Maternal iron status during pregnancy compared with neonatal iron status better predicts placental iron transporter expression in humans.

PubMed

Best, Cora M; Pressman, Eva K; Cao, Chang; Cooper, Elizabeth; Guillet, Ronnie; Yost, Olivia L; Galati, Jonathan; Kent, Tera R; O'Brien, Kimberly O

2016-10-01

The placenta richly expresses nonheme and heme Fe transport proteins. To address the impact of maternal and neonatal Fe status and hepcidin on the regulation of these proteins, mRNA expression and protein abundance of nonheme and heme Fe transport proteins were evaluated in placental tissue from 154 adolescents. Regression analyses found maternal Fe status was significantly associated with multiple placental nonheme and heme transporters, whereas neonatal Fe status was related to only 3 heme transporters. Across statistical analyses, maternal Fe status was consistently associated with the placental nonheme Fe importer transferrin receptor 1 (TfR1). Protein abundance of TfR1 was related to midgestation maternal serum ferritin (SF) (β = -0.32; P = 0.005) and serum TfR (β = 0.25; P = 0.024). Protein abundance of the heme importer, proton-coupled folate transporter, was related to neonatal SF (β = 0.30; P = 0.016) and serum TfR (β = -0.46; P < 0.0001). Neonatal SF was also related to mRNA expression of the heme exporter feline leukemia virus subgroup C receptor 1 (β = -0.30; P = 0.004). In summary, maternal Fe insufficiency during pregnancy predicts increased expression of the placental nonheme Fe transporter TfR1. Associations between placental heme Fe transporters and neonatal Fe status require further study.-Best, C. M., Pressman, E. K., Cao, C., Cooper, E., Guillet, R., Yost, O. L., Galati, J., Kent, T. R., O'Brien, K. O. Maternal iron status during pregnancy compared with neonatal iron status better predicts placental iron transporter expression in humans. © FASEB.

Evidence for two transferrin loci in the Salmo trutta genome.

PubMed

Rozman, T; Dovc, P; Marić, S; Kokalj-Vokac, N; Erjavec-Skerget, A; Rab, P; Snoj, A

2008-12-01

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer.

PubMed

Ceelie, H; Spaargaren-Van Riel, C C; De Jong, M; Bertina, R M; Vos, H L

2003-08-01

Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization

PubMed Central

Murton, Heather E.; Grady, Patrick J. R.; Chan, Tsun Ho; Cam, Hugh P.; Whitehall, Simon K.

2016-01-01

Uncontrolled propagation of retrotransposons is potentially detrimental to host genome integrity. Therefore, cells have evolved surveillance mechanisms to restrict the mobility of these elements. In Schizosaccharomyces pombe the Tf2 LTR retrotransposons are transcriptionally silenced and are also clustered in the nucleus into structures termed Tf bodies. Here we describe the impact of silencing and clustering on the mobility of an endogenous Tf2 element. Deletion of genes such as set1+ (histone H3 lysine 4 methyltransferase) or abp1+ (CENP-B homolog) that both alleviate silencing and clustering, result in a corresponding increase in mobilization. Furthermore, expression of constitutively active Sre1, a transcriptional activator of Tf2 elements, also alleviates clustering and induces mobilization. In contrast, clustering is not disrupted by loss of the HIRA histone chaperone, despite high levels of expression, and in this background, mobilization frequency is only marginally increased. Thus, mutations that compromise transcriptional silencing but not Tf bodies are insufficient to drive mobilization. Furthermore, analyses of mutant alleles that separate the transcriptional repression and clustering functions of Set1 are consistent with control of Tf2 propagation via a combination of silencing and spatial organization. Our results indicate that host surveillance mechanisms operate at multiple levels to restrict Tf2 retrotransposon mobilization. PMID:27343236

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

Transcriptomics hit the target: Monitoring of ligand-activated and stress response pathways for chemical testing.

PubMed

Limonciel, Alice; Moenks, Konrad; Stanzel, Sven; Truisi, Germaine L; Parmentier, Céline; Aschauer, Lydia; Wilmes, Anja; Richert, Lysiane; Hewitt, Philip; Mueller, Stefan O; Lukas, Arno; Kopp-Schneider, Annette; Leonard, Martin O; Jennings, Paul

2015-12-25

High content omic methods provide a deep insight into cellular events occurring upon chemical exposure of a cell population or tissue. However, this improvement in analytic precision is not yet matched by a thorough understanding of molecular mechanisms that would allow an optimal interpretation of these biological changes. For transcriptomics (TCX), one type of molecular effects that can be assessed already is the modulation of the transcriptional activity of a transcription factor (TF). As more ChIP-seq datasets reporting genes specifically bound by a TF become publicly available for mining, the generation of target gene lists of TFs of toxicological relevance becomes possible, based on actual protein-DNA interaction and modulation of gene expression. In this study, we generated target gene signatures for Nrf2, ATF4, XBP1, p53, HIF1a, AhR and PPAR gamma and tracked TF modulation in a large collection of in vitro TCX datasets from renal and hepatic cell models exposed to clinical nephro- and hepato-toxins. The result is a global monitoring of TF modulation with great promise as a mechanistically based tool for chemical hazard identification. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Novel Prothrombotic Pathway in Systemic Sclerosis Patients: Possible Role of Bisphosphonate-Activated γδ T Cells

PubMed Central

Marcu-Malina, Victoria; Balbir-Gurman, Alexandra; Dardik, Rima; Braun-Moscovici, Yolanda; Segel, Michael J.; Bank, Ilan

2014-01-01

Objectives: Infusions of aminobisphonates (ABP) activate Vγ9δ2T cells in vivo and induce an acute inflammatory response in 30% of patients treated for osteoporosis. Following the observation of digital thrombosis in a systemic sclerosis (SSc) patient after treatment with an intravenous ABP, zoledronate (Zol), we evaluated whether patient and control peripheral blood (PB) mononuclear cell (MC, PBMC) acquire a prothrombotic phenotype in response to Zol. Results: Vγ9δ2T cells of both patients and healthy donors (HD) upregulated the CD69 activation antigen and secreted tumor necrosis factor (TNF)α in response to Zol in vitro. In addition, exposure to either Zol or lipopolysaccharide (LPS), or to both additively, induced expression of the highly procoagulant, tissue factor (TF)-1 on CD14+ monocytes. Importantly, only Zol-induced TF-1 was blocked by a monoclonal antibody to TNFα. Interestingly, we found that SSc, but not HD, Vδ1+ T cells were concurrently activated by Zol to produce interleukin (IL)-4. Addition of plasma from the blood of the SSc patient who developed critical digital ischemia after infusion of Zol, but neither plasma from a second patient with no adverse clinical response to Zol infusion nor of a HD, strongly enhanced Zol-induced monocyte TF-1, which could still be blocked by anti-TNFα. Conclusion: Aminobisphonates induced secretion of TNFα by Vγ9δ2+ T cells may lead to TNFα-dependent induction of procoagulant TF-1 induction on monocytes. In certain clinical settings, e.g., SSc, TF-1+ monocytes could play a role in triggering clinically relevant thrombosis. PMID:25250025

COPS: Detecting Co-Occurrence and Spatial Arrangement of Transcription Factor Binding Motifs in Genome-Wide Datasets

PubMed Central

Lohmann, Ingrid

2012-01-01

In multi-cellular organisms, spatiotemporal activity of cis-regulatory DNA elements depends on their occupancy by different transcription factors (TFs). In recent years, genome-wide ChIP-on-Chip, ChIP-Seq and DamID assays have been extensively used to unravel the combinatorial interaction of TFs with cis-regulatory modules (CRMs) in the genome. Even though genome-wide binding profiles are increasingly becoming available for different TFs, single TF binding profiles are in most cases not sufficient for dissecting complex regulatory networks. Thus, potent computational tools detecting statistically significant and biologically relevant TF-motif co-occurrences in genome-wide datasets are essential for analyzing context-dependent transcriptional regulation. We have developed COPS (Co-Occurrence Pattern Search), a new bioinformatics tool based on a combination of association rules and Markov chain models, which detects co-occurring TF binding sites (BSs) on genomic regions of interest. COPS scans DNA sequences for frequent motif patterns using a Frequent-Pattern tree based data mining approach, which allows efficient performance of the software with respect to both data structure and implementation speed, in particular when mining large datasets. Since transcriptional gene regulation very often relies on the formation of regulatory protein complexes mediated by closely adjoining TF binding sites on CRMs, COPS additionally detects preferred short distance between co-occurring TF motifs. The performance of our software with respect to biological significance was evaluated using three published datasets containing genomic regions that are independently bound by several TFs involved in a defined biological process. In sum, COPS is a fast, efficient and user-friendly tool mining statistically and biologically significant TFBS co-occurrences and therefore allows the identification of TFs that combinatorially regulate gene expression. PMID:23272209

A prior-based integrative framework for functional transcriptional regulatory network inference

PubMed Central

Siahpirani, Alireza F.

2017-01-01

Abstract Transcriptional regulatory networks specify regulatory proteins controlling the context-specific expression levels of genes. Inference of genome-wide regulatory networks is central to understanding gene regulation, but remains an open challenge. Expression-based network inference is among the most popular methods to infer regulatory networks, however, networks inferred from such methods have low overlap with experimentally derived (e.g. ChIP-chip and transcription factor (TF) knockouts) networks. Currently we have a limited understanding of this discrepancy. To address this gap, we first develop a regulatory network inference algorithm, based on probabilistic graphical models, to integrate expression with auxiliary datasets supporting a regulatory edge. Second, we comprehensively analyze our and other state-of-the-art methods on different expression perturbation datasets. Networks inferred by integrating sequence-specific motifs with expression have substantially greater agreement with experimentally derived networks, while remaining more predictive of expression than motif-based networks. Our analysis suggests natural genetic variation as the most informative perturbation for network inference, and, identifies core TFs whose targets are predictable from expression. Multiple reasons make the identification of targets of other TFs difficult, including network architecture and insufficient variation of TF mRNA level. Finally, we demonstrate the utility of our inference algorithm to infer stress-specific regulatory networks and for regulator prioritization. PMID:27794550

Transcription Factor Activities Enhance Markers of Drug Sensitivity in Cancer.

PubMed

Garcia-Alonso, Luz; Iorio, Francesco; Matchan, Angela; Fonseca, Nuno; Jaaks, Patricia; Peat, Gareth; Pignatelli, Miguel; Falcone, Fiammetta; Benes, Cyril H; Dunham, Ian; Bignell, Graham; McDade, Simon S; Garnett, Mathew J; Saez-Rodriguez, Julio

2018-02-01

Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies. Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769-80. ©2017 AACR . ©2017 American Association for Cancer Research.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow

PubMed Central

Zhu, Shu; Travers, Richard J.; Morrissey, James H.

2015-01-01

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) –bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm2. Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm2 and sensitive to O1A6 at 0 to 0.2 molecules per µm2. However, neither antibody reduced fibrin generation at ∼2 molecules per µm2 when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm2) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. PMID:26136249

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor*

PubMed Central

Collier, Mary E. W.; Ettelaie, Camille

2011-01-01

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF. PMID:21310953

A Radish Basic Helix-Loop-Helix Transcription Factor, RsTT8 Acts a Positive Regulator for Anthocyanin Biosynthesis

PubMed Central

Lim, Sun-Hyung; Kim, Da-Hye; Kim, Jae K.; Lee, Jong-Yeol; Ha, Sun-Hwa

2017-01-01

The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis. PMID:29167678

N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth)cells using the LdMNPV expression system

Treesearch

One Choi; Noboru Tomiya; Jung H. Kim; James M. Slavicek; Michael J. Betenbaugh; Yuan C. Lee

2003-01-01

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then...

Transfer factor - hypotheses for its structure and function.

PubMed

Shifrine, M; Scibienski, R

1975-01-01

Transfer factor (TF) is a dialyzable extract from primed lymphocytes that is able to transfer specific delayed hypersensitivity from one animal to another. On the basis of available data we suggest that TF is a polypeptide with a molecular weight below 15,000 daltons. We hypothesize that TF is the variable light or heavy chain domain of immunoglobulin: such a molecule conforms with the accepted properties of TF and also has the necessary specificity requirements. We also hypothesize that TF is part of a receptor site. beta-2-microglobulin, a molecule that is an integral part of cell surfaces, could be the anchor for TF. beta-2-microglobulin has homologies with the constant portion of immunoglobulin light or heavy chain and thus would combine with the variable domain (TF) to form a complete receptor site for a specific antigen. The properties of TF suggest its mode of action, which is discussed in detail in the text. The biologic advantages of TF is its ability to confer immediate (immunologie specific) protection while the 'normal' immune response develops.

"Hit-and-Run" leaves its mark: catalyst transcription factors and chromatin modification.

PubMed

Varala, Kranthi; Li, Ying; Marshall-Colón, Amy; Para, Alessia; Coruzzi, Gloria M

2015-08-01

Understanding how transcription factor (TF) binding is related to gene regulation is a moving target. We recently uncovered genome-wide evidence for a "Hit-and-Run" model of transcription. In this model, a master TF "hits" a target promoter to initiate a rapid response to a signal. As the "hit" is transient, the model invokes recruitment of partner TFs to sustain transcription over time. Following the "run", the master TF "hits" other targets to propagate the response genome-wide. As such, a TF may act as a "catalyst" to mount a broad and acute response in cells that first sense the signal, while the recruited TF partners promote long-term adaptive behavior in the whole organism. This "Hit-and-Run" model likely has broad relevance, as TF perturbation studies across eukaryotes show small overlaps between TF-regulated and TF-bound genes, implicating transient TF-target binding. Here, we explore this "Hit-and-Run" model to suggest molecular mechanisms and its biological relevance. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

Evolutionary and Expression Analyses of the Apple Basic Leucine Zipper Transcription Factor Family

PubMed Central

Zhao, Jiao; Guo, Rongrong; Guo, Chunlei; Hou, Hongmin; Wang, Xiping; Gao, Hua

2016-01-01

Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling, and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in 10 different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones. PMID:27066030

Evolutionary and Expression Analyses of the Apple Basic Leucine Zipper Transcription Factor Family.

PubMed

Zhao, Jiao; Guo, Rongrong; Guo, Chunlei; Hou, Hongmin; Wang, Xiping; Gao, Hua

2016-01-01

Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling, and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in 10 different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones.

The Long Terminal Repeat Retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe.

PubMed

Esnault, Caroline; Levin, Henry L

2015-08-01

The long terminal repeat (LTR) retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe are active mobile elements of the Ty3/gypsy family. The mobilization of these retrotransposons depends on particle formation, reverse transcription and integration, processes typical of other LTR retrotransposons. However, Tf1 and Tf2 are distinct from other LTR elements in that they assemble virus-like particles from a single primary translation product, initiate reverse transcription with an unusual self-priming mechanism, and, in the case of Tf1, integrate with a pattern that favors specific promoters of RNA pol II-transcribed genes. To avoid the chromosome instability and genome damage that results from increased copy number, S. pombe applies a variety of defense mechanisms that restrict Tf1 and Tf2 activity. The mRNA of the Tf elements is eliminated by an exosome-based pathway when cells are in favorable conditions whereas nutrient deprivation triggers an RNA interference-dependent pathway that results in the heterochromatization of the elements. Interestingly, Tf1 integrates into the promoters of stress-induced genes and these insertions are capable of increasing the expression of adjacent genes. These properties of Tf1 transposition raise the possibility that Tf1 benefits cells with specific insertions by providing resistance to environmental stress.

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Isolation and characterization of GmMYBJ3, an R2R3-MYB transcription factor that affects isoflavonoids biosynthesis in soybean

PubMed Central

Zhao, Mingzhu; Wang, Tianliang; Wu, Ping; Guo, Wenyun; Su, Liantai; Wang, Ying; Liu, Yajing; Yan, Fan

2017-01-01

Isoflavonoids are secondary metabolites that play a variety of roles in plant-microbe interactions and plant defenses against abiotic stresses. Here we report a new MYB transcription factor (TF) gene, GmMYBJ3, that is involved in the isoflavonoids biosynthesis. The GmMYBJ3 gene is 1,002 bp long and encodes a protein of 333 amino acids. Amino acid sequence analysis showed that GmMYBJ3 is a typical R2R3 MYB TF. Yeast expression experiment demonstrated that GmMYBJ3 has its transcription activity in the nucleus and is transiently expressed in onion epidermal cells. The GmMYBJ3 gene was transformed into soybean and the expression activity of the GmMYBJ3 gene was significantly positively correlated with total isoflavonoid accumulation in soybean. Transient expression assays indicated that GmMYBJ3 can activate CHS8 expression. Furthermore, we analyzed the expressions of several genes known involved in the isoflavonoid biosynthesis, including CHS8, CHI1A, PAL1, IFS2 and F3H, in the GmMYBJ3 transgenic plants. The results showed that the expression levels of CHS8 and CHI1A were significantly increased in the transgenic plants compared to wild-type plants, but those of PAL1, IFS2 and F3H remained similar between the transgenic and wild-type plants. These results suggest that GmMYBJ3 participates in the isoflavonoid biosynthesis through regulation of CHS8 and CHI1A in soybean. PMID:28654660

The Effect of Short-term Intra-arterial Delivery of Paclitaxel on Neointimal Hyperplasia and the Local Thrombotic Environment after Angioplasty

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yajun, E, E-mail: eyj7681@yahoo.com.cn; He Nengshu, E-mail: eyajun@hotmail.com; Fan Hailun, E-mail: mydream510@yahoo.com.cn

2013-08-01

PurposeTo evaluate the effects of short-term intra-arterial delivery of paclitaxel on neointimal hyperplasia and the local thrombotic environment after angioplasty.MethodsAn experimental common carotid artery injury model was established in 60 rats, which were divided into experimental groups (40 rats) and controls (20 rats). Local intra-arterial administration of paclitaxel was applied at 2 doses (90 and 180 {mu}g/30 {mu}l), and the effects of short-term delivery of paclitaxel on neointimal hyperplasia and the expression of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated at days 15 and 30 by hematoxylin and eosin staining and immunohistochemistry.ResultsAt 15more » and 30 days after injury, neointimal thickness and area, the ratio of intimal area to medial area and the stenotic rate were all significantly decreased in the group provided the high concentrations (180 {mu}g/30 {mu}l) of paclitaxel for 2 min or 10 min and in the group provided the low concentration (90 {mu}g/30 {mu}l) of paclitaxel for 10 min (p < 0.05). At 30 days after injury, there were no significant changes in TF expression among all experimental groups. PAI-1 expression increased in the neointima of the high concentration 10 min group (p < 0.05), while t-PA expression decreased in the neointima of the high concentration 2 min group (p < 0.05).ConclusionIn the rat common carotid artery injury model, the short-term delivery of paclitaxel could effectively inhibit neointimal hyperplasia in the long term, with very little influence on the local expression of TF and PAI-1.« less

Beyond generalized hair cells: Molecular cues for hair cell types

PubMed Central

Jahan, Israt; Pan, Ning; Kersigo, Jennifer; Fritzsch, Bernd

2012-01-01

Basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial for inner ear neurosensory development. The proneural TF Atoh1 regulates the differentiation of hair cells (HCs) whereas Neurog1 and Neurod1 regulate specification and differentiation of neurons, respectively, but also affect HC development. Expression of Delta and Jagged ligands in nascent HCs and Notch receptors in supporting cells induce supporting cell differentiation through the regulation of neurogenic bHLH TFs (such as Hes1, Hes5) and suppression of limited Atoh1 expression. In sensorineural hearing loss, HCs are lost followed by supporting cells and progressive degeneration of neurons, at least in rodents. Regaining complete hearing may require reconstituting the organ of Corti (OC) from scratch, including the two types of HCs, inner (IHC) and outer (OHC) hair cells with the precise sorting of two types of afferent (type I and II) and efferent (lateral, LOC and medial, MOC olivo-cochlear) innervation. We review effects of bHLH TF dosage and their cross-regulation to differentiate HC types in the OC. We categorize findings of specific gene expressions in HCs: 1. as markers without meaning for the regeneration task, 2. as stabilizers who are needed to maintain or complete differentiation, and 3. as decision making genes, expressed and acting early enough to be useful in this process. Only one TF has been characterized that fits the last aspect: Atoh1. We propose that temporal and intensity variations of Atoh1 are naturally modulated to differentiate specific types of HCs. Importantly, the molecular means to modify the Atoh1 expression are at least partially understood and can be readily implemented in the attempts to regenerate specific types of HCs. PMID:23201032

The G-Box Transcriptional Regulatory Code in Arabidopsis1[OPEN

PubMed Central

Shepherd, Samuel J.K.; Brestovitsky, Anna; Dickinson, Patrick; Biswas, Surojit

2017-01-01

Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes; these expansions are linked to adaptation to environmental stressors. Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determined that the flanking sequences near G-boxes help determine in vitro specificity but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we constructed a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a Web site that provides interactive visualizations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations. PMID:28864470

Transcriptional network control of normal and leukaemic haematopoiesis

PubMed Central

Sive, Jonathan I.; Göttgens, Berthold

2014-01-01

Transcription factors (TFs) play a key role in determining the gene expression profiles of stem/progenitor cells, and defining their potential to differentiate into mature cell lineages. TF interactions within gene-regulatory networks are vital to these processes, and dysregulation of these networks by TF overexpression, deletion or abnormal gene fusions have been shown to cause malignancy. While investigation of these processes remains a challenge, advances in genome-wide technologies and growing interactions between laboratory and computational science are starting to produce increasingly accurate network models. The haematopoietic system provides an attractive experimental system to elucidate gene regulatory mechanisms, and allows experimental investigation of both normal and dysregulated networks. In this review we examine the principles of TF-controlled gene regulatory networks and the key experimental techniques used to investigate them. We look in detail at examples of how these approaches can be used to dissect out the regulatory mechanisms controlling normal haematopoiesis, as well as the dysregulated networks associated with haematological malignancies. PMID:25014893

Transcriptional network control of normal and leukaemic haematopoiesis.

PubMed

Sive, Jonathan I; Göttgens, Berthold

2014-12-10

Transcription factors (TFs) play a key role in determining the gene expression profiles of stem/progenitor cells, and defining their potential to differentiate into mature cell lineages. TF interactions within gene-regulatory networks are vital to these processes, and dysregulation of these networks by TF overexpression, deletion or abnormal gene fusions have been shown to cause malignancy. While investigation of these processes remains a challenge, advances in genome-wide technologies and growing interactions between laboratory and computational science are starting to produce increasingly accurate network models. The haematopoietic system provides an attractive experimental system to elucidate gene regulatory mechanisms, and allows experimental investigation of both normal and dysregulated networks. In this review we examine the principles of TF-controlled gene regulatory networks and the key experimental techniques used to investigate them. We look in detail at examples of how these approaches can be used to dissect out the regulatory mechanisms controlling normal haematopoiesis, as well as the dysregulated networks associated with haematological malignancies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

SRF modulates seizure occurrence, activity induced gene transcription and hippocampal circuit reorganization in the mouse pilocarpine epilepsy model.

PubMed

Lösing, Pascal; Niturad, Cristina Elena; Harrer, Merle; Reckendorf, Christopher Meyer Zu; Schatz, Theresa; Sinske, Daniela; Lerche, Holger; Maljevic, Snezana; Knöll, Bernd

2017-07-17

A hallmark of temporal lobe epilepsy (TLE) is hippocampal neuronal demise and aberrant mossy fiber sprouting. In addition, unrestrained neuronal activity in TLE patients induces gene expression including immediate early genes (IEGs) such as Fos and Egr1.We employed the mouse pilocarpine model to analyze the transcription factor (TF) serum response factor (SRF) in epileptogenesis, seizure induced histopathology and IEG induction. SRF is a neuronal activity regulated TF stimulating IEG expression as well as nerve fiber growth and guidance. Adult conditional SRF deficient mice (Srf CaMKCreERT2 ) were more refractory to initial status epilepticus (SE) acquisition. Further, SRF deficient mice developed more spontaneous recurrent seizures (SRS). Genome-wide transcriptomic analysis uncovered a requirement of SRF for SE and SRS induced IEG induction (e.g. Fos, Egr1, Arc, Npas4, Btg2, Atf3). SRF was required for epilepsy associated neurodegeneration, mossy fiber sprouting and inflammation. We uncovered MAP kinase signaling as SRF target during epilepsy. Upon SRF ablation, seizure evoked induction of dual specific phosphatases (Dusp5 and Dusp6) was reduced. Lower expression of these negative ERK kinase regulators correlated with altered P-ERK levels in epileptic Srf mutant animals.Overall, this study uncovered an SRF contribution to several processes of epileptogenesis in the pilocarpine model.

Combinatorial Regulation of Stilbene Synthase Genes by WRKY and MYB Transcription Factors in Grapevine (Vitis vinifera L.).

PubMed

Vannozzi, Alessandro; Wong, Darren Chern Jan; Höll, Janine; Hmmam, Ibrahim; Matus, José Tomás; Bogs, Jochen; Ziegler, Tobias; Dry, Ian; Barcaccia, Gianni; Lucchin, Margherita

2018-05-01

Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter-luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.

Increased tissue factor, MMP-8, and D-dimer expression in diabetic patients with unstable advanced carotid atherosclerosis.

PubMed

Krupinski, Jerzy; Turu, Marta M; Font, M Angels; Ahmed, Nesser; Sullivan, Matthew; Rubio, Francisco; Badimon, Lina; Slevin, Mark

2007-01-01

Advanced atherogenesis is characterized by the presence of markers of enhanced prothrombotic capacity, attenuated fibrinolysis, and by clinical conditions associated with defective coagulation. Diabetes may be associated with enhanced lesion instability and atherosclerotic plaque rupture. Plaques obtained from 206 patients undergoing carotid endarterectomy were divided into diabetic (type 2) and nondiabetic and analyzed by Western blotting and immunohistochemistry to detect tissue factor (TF), metalloproteinases (MMP)-2, -8, -9, and fibrin/fibrinogen related antigens, and in situ zymography to detect MMP activity. Plasma samples were quantified for TF procoagulant activity, C-reactive protein, fibrinogen and D-dimer. Diabetic and symptomatic patients with hypoechogenic plaques had increased plasma TF activity and D-dimer, compared with those with hyperechogenic plaques (p = 0.03, p = 0.007, respectively). Diabetic, symptomatic patients had higher plasma D-dimer levels than asymptomatic patients (p = 0.03). There was a significant correlation between intramural TF levels and D-dimer in diabetic patients with symptomatic disease (p = 0.001, r2 = 0.4). In diabetic patients, plasma fibrinogen levels were higher in patients with hypoechogenic plaques (p = 0.007). Diabetic patients with ulcerated plaques had higher plasma D-dimer and MMP-8 levels than those with fibrous plaques (p = 0.02, p = 0.01, respectively). This data suggests that currently available circulating markers may be clinically useful to select diabetic patients at higher risk of atherothrombosis. Increased procoagulant activity in diabetic patients may be linked to increased mural remodeling.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

PubMed

Marone, M; Scambia, G; Bonanno, G; Rutella, S; de Ritis, D; Guidi, F; Leone, G; Pierelli, L

2002-01-01

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

COUP-TF1 antagonizes Nkx2.5-mediated activation of the calreticulin gene during cardiac development.

PubMed

Guo, L; Lynch, J; Nakamura, K; Fliegel, L; Kasahara, H; Izumo, S; Komuro, I; Agellon, L B; Michalak, M

2001-01-26

Calreticulin, a Ca(2+) binding chaperone of the endoplasmic reticulum, is also highly expressed in the embryonic heart, and knockout of the calreticulin gene is lethal during embryogenesis because of impaired cardiac development. The protein is down-regulated after birth, and elevated expression of calreticulin in newborn hearts is associated with severe cardiac pathology and death. Here we show that the transcription factor Nkx2.5 activates expression of the calreticulin gene in the heart. Binding of chicken ovalbumin upstream promoter-transcription factor 1 to the Nkx2.5 binding site suppresses transcription from the calreticulin promoter. Nkx2.5 and chicken ovalbumin upstream promoter-transcription factor 1 play antagonistic roles in regulating the expression of calreticulin during cardiac development. These studies indicate that cardiac-specific transcription factor Nkx2.5 plays a central role in activating calreticulin expression and that there is a cooperation between chicken ovalbumin upstream promoter-transcription factor 1 and Nkx2.5 at the calreticulin promoter.

Regulation of theta-antigen expression by agents altering cyclic AMP level and by thymic factor.

PubMed

Bach, M A; Fournier, C; Bach, J F

1975-02-28

Thymic factor, cyclic AMP, and products increasing its cellular level, such as Prostaglandin E1, induce the appearance of the theta-antigen on T-cell precursors whether assessed by a rossette-inhibition assay or a cytotoxic assay after cell fractionation on BSA discontinuous gradiet. Synergism has been demonstrated between cyclic AMPT and TF for that effect. Conversely, decrease of theta expression has been obtained by altering cyclic AMP level in theta-positive cells either increasing it by dibutyryl cAMP treatment or decreasing it by indomethacin treatment. Finally, these data suggest the involvement of cyclic AMP in the regulation of theta expression under thymic hormone control.

Maternal iron status during pregnancy compared with neonatal iron status better predicts placental iron transporter expression in humans

PubMed Central

Best, Cora M.; Pressman, Eva K.; Cao, Chang; Cooper, Elizabeth; Guillet, Ronnie; Yost, Olivia L.; Galati, Jonathan; Kent, Tera R.; O’Brien, Kimberly O.

2016-01-01

The placenta richly expresses nonheme and heme Fe transport proteins. To address the impact of maternal and neonatal Fe status and hepcidin on the regulation of these proteins, mRNA expression and protein abundance of nonheme and heme Fe transport proteins were evaluated in placental tissue from 154 adolescents. Regression analyses found maternal Fe status was significantly associated with multiple placental nonheme and heme transporters, whereas neonatal Fe status was related to only 3 heme transporters. Across statistical analyses, maternal Fe status was consistently associated with the placental nonheme Fe importer transferrin receptor 1 (TfR1). Protein abundance of TfR1 was related to midgestation maternal serum ferritin (SF) (β = −0.32; P = 0.005) and serum TfR (β = 0.25; P = 0.024). Protein abundance of the heme importer, proton-coupled folate transporter, was related to neonatal SF (β = 0.30; P = 0.016) and serum TfR (β = −0.46; P < 0.0001). Neonatal SF was also related to mRNA expression of the heme exporter feline leukemia virus subgroup C receptor 1 (β = −0.30; P = 0.004). In summary, maternal Fe insufficiency during pregnancy predicts increased expression of the placental nonheme Fe transporter TfR1. Associations between placental heme Fe transporters and neonatal Fe status require further study.—Best, C. M., Pressman, E. K., Cao, C., Cooper, E., Guillet, R., Yost, O. L., Galati, J., Kent, T. R., O’Brien, K. O. Maternal iron status during pregnancy compared with neonatal iron status better predicts placental iron transporter expression in humans. PMID:27402672

Finding biomarkers in non-model species: literature mining of transcription factors involved in bovine embryo development

PubMed Central

2012-01-01

Background Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries). Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i) trophoblast proliferation and differentiation or (ii) embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i) stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii) mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this “mining”, we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n = 64) that we used as biological “gold standard”. When successful, the “mining” approach would identify them all, as well as novel ones. Methods To gain knowledge on molecular-genetic regulations in a non model organism, we offer an approach based on literature-mining and score arrangement of data from model organisms. This approach was applied to identify novel transcription factors during bovine blastocyst elongation, a process that is not observed in rodents and primates. As a result, searching through human and mouse corpuses, we identified numerous bovine homologs, among which 11 to 14% of transcription factors including the gold standard TF as well as novel TF potentially important to gene regulation in ruminant embryo development. The scripts of the workflow are written in Perl and available on demand. They require data input coming from all various databases for any kind of biological issue once the data has been prepared according to keywords for the studied topic and species; we can provide data sample to illustrate the use and functionality of the workflow. Results To do so, we created a workflow that allowed the pipeline processing of literature data and biological data, extracted from Web of Science (WoS) or PubMed but also from Gene Expression Omnibus (GEO), Gene Ontology (GO), Uniprot, HomoloGene, TcoF-DB and TFe (TF encyclopedia). First, the human and mouse homologs of the bovine proteins were selected, filtered by text corpora and arranged by score functions. The score functions were based on the gene name frequencies in corpora. Then, transcription factors were identified using TcoF-DB and double-checked using TFe to characterise TF groups and families. Thus, among a search space of 18,670 bovine homologs, 489 were identified as transcription factors. Among them, 243 were absent from the high-throughput data available at the time of the study. They thus stand so far for putative TF acting during bovine embryo elongation, but might be retrieved from a recent RNA sequencing dataset (Mamo et al. , 2012). Beyond the 246 TF that appeared expressed in bovine elongating tissues, we restricted our interpretation to those occurring within a list of 50 top-ranked genes. Among the transcription factors identified therein, half belonged to the gold standard (ASCL2, c-FOS, ETS2, GATA3, HAND1) and half did not (ESR1, HES1, ID2, NANOG, PHB2, TP53, STAT3). Conclusions A workflow providing search for transcription factors acting in bovine elongation was developed. The model assumed that proteins sharing the same protein domains in closely related species had the same protein functionalities, even if they were differently regulated among species or involved in somewhat different pathways. Under this assumption, we merged the information on different mammalian species from different databases (literature and biology) and proposed 489 TF as potential participants of embryo proliferation and differentiation, with (i) a recall of 95% with regard to a biological gold standard defined in 2011 and (ii) an extension of more than 3 times the gold standard of TF detected so far in elongating tissues. The working capacity of the workflow was supported by the manual expertise of the biologists on the results. The workflow can serve as a new kind of bioinformatics tool to work on fused data sources and can thus be useful in studies of a wide range of biological processes. PMID:22931563

Disruption of sex-hormone levels and steroidogenic-related gene expression on Mongolia Racerunner (Eremias argus) after exposure to triadimefon and its enantiomers.

PubMed

Li, Jitong; Chang, Jing; Li, Wei; Guo, Baoyuan; Li, Jianzhong; Wang, Huili

2017-03-01

Triadimefon (TF) is a widely used chiral fungicide with one chiral centre and two enantiomers (TF 1 and TF 2 ). However, little is reported about the ecological toxicity of reptiles on an enantioselective level. TF is a potential endocrine disruptor that may interfere with sex steroid hormones, such as testosterone (T) and 17beta-estradiol (E 2 ). In our study, the lizards Mongolia Racerunner (Eremias argus) were orally exposed to TF and its enantiomers for 21 days. Plasma sex steroid hormones and steroidogenic-related genes, including 17-beta-hydroxysteroid (hsd17β), cytochrome P450 enzymes (cyp19 and cyp17), and steroid hormone receptors (erα and Ar) were evaluated. After exposure, the plasma testosterone level in the 100 mg/kg bw group was elevated, while the oestradiol level was reduced. This phenomenon may be caused by the transformation of cyp19, which may inhibit the conversion of testosterone to oestradiol and affect sexual behaviour. In addition, the two enantiomers have different effects on hormone levels, which testified to the previously reported biotoxic dissimilarity between TF 1 and TF 2 in organisms. Furthermore, the cyp19 mRNA level in liver and gonad of the TF 2 and TF group (100 mg/kg bw ) were significantly down-regulated, while the cyp17 and hsd17β mRNA levels were up-regulated. The expression of erα and Ar mRNA levels were up-regulated in males but not in females, which may indicate that TF has sex differences on these two genes. As seen from the above results, TF and its enantiomers may have endocrine-disrupting effects on lizards (E. argus) by acting sensitively on sex steroid hormones and steroidogenic-related genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

2016-03-01

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Angiotensin II inhibits uptake of transferrin-bound iron but not non-transferrin-bound iron by cultured astrocytes.

PubMed

Huang, Suna; Du, Fang; Li, Lan; Liu, Yong; Liu, Yuhong; Zhang, Chao; Qian, Zhong Ming

2014-06-01

The existence of all components of the renin-angiotensin system (RAS) and the iron metabolism system, and the recent findings on the functions of angiotensin II (ANGII) in peripheral iron metabolism imply that ANGII might play a role in iron homeostasis by regulating expression of iron transport proteins in the brain. Here, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes. We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as the expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes. This indicated that the inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on the expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in the cells. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

PubMed Central

Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

2013-01-01

Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo. PMID:24089666

Measurement of microparticle tissue factor activity in clinical samples: A summary of two tissue factor-dependent FXa generation assays.

PubMed

Hisada, Yohei; Alexander, Wyeth; Kasthuri, Raj; Voorhees, Peter; Mobarrez, Fariborz; Taylor, Angela; McNamara, Coleen; Wallen, Hakan; Witkowski, Marco; Key, Nigel S; Rauch, Ursula; Mackman, Nigel

2016-03-01

Thrombosis is a leading cause of morbidity and mortality. Detection of a prothrombotic state using biomarkers would be of great benefit to identify patients at risk of thrombosis that would benefit from thromboprophylaxis. Tissue factor (TF) is a highly procoagulant protein that under normal conditions is not present in the blood. However, increased levels of TF in the blood in the form of microparticles (MPs) (also called extracellular vesicles) are observed under various pathological conditions. In this review, we will discuss studies that have measured MP-TF activity in a variety of diseases using two similar FXa generation assay. One of the most robust signals for MP-TF activity (16-26 fold higher than healthy controls) is observed in pancreatic cancer patients with venous thromboembolism. In this case, the TF+ MPs appear to be derived from the cancer cells. Surprisingly, cirrhosis and acute liver injury are associated with 17-fold and 38-fold increases in MP-TF activity, respectively. Based on mouse models, we speculate that the TF+ MPs are derived from hepatocytes. More modest increases are observed in patients with urinary tract infections (6-fold) and in a human endotoxemia model (9-fold) where monocytes are the likely source of the TF+ MPs. Finally, there is no increase in MP-TF activity in the majority of cardiovascular disease patients. These studies indicate that MP-TF activity may be a useful biomarker to identify patients with particular diseases that have an increased risk of thrombosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abraham, Jonathan; Corbett, Kevin D.; Farzan, Michael

New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip ofmore » the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.« less

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

PubMed

Cui, X Y; Tinholt, M; Stavik, B; Dahm, A E A; Kanse, S; Jin, Y; Seidl, S; Sahlberg, K K; Iversen, N; Skretting, G; Sandset, P M

2016-02-01

ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients. © 2015 International Society on Thrombosis and Haemostasis.

The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

PubMed

Galson, D L; Tsuchiya, T; Tendler, D S; Huang, L E; Ren, Y; Ogura, T; Bunn, H F

1995-04-01

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Dietary exposure to the endocrine disruptor tolylfluanid promotes global metabolic dysfunction in male mice.

PubMed

Regnier, Shane M; Kirkley, Andrew G; Ye, Honggang; El-Hashani, Essam; Zhang, Xiaojie; Neel, Brian A; Kamau, Wakanene; Thomas, Celeste C; Williams, Ayanna K; Hayes, Emily T; Massad, Nicole L; Johnson, Daniel N; Huang, Lei; Zhang, Chunling; Sargis, Robert M

2015-03-01

Environmental endocrine disruptors are implicated as putative contributors to the burgeoning metabolic disease epidemic. Tolylfluanid (TF) is a commonly detected fungicide in Europe, and previous in vitro and ex vivo work has identified it as a potent endocrine disruptor with the capacity to promote adipocyte differentiation and induce adipocytic insulin resistance, effects likely resulting from activation of glucocorticoid receptor signaling. The present study extends these findings to an in vivo mouse model of dietary TF exposure. After 12 weeks of consumption of a normal chow diet supplemented with 100 parts per million TF, mice exhibited increased body weight gain and an increase in total fat mass, with a specific augmentation in visceral adipose depots. This increased adipose accumulation is proposed to occur through a reduction in lipolytic and fatty acid oxidation gene expression. Dietary TF exposure induced glucose intolerance, insulin resistance, and metabolic inflexibility, while also disrupting diurnal rhythms of energy expenditure and food consumption. Adipose tissue endocrine function was also impaired with a reduction in serum adiponectin levels. Moreover, adipocytes from TF-exposed mice exhibited reduced insulin sensitivity, an effect likely mediated through a specific down-regulation of insulin receptor substrate-1 expression, mirroring effects of ex vivo TF exposure. Finally, gene set enrichment analysis revealed an increase in adipose glucocorticoid receptor signaling with TF treatment. Taken together, these findings identify TF as a novel in vivo endocrine disruptor and obesogen in mice, with dietary exposure leading to alterations in energy homeostasis that recapitulate many features of the metabolic syndrome.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow.

PubMed

Zhu, Shu; Travers, Richard J; Morrissey, James H; Diamond, Scott L

2015-09-17

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) -bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm(2). Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm(2) and sensitive to O1A6 at 0 to 0.2 molecules per µm(2). However, neither antibody reduced fibrin generation at ∼2 molecules per µm(2) when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm(2)) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. © 2015 by The American Society of Hematology.

From DNA binding to transcriptional activation: Is the TALE complete?

PubMed

Bobola, Nicoletta

2017-09-04

How transcription factors (TFs) control enhancer and promoter functions to effect changes in gene expression is an important question. In this issue, Hau et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701154) show that the TALE TF MEIS recruits the histone modifier PARP1/ARTD1 at promoters to decompact chromatin and activate transcription. © 2017 Bobola.

A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development

PubMed Central

Schertel, Claus; Albarca, Monica; Rockel-Bauer, Claudia; Kelley, Nicholas W.; Bischof, Johannes; Hens, Korneel

2015-01-01

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such “bivalent” chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue. PMID:25568052

Treatment processes and demographic variables as predictors of dropout from trauma-focused cognitive behavioral therapy (TF-CBT) for youth.

PubMed

Yasinski, Carly; Hayes, Adele M; Alpert, Elizabeth; McCauley, Thomas; Ready, C Beth; Webb, Charles; Deblinger, Esther

2018-05-22

Premature dropout is a significant concern in trauma-focused psychotherapy for youth. Previous studies have primarily examined pre-treatment demographic and symptom-related predictors of dropout, but few consistent findings have been reported. The current study examined demographic, symptom, and in-session process variables as predictors of dropout from Trauma-Focused Cognitive Behavioral Therapy (TF-CBT) for youth. Participants were a diverse sample of Medicaid-eligible youth (ages 7-17; n = 108) and their nonoffending caregivers (n = 86), who received TF-CBT through an effectiveness study in a community setting. In-session process variables were coded from audio-recorded sessions, and these and pre-treatment demographic variables and symptom levels were examined as predictors of dropout prior to receiving an adequate dose of TF-CBT (<7 sessions). Twenty-nine children were classified as dropouts and 79 as completers. Binary logistic regression analyses revealed that higher levels of child and caregiver avoidance expressed during early sessions, as well as greater relationship difficulties between the child and therapist, predicted dropout. Those children who were in foster care during treatment were less likely to drop out than children living with parents or relatives. No other demographic or symptom-related factors predicted dropout. These findings highlight the importance of addressing avoidance and therapeutic relationship difficulties in early sessions of TF-CBT to help reduce dropout, and they have implications for improving efforts to disseminate evidence-based trauma-focused treatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cancer cell–derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

PubMed Central

Thomas, Grace M.; Panicot-Dubois, Laurence; Lacroix, Romaric; Dignat-George, Françoise; Lombardo, Dominique

2009-01-01

Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients. PMID:19667060

Ptpmt1 induced by HIF-2α regulates the proliferation and glucose metabolism in erythroleukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qin-Qin; Qinghai Provincial People's Hospital, Xining; Xiao, Feng-Jun

Hypoxia provokes metabolism misbalance, mitochondrial dysfunction and oxidative stress in both human and animal cells. However, the mechanisms which hypoxia causes mitochondrial dysfunction and energy metabolism misbalance still remain unclear. In this study, we presented evidence that mitochondrial phosphatase Ptpmt1 is a hypoxia response molecule that regulates cell proliferation, survival and glucose metabolism in human erythroleukemia TF-1 cells. Exposure to hypoxia or DFO treatment results in upregulation of HIF1-α, HIF-2α and Ptpmt1. Only inhibition of HIF-2α by shRNA transduction reduces Ptpmt1 expression in TF-1 cells under hypoxia. Ptpmt1 inhibitor suppresses the growth and induces apoptosis of TF-1 cells. Furthermore, we demonstrated that Ptpmt1more » inhibition reduces the Glut1 and Glut3 expression and decreases the glucose consumption in TF-1 cells. In additional, Ptpmt1 knockdown also results in the mitochondrial dysfunction determined by JC1 staining. These results delineate a key role for HIF-2α-induced Ptpmt1 upregulation in proliferation, survival and glucose metabolism of erythroleukemia cells. It is indicated that Ptpmt1 plays important roles in hypoxia-induced cell metabolism and mitochondrial dysfunction. - Highlights: • Hypoxia induces upregulation of HIF-1α, HIF-2α and Ptpmt1; HIF-2a induces Ptpmt1 upregulation in TF-1 cells. • PTPMT-1 inhibition reduces growth and induces apoptosis of TF-1 cells. • PTPMT1 inhibition downregulates Glut-1, Glut-3 expression and reduces glucose consumption.« less

Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology

PubMed Central

Moreno, Marta; Fernández, Virginia; Monllau, Josep M.; Borrell, Víctor; Lerin, Carles; de la Iglesia, Núria

2015-01-01

Summary Neural stem cells (NSCs) reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs) that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA) of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT) and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state. PMID:26235896

Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

PubMed

Mast, Alan E

2016-01-01

Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients. © 2015 American Heart Association, Inc.

Nanopore sensing of individual transcription factors bound to DNA

PubMed Central

Squires, Allison; Atas, Evrim; Meller, Amit

2015-01-01

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes. PMID:26109509

Nanopore sensing of individual transcription factors bound to DNA

NASA Astrophysics Data System (ADS)

Squires, Allison; Atas, Evrim; Meller, Amit

2015-06-01

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.

System analysis identifies distinct and common functional networks governed by transcription factor ASCL1, in glioma and small cell lung cancer.

PubMed

Donakonda, Sainitin; Sinha, Swati; Dighe, Shrinivas Nivrutti; Rao, Manchanahalli R Satyanarayana

2017-07-25

ASCL1 is a basic Helix-Loop-Helix transcription factor (TF), which is involved in various cellular processes like neuronal development and signaling pathways. Transcriptome profiling has shown that ASCL1 overexpression plays an important role in the development of glioma and Small Cell Lung Carcinoma (SCLC), but distinct and common molecular mechanisms regulated by ASCL1 in these cancers are unknown. In order to understand how it drives the cellular functional network in these two tumors, we generated a gene expression profile in a glioma cell line (U87MG) to identify ASCL1 gene targets by an si RNA silencing approach and then compared this with a publicly available dataset of similarly silenced SCLC (NCI-H1618 cells). We constructed TF-TF and gene-gene interactions, as well as protein interaction networks of ASCL1 regulated genes in glioma and SCLC cells. Detailed network analysis uncovered various biological processes governed by ASCL1 target genes in these two tumor cell lines. We find that novel ASCL1 functions related to mitosis and signaling pathways influencing development and tumor growth are affected in both glioma and SCLC cells. In addition, we also observed ASCL1 governed functional networks that are distinct to glioma and SCLC.

Diversity, expansion, and evolutionary novelty of plant DNA-binding transcription factor families.

PubMed

Lehti-Shiu, Melissa D; Panchy, Nicholas; Wang, Peipei; Uygun, Sahra; Shiu, Shin-Han

2017-01-01

Plant transcription factors (TFs) that interact with specific sequences via DNA-binding domains are crucial for regulating transcriptional initiation and are fundamental to plant development and environmental response. In addition, expansion of TF families has allowed functional divergence of duplicate copies, which has contributed to novel, and in some cases adaptive, traits in plants. Thus, TFs are central to the generation of the diverse plant species that we see today. Major plant agronomic traits, including those relevant to domestication, have also frequently arisen through changes in TF coding sequence or expression patterns. Here our goal is to provide an overview of plant TF evolution by first comparing the diversity of DNA-binding domains and the sizes of these domain families in plants and other eukaryotes. Because TFs are among the most highly expanded gene families in plants, the birth and death process of TFs as well as the mechanisms contributing to their retention are discussed. We also provide recent examples of how TFs have contributed to novel traits that are important in plant evolution and in agriculture.This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer. Copyright © 2016 Elsevier B.V. All rights reserved.

Nucleosome regulatory dynamics in response to TGFβ

PubMed Central

Enroth, Stefan; Andersson, Robin; Bysani, Madhusudhan; Wallerman, Ola; Termén, Stefan; Tuch, Brian B.; De La Vega, Francisco M.; Heldin, Carl-Henrik; Moustakas, Aristidis; Komorowski, Jan; Wadelius, Claes

2014-01-01

Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44–78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing. PMID:24771338

[Trigger factor dependent refolding of bacterial luciferases in Escherichia coli cells: kinetics, efficiency and effect of the bichaperone system, DnaKJE-ClpB].

PubMed

Mel'kina, O E; Gorianin, I I; Manukhov, I V; Zavil'gel'skiĭ, G B

2013-01-01

Here were determined the basic parameters of the Tigger Factor (TF) -dependent refolding of thermal inactivated bacterial luciferases. The TF-dependent refolding is less efficient and requires more time than DnaKJE-dependent refolding. The increase in the intracellular concentration of TF leads to an apparent decrease in the level of the thermal inactivated bacterial luciferase refolding. For thermolabile luciferases, the level of TF-dependent refolding is significantly higher, than for thermostable luciferases: 30-40%--for the thermolabile Aliivibrio fischeri and Photobacterium leiognathi luciferases, and 10 and 0.5% for the thermostable Vibrio harveyi and Photorhabdus luminescens luciferases, respectively. The negative effect of the ClpB protein on the TF-dependent refolding was shown: in Escherichia coli clpB::kan TF-dependent refolding is more efficient than in the E. coli clpB+.

Dexamethasone enhances agonist induction of tissue factor in monocytes but not in endothelial cells.

PubMed

Bottles, K D; Morrissey, J H

1993-06-01

Stimulation of monocytic cells by inflammatory agents such as bacterial lipopolysaccharide or tumour necrosis factor-alpha leads to the rapid and transient expression of tissue factor, the major cellular initiator of the extrinsic coagulation cascade in both haemostasis and tissue inflammation. In this study we investigated whether the synthetic anti-inflammatory glucocorticoid, dexamethasone, would inhibit agonist induction of tissue factor expression in both monocytes and endothelial cells. Surprisingly, dexamethasone significantly enhanced the induction of tissue factor expression by peripheral blood mononuclear cells and an established monocytic cell line, THP-1, in response to lipopolysaccharide or tumour necrosis factor-alpha. However, unlike monocytic cells, dexamethasone did not enhance agonist induction of tissue factor in endothelial cells. Synergistic enhancement of tissue factor expression by dexamethasone was also reflected in tissue factor mRNA levels in THP-1 cells, but was not the result of improved TF mRNA stability. Synergism between bacterial lipopolysaccharide and glucocorticoid in the induction of monocyte effector function is extremely unusual and may help to explain the variable outcome of glucocorticoid treatment of septic shock.

Genome-wide transcriptional responses of Alteromonas naphthalenivorans SN2 to contaminated seawater and marine tidal flat sediment.

PubMed

Jin, Hyun Mi; Jeong, Hye Im; Kim, Kyung Hyun; Hahn, Yoonsoo; Madsen, Eugene L; Jeon, Che Ok

2016-02-18

A genome-wide transcriptional analysis of Alteromonas naphthalenivorans SN2 was performed to investigate its ecophysiological behavior in contaminated tidal flats and seawater. The experimental design mimicked these habitats that either added naphthalene or pyruvate; tidal flat-naphthalene (TF-N), tidal flat-pyruvate (TF-P), seawater-naphthalene (SW-N), and seawater-pyruvate (SW-P). The transcriptional profiles clustered by habitat (TF-N/TF-P and SW-N/SW-P), rather than carbon source, suggesting that the former may exert a greater influence on genome-wide expression in strain SN2 than the latter. Metabolic mapping of cDNA reads from strain SN2 based on KEGG pathway showed that metabolic and regulatory genes associated with energy metabolism, translation, and cell motility were highly expressed in all four test conditions, probably highlighting the copiotrophic properties of strain SN2 as an opportunistic marine r-strategist. Differential gene expression analysis revealed that strain SN2 displayed specific cellular responses to environmental variables (tidal flat, seawater, naphthalene, and pyruvate) and exhibited certain ecological fitness traits -- its notable PAH degradation capability in seasonally cold tidal flat might be reflected in elevated expression of stress response and chaperone proteins, while fast growth in nitrogen-deficient and aerobic seawater probably correlated with high expression of glutamine synthetase, enzymes utilizing nitrite/nitrate, and those involved in the removal of reactive oxygen species.

Genome-wide transcriptional responses of Alteromonas naphthalenivorans SN2 to contaminated seawater and marine tidal flat sediment

PubMed Central

Jin, Hyun Mi; Jeong, Hye Im; Kim, Kyung Hyun; Hahn, Yoonsoo; Madsen, Eugene L.; Jeon, Che Ok

2016-01-01

A genome-wide transcriptional analysis of Alteromonas naphthalenivorans SN2 was performed to investigate its ecophysiological behavior in contaminated tidal flats and seawater. The experimental design mimicked these habitats that either added naphthalene or pyruvate; tidal flat-naphthalene (TF-N), tidal flat-pyruvate (TF-P), seawater-naphthalene (SW-N), and seawater-pyruvate (SW-P). The transcriptional profiles clustered by habitat (TF-N/TF-P and SW-N/SW-P), rather than carbon source, suggesting that the former may exert a greater influence on genome-wide expression in strain SN2 than the latter. Metabolic mapping of cDNA reads from strain SN2 based on KEGG pathway showed that metabolic and regulatory genes associated with energy metabolism, translation, and cell motility were highly expressed in all four test conditions, probably highlighting the copiotrophic properties of strain SN2 as an opportunistic marine r-strategist. Differential gene expression analysis revealed that strain SN2 displayed specific cellular responses to environmental variables (tidal flat, seawater, naphthalene, and pyruvate) and exhibited certain ecological fitness traits –– its notable PAH degradation capability in seasonally cold tidal flat might be reflected in elevated expression of stress response and chaperone proteins, while fast growth in nitrogen-deficient and aerobic seawater probably correlated with high expression of glutamine synthetase, enzymes utilizing nitrite/nitrate, and those involved in the removal of reactive oxygen species. PMID:26887987

Model of a ternary complex between activated factor VII, tissue factor and factor IX.

PubMed

Chen, Shu-wen W; Pellequer, Jean-Luc; Schved, Jean-François; Giansily-Blaizot, Muriel

2002-07-01

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

PvTFDB: a Phaseolus vulgaris transcription factors database for expediting functional genomics in legumes

PubMed Central

Bhawna; Bonthala, V.S.; Gajula, MNV Prasad

2016-01-01

The common bean [Phaseolus vulgaris (L.)] is one of the essential proteinaceous vegetables grown in developing countries. However, its production is challenged by low yields caused by numerous biotic and abiotic stress conditions. Regulatory transcription factors (TFs) symbolize a key component of the genome and are the most significant targets for producing stress tolerant crop and hence functional genomic studies of these TFs are important. Therefore, here we have constructed a web-accessible TFs database for P. vulgaris, called PvTFDB, which contains 2370 putative TF gene models in 49 TF families. This database provides a comprehensive information for each of the identified TF that includes sequence data, functional annotation, SSRs with their primer sets, protein physical properties, chromosomal location, phylogeny, tissue-specific gene expression data, orthologues, cis-regulatory elements and gene ontology (GO) assignment. Altogether, this information would be used in expediting the functional genomic studies of a specific TF(s) of interest. The objectives of this database are to understand functional genomics study of common bean TFs and recognize the regulatory mechanisms underlying various stress responses to ease breeding strategy for variety production through a couple of search interfaces including gene ID, functional annotation and browsing interfaces including by family and by chromosome. This database will also serve as a promising central repository for researchers as well as breeders who are working towards crop improvement of legume crops. In addition, this database provide the user unrestricted public access and the user can download entire data present in the database freely. Database URL: http://www.multiomics.in/PvTFDB/ PMID:27465131

Prognostic Value of EMT-inducing Transcription Factors (EMT-TFs) in Metastatic Breast Cancer: A Systematic Review and Meta-analysis

PubMed Central

Imani, Saber; Hosseinifard, Hossein; Cheng, Jingliang; Wei, Chunli; Fu, Junjiang

2016-01-01

The epithelial-to-mesenchymal transition (EMT) is a vital control point in metastatic breast cancer (MBC). TWIST1, SNAIL1, SLUG, and ZEB1, as key EMT-inducing transcription factors (EMT-TFs), are involved in MBC through different signaling cascades. This updated meta-analysis was conducted to assess the correlation between the expression of EMT-TFs and prognostic value in MBC patients. A total of 3,218 MBC patients from fourteen eligible studies were evaluated. The pooled hazard ratios (HR) for EMT-TFs suggested that high EMT-TF expression was significantly associated with poor prognosis in MBC patients (HRs = 1.72; 95% confidence intervals (CIs) = 1.53–1.93; P = 0.001). In addition, the overexpression of SLUG was the most impactful on the risk of MBC compared with TWIST1 and SNAIL1, which sponsored fixed models. Strikingly, the increased risk of MBC was less associated with ZEB1 expression. However, the EMT-TF expression levels significantly increased the risk of MBC in the Asian population (HR = 2.11, 95% CI = 1.70–2.62) without any publication bias (t = 1.70, P = 0.11). These findings suggest that the overexpression of potentially TWIST1, SNAIL1 and especially SLUG play a key role in the aggregation of MBC treatment as well as in the improvement of follow-up plans in Asian MBC patients. PMID:27335258

The Arabidopsis Transcription Factor MYB112 Promotes Anthocyanin Formation during Salinity and under High Light Stress1[OPEN

PubMed Central

Lotkowska, Magda E.; Tohge, Takayuki; Fernie, Alisdair R.; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

2015-01-01

MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions. PMID:26378103

Gonad Transcriptome Analysis of High-Temperature-Treated Females and High-Temperature-Induced Sex-Reversed Neomales in Nile Tilapia.

PubMed

Sun, Li Xue; Teng, Jian; Zhao, Yan; Li, Ning; Wang, Hui; Ji, Xiang Shan

2018-02-28

Nowadays, the molecular mechanisms governing TSD (temperature-dependent sex determination) or GSD + TE (genotypic sex determination + temperature effects) remain a mystery in fish. We developed three all-female families of Nile tilapia ( Oreochromis niloticus ), and the family with the highest male ratio after high-temperature treatment was used for transcriptome analysis. First, gonadal histology analysis indicated that the histological morphology of control females (CF) was not significantly different from that of high-temperature-treated females (TF) at various development stages. However, the high-temperature treatment caused a lag of spermatogenesis in high-temperature-induced neomales (IM). Next, we sequenced the transcriptome of CF, TF, and IM Nile tilapia. 79, 11,117, and 11,000 differentially expressed genes (DEGs) were detected in the CF-TF, CF-IM, and TF-IM comparisons, respectively, and 44 DEGs showed identical expression changes in the CF-TF and CF-IM comparisons. Principal component analysis (PCA) indicated that three individuals in CF and three individuals in TF formed a cluster, and three individuals in IM formed a distinct cluster, which confirmed that the gonad transcriptome profile of TF was similar to that of CF and different from that of IM. Finally, six sex-related genes were validated by qRT-PCR. This study identifies a number of genes that may be involved in GSD + TE, which will be useful for investigating the molecular mechanisms of TSD or GSD + TE in fish.

The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.

PubMed

Persson, Egon; Madsen, Jesper J; Olsen, Ole H

2014-12-01

Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity. © 2014 The Protein Society.

Nup124p Is a Nuclear Pore Factor of Schizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1

PubMed Central

Balasundaram, David; Benedik, Michael J.; Morphew, Mary; Dang, Van-Dinh; Levin, Henry L.

1999-01-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag. PMID:10409764

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

PubMed

Balasundaram, D; Benedik, M J; Morphew, M; Dang, V D; Levin, H L

1999-08-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

PubMed Central

Nielsen, Anders L.; Sorensen, Anders B.; Holmberg, Heidi L.; Gandhi, Prafull S.; Karlsson, Johan; Buchardt, Jens; Lamberth, Kasper; Kjelgaard-Hansen, Mads; Ley, Carsten Dan; Sørensen, Brit B.; Ruf, Wolfram; Olsen, Ole H.; Østergaard, Henrik

2017-01-01

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa. PMID:29109275

PCTFPeval: a web tool for benchmarking newly developed algorithms for predicting cooperative transcription factor pairs in yeast.

PubMed

Lai, Fu-Jou; Chang, Hong-Tsun; Wu, Wei-Sheng

2015-01-01

Computational identification of cooperative transcription factor (TF) pairs helps understand the combinatorial regulation of gene expression in eukaryotic cells. Many advanced algorithms have been proposed to predict cooperative TF pairs in yeast. However, it is still difficult to conduct a comprehensive and objective performance comparison of different algorithms because of lacking sufficient performance indices and adequate overall performance scores. To solve this problem, in our previous study (published in BMC Systems Biology 2014), we adopted/proposed eight performance indices and designed two overall performance scores to compare the performance of 14 existing algorithms for predicting cooperative TF pairs in yeast. Most importantly, our performance comparison framework can be applied to comprehensively and objectively evaluate the performance of a newly developed algorithm. However, to use our framework, researchers have to put a lot of effort to construct it first. To save researchers time and effort, here we develop a web tool to implement our performance comparison framework, featuring fast data processing, a comprehensive performance comparison and an easy-to-use web interface. The developed tool is called PCTFPeval (Predicted Cooperative TF Pair evaluator), written in PHP and Python programming languages. The friendly web interface allows users to input a list of predicted cooperative TF pairs from their algorithm and select (i) the compared algorithms among the 15 existing algorithms, (ii) the performance indices among the eight existing indices, and (iii) the overall performance scores from two possible choices. The comprehensive performance comparison results are then generated in tens of seconds and shown as both bar charts and tables. The original comparison results of each compared algorithm and each selected performance index can be downloaded as text files for further analyses. Allowing users to select eight existing performance indices and 15 existing algorithms for comparison, our web tool benefits researchers who are eager to comprehensively and objectively evaluate the performance of their newly developed algorithm. Thus, our tool greatly expedites the progress in the research of computational identification of cooperative TF pairs.

PCTFPeval: a web tool for benchmarking newly developed algorithms for predicting cooperative transcription factor pairs in yeast

PubMed Central

2015-01-01

Background Computational identification of cooperative transcription factor (TF) pairs helps understand the combinatorial regulation of gene expression in eukaryotic cells. Many advanced algorithms have been proposed to predict cooperative TF pairs in yeast. However, it is still difficult to conduct a comprehensive and objective performance comparison of different algorithms because of lacking sufficient performance indices and adequate overall performance scores. To solve this problem, in our previous study (published in BMC Systems Biology 2014), we adopted/proposed eight performance indices and designed two overall performance scores to compare the performance of 14 existing algorithms for predicting cooperative TF pairs in yeast. Most importantly, our performance comparison framework can be applied to comprehensively and objectively evaluate the performance of a newly developed algorithm. However, to use our framework, researchers have to put a lot of effort to construct it first. To save researchers time and effort, here we develop a web tool to implement our performance comparison framework, featuring fast data processing, a comprehensive performance comparison and an easy-to-use web interface. Results The developed tool is called PCTFPeval (Predicted Cooperative TF Pair evaluator), written in PHP and Python programming languages. The friendly web interface allows users to input a list of predicted cooperative TF pairs from their algorithm and select (i) the compared algorithms among the 15 existing algorithms, (ii) the performance indices among the eight existing indices, and (iii) the overall performance scores from two possible choices. The comprehensive performance comparison results are then generated in tens of seconds and shown as both bar charts and tables. The original comparison results of each compared algorithm and each selected performance index can be downloaded as text files for further analyses. Conclusions Allowing users to select eight existing performance indices and 15 existing algorithms for comparison, our web tool benefits researchers who are eager to comprehensively and objectively evaluate the performance of their newly developed algorithm. Thus, our tool greatly expedites the progress in the research of computational identification of cooperative TF pairs. PMID:26677932

A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

DOE PAGES

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang; ...

2016-08-30

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

Transcription factors for modification of lignin content in plants

DOEpatents

Wang, Huanzhong; Chen, Fang; Dixon, Richard A.

2015-06-02

The invention provides methods for modifying lignin, cellulose, xylan, and hemicellulose content in plants, and for achieving ectopic lignification and, for instance, secondary cell wall synthesis in pith cells, by altered regulation of a WRKY transcription factor. Nucleic acid constructs for altered WRKY-TF expression are described. Transgenic plants are provided that comprise modified pith cell walls, and lignin, cellulose, and hemicellulose content. Plants described herein may be used, for example, as improved biofuel feedstock and as highly digestible forage crops.

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

PubMed Central

Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

2016-01-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

The Novel Endocrine Disruptor Tolylfluanid Impairs Insulin Signaling in Primary Rodent and Human Adipocytes through a Reduction in Insulin Receptor Substrate-1 Levels

PubMed Central

Sargis, Robert M.; Neel, Brian A.; Brock, Clifton O.; Lin, Yuxi; Hickey, Allison T.; Carlton, Daniel A.; Brady, Matthew J.

2012-01-01

Emerging data suggest that environmental endocrine disrupting chemicals (EDCs) may contribute to the pathophysiology of obesity and diabetes. In prior work, the phenylsulfamide fungicide tolylfluanid (TF) was shown to augment adipocyte differentiation, yet its effects on mature adipocyte metabolism remain unknown. Because of the central role of adipose tissue in global energy regulation, the present study tested the hypothesis that TF modulates insulin action in primary rodent and human adipocytes. Alterations in insulin signaling in primary mammalian adipocytes were determined by the phosphorylation of Akt, a critical insulin signaling intermediate. Treatment of primary murine adipose tissue in vitro with 100 nM TF for 48 h markedly attenuated acute insulin-stimulated Akt phosphorylation in a strain- and species-independent fashion. Perigonadal, perirenal, and mesenteric fat were all sensitive to TF-induced insulin resistance. A similar TF-induced reduction in insulin-stimulated Akt phosphorylation was observed in primary human subcutaneous adipose tissue. TF-treatment led to a potent and specific reduction in insulin receptor substrate-1 (IRS-1) mRNA and protein levels, a key upstream mediator of insulin’s diverse metabolic effects. In contrast, insulin receptor-β, phosphatidylinositol 3-kinase, and Akt expression were unchanged, indicating a specific abrogation of insulin signaling. Additionally, TF-treated adipocytes exhibited altered endocrine function with a reduction in both basal and insulin-stimulated leptin secretion. These studies demonstrate that TF induces cellular insulin resistance in primary murine and human adipocytes through a reduction of IRS-1 expression and protein stability, raising concern about the potential for this fungicide to disrupt metabolism and thereby contribute to the pathogenesis of diabetes. PMID:22387882

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach

PubMed Central

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions. PMID:29033978

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach.

PubMed

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions.

JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles

PubMed Central

Mathelier, Anthony; Fornes, Oriol; Arenillas, David J.; Chen, Chih-yu; Denay, Grégoire; Lee, Jessica; Shi, Wenqiang; Shyr, Casper; Tan, Ge; Worsley-Hunt, Rebecca; Zhang, Allen W.; Parcy, François; Lenhard, Boris; Sandelin, Albin; Wasserman, Wyeth W.

2016-01-01

JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. We updated the structural annotation of the TF DNA binding domains (DBDs) following a published hierarchical structural classification. In addition, we introduced 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites. This new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, we provide the users with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. Finally, we provide the JASPAR2016 R/Bioconductor data package with the data of this release. PMID:26531826

Detection of von Willebrand factor and tissue factor in platelets-fibrin rich coronary thrombi in acute myocardial infarction.

PubMed

Yamashita, Atsushi; Sumi, Takahiro; Goto, Shinya; Hoshiba, Yasunari; Nishihira, Kensaku; Kawamoto, Riichirou; Hatakeyama, Kinta; Date, Haruhiko; Imamura, Takuroh; Ogawa, Hisao; Asada, Yujiro

2006-01-01

The rapid closure of coronary arteries due to occlusive thrombi is the major cause of acute myocardial infarction. However, the mechanisms of coronary thrombus formation have not been elucidated. We immunohistochemically assessed the localizations and their changes over time of glycoprotein IIb/IIIa, fibrin, von Willebrand factor (vWF), and tissue factor (TF), after the onset of chest pain (<4, 4 to 6, or 6 to 12 hours), in fresh coronary thrombi causing acute myocardial infarction. The occlusive thrombi were consistently composed of platelets, fibrin, vWF, and TF from the early phase of onset, and glycoprotein IIb/IIIa and fibrin were closely associated with vWF and TF, respectively. vWF and/or TF may contribute to occlusive thrombus formation and be novel therapeutic candidates for treating patients with coronary thrombosis.

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

PubMed Central

de las Heras, Aitor; Fraile, Sofia; de Lorenzo, Victor

2012-01-01

Prokaryotic transcription factors (TFs) that bind small xenobiotic molecules (e.g., TFs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. XylR, the master TF for expression of the m-xylene biodegradation operons encoded in the TOL plasmid pWW0 of Pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbenzylalcohol (3MBA). We have examined whether such a relaxed regulatory scenario can be reshaped into a high-capacity/high-specificity regime by changing the connectivity of this effector-sensing TF within the rest of the circuit rather than modifying XylR structure itself. To this end, the natural negative feedback loop that operates on xylR transcription was modified with a translational attenuator that brings down the response to 3MBA while maintaining the transcriptional output induced by m-xylene (as measured with a luxCDABE reporter system). XylR expression was then subject to a positive feedback loop in which the TF was transcribed from its own target promoters, each known to hold different input/output transfer functions. In the first case (xylR under the strong promoter of the upper TOL operon, Pu), the reporter system displayed an increased transcriptional capacity in the resulting network for both the optimal and the suboptimal XylR effectors. In contrast, when xylR was expressed under the weaker Ps promoter, the resulting circuit unmistakably discriminated m-xylene from 3MBA. The non-natural connectivity engineered in the network resulted both in a higher promoter activity and also in a much-increased signal-to-background ratio. These results indicate that the working regimes of given genetic circuits can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops. PMID:23071444

Novel gene expression mechanism in a fission yeast retroelement: Tf1 proteins are derived from a single primary translation product.

PubMed

Levin, H L; Weaver, D C; Boeke, J D

1993-12-01

In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol proteins. The different ORFs are thought to allow for overexpression of the Gag protein relative to Pol protein presumed necessary for the assembly of functional retrovirus particles and virus-like particles (VLPs). The results of in vivo experiments designed to detect the transposition of Tf1 show that Tf1 is indeed active and can insert itself into the host genome via a true retrotransposition process. Thus, a paradox emerged between the lack of any obvious means of overexpressing Tf1 Gag protein and the demonstrated functionality of the element. Epitope tagging experiments described here confirm that the Tf1 large ORF is intact and that there is no translational or transcriptional mechanism used to overexpress the Tf1 Gag protein. In addition, we used sucrose gradients and antisera specific for Tf1 capsid (CA) and integrase (IN) to show that the Tf1 proteins do assemble into uniform populations of macromolecular particles that also cosediment with Tf1 reverse transcription products. This evidence suggests that Tf1 proteins form VLPs without using the previously described mechanisms that retroviruses and retrotransposons require to overexpress Gag proteins.

Conjunctival transcriptome profiling of Solomon Islanders with active trachoma in the absence of Chlamydia trachomatis infection.

PubMed

Vasileva, Hristina; Butcher, Robert; Pickering, Harry; Sokana, Oliver; Jack, Kelvin; Solomon, Anthony W; Holland, Martin J; Roberts, Chrissy H

2018-02-21

Clinical signs of active (inflammatory) trachoma are found in many children in the Solomon Islands, but the majority of these individuals have no serological evidence of previous infection with Chlamydia trachomatis. In Temotu and Rennell and Bellona provinces, ocular infections with C. trachomatis were seldom detected among children with active trachoma; a similar lack of association was seen between active trachoma and other common bacterial and viral causes of follicular conjunctivitis. Here, we set out to characterise patterns of gene expression at the conjunctivae of children in these provinces with and without clinical signs of trachomatous inflammation-follicular (TF) and C. trachomatis infection. Purified RNA from children with and without active trachoma was run on Affymetrix GeneChip Human Transcriptome Array 2.0 microarrays. Profiles were compared between individuals with ocular C. trachomatis infection and TF (group DI; n = 6), individuals with TF but no C. trachomatis infection (group D; n = 7), and individuals without TF or C. trachomatis infection (group N; n = 7). Differential gene expression and gene set enrichment for pathway membership were assessed. Conjunctival gene expression profiles were more similar within-group than between-group. Principal components analysis indicated that the first and second principal components combined explained almost 50% of the variance in the dataset. When comparing the DI group to the N group, genes involved in T-cell proliferation, B-cell signalling and CD8+ T cell signalling pathways were differentially regulated. When comparing the DI group to the D group, CD8+ T-cell regulation, interferon-gamma and IL17 production pathways were enriched. Genes involved in RNA transcription and translation pathways were upregulated when comparing the D group to the N group. Gene expression profiles in children in the Solomon Islands indicate immune responses consistent with bacterial infection when TF and C. trachomatis infection are concurrent. The transcriptomes of children with TF but without identified infection were not consistent with allergic or viral conjunctivitis.

Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

PubMed Central

Ullers, Ronald S.; Ang, Debbie; Schwager, Françoise; Georgopoulos, Costa; Genevaux, Pierre

2007-01-01

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30°C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. PMID:17360615

Activated factor XI and tissue factor in aortic stenosis: Links with thrombin generation

PubMed Central

Luszczak, Joanna; Undas, Anetta; Gissel, Matthew; Olszowska, Maria; Butenas, Saulius

2011-01-01

Introduction In our previous studies we showed that a significant proportion of patients with various cardiovascular diseases have active tissue factor (TF) and factor (F)XIa in their plasma. Objective To evaluate these two proteins in plasma from patients with aortic stenosis (AS) and established their relationship with the severity of the disease. Methods Fifty-four consecutive patients with AS, including 38 (70.4%) severe AS patients, were studied. Plasma FXIa and TF activity were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies. Results TF activity was detectible in plasma from 14 of 54 patients (25.9%), including 13 of 38 with severe AS (34.2%) and 1 of 16 (6.25%) with moderate AS (p=0.052). FXIa activity was found in 12 (22.2%) patients, mostly in individuals with severe AS (11 of 38, 28.9%, p=0.067). All 12 patients with circulating FXIa had active TF in their plasma as well. Severe AS patients with detectable TF had higher maximal (111±20 vs 97±16 mm Hg, p=0.02) and mean (61±12 vs 53±8 mm Hg, p=0.02) transvalvular gradient, compared with those without such activity in plasma. In severe AS patients with detectable active TF, prothrombin fragment 1.2, a thrombin generation marker, was higher than in patients without TF (375±122 vs. 207±64 pM, p<0.001). Conclusions Detectable FXIa and TF activity was observed for the first time in AS patients, primarily in severe ones. This activity correlates with thrombin generation in those patients. PMID:21519234

Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

PubMed

Brand, Caroline; Dencks, Stefanie; Schmitz, Georg; Mühlmeister, Mareike; Stypmann, Jörg; Ross, Rebecca; Hintelmann, Heike; Schliemann, Christoph; Müller-Tidow, Carsten; Mesters, Rolf M; Berdel, Wolfgang E; Schwöppe, Christian

2015-07-01

To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy. © 2015 by the American Institute of Ultrasound in Medicine.

Expression of the carbohydrate tumour marker Sialyl Lewis A, Sialyl Lewis X, Lewis Y and Thomsen-Friedenreich antigen in normal squamous epithelium of the uterine cervix, cervical dysplasia and cervical cancer.

PubMed

Engelstaedter, V; Fluegel, B; Kunze, S; Mayr, D; Friese, K; Jeschke, U; Bergauer, F

2012-04-01

The carbohydrate molecules Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA), Lewis Y (LeY) and Thomsen-Friedenreich antigen (TF) are known to mediate the adhesion between tumor cells and endothelium. They are used as serum markers in diagnosis and treatment in a broad spectrum of human carcinomas, but their expression profile and role in the development of cervical cancer remains unclear. The aim of this study was to investigate the expression of SLeX, SLeA, LeY and TF in normal cervical squamous epithelium, cervical dysplasia and cervical cancer. Slides of paraffin-embedded tissue were fixed and incubated with monoclonal antibodies against SLeX, SLeA, LeY and TF. Immunohistochemical staining was evaluated by using a semi-quantitative score (IRS Score). We found a significant difference of SLeA expression in invasive cervical cancer compared to normal epithelium (p=0.006) and all grades of dysplasia (p=0.002). The expression of SLeX in normal epithelium was less intense than in carcinoma in situ (p=0.036). Staining for LeY showed the weakest results of the investigated markers. Significant differences were found when normal epithelium was compared to CIN I (p=0.011), to CIN II (p=0.013) and to invasive cervical cancer (p=0.005). For TF, significant differences were found in normal epithelium compared to CIN I (p=0.011), CIN II (p=0.013) and compared to invasive cervical cancer (p=0.005). This is the first study on the expression of SLeA, SLeX, LeY and TF in normal cervical endothelium, cervical dysplasia, carcinoma in situ and invasive cervical cancer. Further studies and higher numbers are desirable.

Matrix metalloproteinase-2: A key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling.

PubMed

Das, Kaushik; Prasad, Ramesh; Ansari, Shabbir Ahmed; Roy, Abhishek; Mukherjee, Ashis; Sen, Prosenjit

2018-06-02

Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic manipulation of AKT or inhibition of NF-ĸB suggested that PAR2-mediated enhanced MMP-2 expression is dependent on the PI3K-AKT-NF-ĸB pathway. We also reveal that TF, PAR2, and MMP-2 are over-expressed in invasive breast carcinoma tissues as compared to normal. Knock-down of MMP-2 significantly impeded TF-FVIIa/trypsin-induced cell invasion. Further, we report that MMP-2 activates p38 MAPK-MK2-HSP27 signaling axis that leads to actin polymerization and induces cell migration. Pharmacological inhibition of p38 MAPK or MK2 attenuates MMP-2-induced cell migration. The study delineates a novel signaling pathway by which PAR2-induced MMP-2 expression regulates human breast cancer cell migration/invasion. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in breast cancer metastasis. Copyright © 2018. Published by Elsevier Masson SAS.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

PubMed Central

Gaul, Daniel S; Weber, Julien; van Tits, Lambertus J; Sluka, Susanna; Pasterk, Lisa; Reiner, Martin F; Calatayud, Natacha; Lohmann, Christine; Klingenberg, Roland; Pahla, Jürgen; Vdovenko, Daria; Tanner, Felix C; Camici, Giovanni G; Eriksson, Urs; Auwerx, Johan; Mach, François; Windecker, Stephan; Rodondi, Nicolas; Lüscher, Thomas F; Winnik, Stephan; Matter, Christian M

2018-01-01

Abstract Aims Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke. PMID:29444200

Factor VIII-bypassing activity of bovine tissue factor using the canine hemophilic model.

PubMed Central

O'Brien, D P; Giles, A R; Tate, K M; Vehar, G A

1988-01-01

The bleeding disorder of hemophilia A currently treated by replacement therapy of the missing coagulation factor, factor VIII, is frequently complicated by the development of neutralizing antibodies. The therapeutic potential of attenuated forms of the lipid-associated glycoprotein tissue factor, a known initiator of coagulation, was investigated as a factor VIII-by-passing activity. The protein moiety of tissue factor (Apo-TF) was partially purified and exhibited minimal procoagulant activity before relipidation in vitro. In pilot studies, Apo-TF injection into rabbits previously anticoagulated with an antibody to factor VIII was found to have a procoagulant effect. The efficacy of the material was further demonstrated when injection of Apo-TF in hemophilic dogs resulted in a normalization of the cuticle bleeding time. Little or no change in the blood parameters associated with disseminated intravascular coagulation was observed at lower doses, although mild to moderate effects were seen at higher doses. These data suggest a novel role for Apo-TF preparations as a potential therapeutic agent for hemophiliacs with antibodies to factor VIII once the potential thrombogenicity of such materials is evaluated. Images PMID:3134399

[Atelocollagen-mediated small interfering RNA delivery for effective gene silencing in rat vein grafts].

PubMed

Qiu, Xue-feng; Dong, Nian-guo; Sun, Zong-quan; Su, Wei; Shi, Jia-wei

2009-07-01

To evaluate the efficacy of using small interfering RNA targeting TF as a therapy for vein graft failure. External jugular vein to carotid artery interposition vein grafts, which were applied to a low flow condition, were made in 120 Sprague-Dawley rats weighing 260 to 300 g. These rats were randomly divided into 4 groups, 30 rats each group. Group A was atelocollagen-TF Stealth Select RNAi group. Group B was atelocollagen-TF Stealth RNAi group. Group C was atelocollagen group. Group D was control group. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. The TF protein expression of vein grafts was analyzed by Western blot at 1, 3, 7, 14, and 28 d postoperatively, and by immunochemistry at 3 d postoperatively. The proliferation index was determined at 14 d postoperatively. Neointimal hyperplasia was evaluated at 28 d postoperatively. BLOCK-iT fluorescent oligo was used to confirm its stability and successful transfer into the vein graft wall at 3 and 7 d postoperatively for another group (n=12). Fluorescence of BLOCK-iT fluorescent oligo could be detected in the graft wall even at 7 d postoperatively. Knockdown of the TF expression was achieved by perivascular application of siRNA using atelocollagen. Compared with control group, the intima thickness at 28 d after grafting was significantly reduced (P < 0.05). This phenomenon was preceded by significant reduction of cell proliferation in siRNA-treated grafts at 14 d postoperatively (P < 0.05). The expression of TF in vein grafts can be effectively inhibited by specific siRNAs using a atelocollagen-based nonviral delivery approach in vivo, so that the neointimal thickening can be prevented. Transplants;

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants.

PubMed

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A M; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram ( Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY 3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut ( Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H 2 O 2 ), and superoxide anion (O 2 ∙- ), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD , and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants.

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants

PubMed Central

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A. M.; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram (Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut (Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion (O2∙-), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD, and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants. PMID:29616059

A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells

PubMed Central

2011-01-01

Background Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. Methods In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. Results The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC50 values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC50 values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47D cancer cells just in 10 min, to effectively kill these cells and induce approximately 80% apoptotic cell death but not in normal cells. The intravenous administration of TfR-lytic peptide in the athymic mice model significantly inhibited tumor progression. Conclusions TfR-lytic peptide might provide a potent and selective anticancer therapy for patients. PMID:21849092

Biophysics and bioinformatics of transcription regulation in bacteria and bacteriophages

NASA Astrophysics Data System (ADS)

Djordjevic, Marko

2005-11-01

Due to rapid accumulation of biological data, bioinformatics has become a very important branch of biological research. In this thesis, we develop novel bioinformatic approaches and aid design of biological experiments by using ideas and methods from statistical physics. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of the regulatory circuits that control expression of genes. We propose a novel, biophysics based algorithm, for the supervised detection of transcription factor (TF) binding sites. The method classifies potential binding sites by explicitly estimating the sequence-specific binding energy and the chemical potential of a given TF. In contrast with the widely used information theory based weight matrix method, our approach correctly incorporates saturation in the transcription factor/DNA binding probability. This results in a significant reduction in the number of expected false positives, and in the explicit appearance---and determination---of a binding threshold. The new method was used to identify likely genomic binding sites for the Escherichia coli TFs, and to examine the relationship between TF binding specificity and degree of pleiotropy (number of regulatory targets). We next address how parameters of protein-DNA interactions can be obtained from data on protein binding to random oligos under controlled conditions (SELEX experiment data). We show that 'robust' generation of an appropriate data set is achieved by a suitable modification of the standard SELEX procedure, and propose a novel bioinformatic algorithm for analysis of such data. Finally, we use quantitative data analysis, bioinformatic methods and kinetic modeling to analyze gene expression strategies of bacterial viruses. We study bacteriophage Xp10 that infects rice pathogen Xanthomonas oryzae. Xp10 is an unusual bacteriophage, which has morphology and genome organization that most closely resembles temperate phages, such as lambda. It, however, encodes its own T7-like RNA polymerase (characteristic of virulent phages), whose role in gene expression was unclear. Our analysis resulted in quantitative understanding of the role of both host and phage RNA polymerase, and in the identification of the previously unknown promoter sequence for Xp10 RNA polymerase. More generally, an increasing number of phage genomes are being sequenced every year, and we expect that methods of quantitative data analysis that we introduced will provide an efficient way to study gene expression strategies of novel bacterial viruses.

ROBNCA: robust network component analysis for recovering transcription factor activities.

PubMed

Noor, Amina; Ahmad, Aitzaz; Serpedin, Erchin; Nounou, Mohamed; Nounou, Hazem

2013-10-01

Network component analysis (NCA) is an efficient method of reconstructing the transcription factor activity (TFA), which makes use of the gene expression data and prior information available about transcription factor (TF)-gene regulations. Most of the contemporary algorithms either exhibit the drawback of inconsistency and poor reliability, or suffer from prohibitive computational complexity. In addition, the existing algorithms do not possess the ability to counteract the presence of outliers in the microarray data. Hence, robust and computationally efficient algorithms are needed to enable practical applications. We propose ROBust Network Component Analysis (ROBNCA), a novel iterative algorithm that explicitly models the possible outliers in the microarray data. An attractive feature of the ROBNCA algorithm is the derivation of a closed form solution for estimating the connectivity matrix, which was not available in prior contributions. The ROBNCA algorithm is compared with FastNCA and the non-iterative NCA (NI-NCA). ROBNCA estimates the TF activity profiles as well as the TF-gene control strength matrix with a much higher degree of accuracy than FastNCA and NI-NCA, irrespective of varying noise, correlation and/or amount of outliers in case of synthetic data. The ROBNCA algorithm is also tested on Saccharomyces cerevisiae data and Escherichia coli data, and it is observed to outperform the existing algorithms. The run time of the ROBNCA algorithm is comparable with that of FastNCA, and is hundreds of times faster than NI-NCA. The ROBNCA software is available at http://people.tamu.edu/∼amina/ROBNCA

Inhibition of liver metastases from neuraminidase-treated colon 26 cells by an anti-Thomsen-Friedenreich-specific monoclonal antibody.

PubMed

Shigeoka, H; Karsten, U; Okuno, K; Yasutomi, M

1999-01-01

Thomsen-Friedenreich antigen (TF; Galbeta1-3GalNAcalpha1-) is expressed on many human carcinomas. Evidence suggests that TF-carrying tumor cells specifically bind asialoglycoprotein receptors on hepatocytes resulting in metastasis formation in the liver. We used an animal model to examine the feasibility of preventing metastasis formation by an antibody to TF. Treatment of Colon 26 cells with neuraminidase led to the exposure of TF, and consequently to a higher frequency of liver metastases in syngeneic Balb/c mice. This could be prevented by an antibody to TF (A78-G/A7), but not by a control antibody. The results may open up a new strategy for the prophylaxis of metastatic spread to the liver.

The spatial patterning of mouse cone opsin expression is regulated by BMP signaling through downstream effector COUP-TF nuclear receptors

PubMed Central

Satoh, Shinya; Tang, Ke; Iida, Atsumi; Inoue, Mariko; Kodama, Tatsuhiko; Tsai, Sophia Y.; Tsai, Ming-Jer; Furuta, Yasuhide; Watanabe, Sumiko

2009-01-01

Cone photopigments, known as opsins, are pivotal elements and the first detection module employed in color vision. In mice, cone photoreceptors are distributed throughout the retina, and S- and M-opsins have unique expression patterns in the retina with a gradient along the dorsoventral axis; however, the mechanisms regulating the spatial patterning of cone opsin expression have not been well documented. The purpose of this study was to define the mechanisms regulating the spatial patterning of cone opsin expression. By analyzing knockouts for bone morphogenetic protein (BMP) signaling, we found an essential role for BMP in forming cone opsin expression patterns in the retina; however, BMP signaling is activated only transiently in the dorsal half of the retina during early retinal development. Thus, BMP is not likely to play a direct role in opsin gene expression, which starts at a later stage of retinal development. We identified the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) nuclear receptor as a link between BMP and opsin expression. BMP signaling is essential for the correct dorsoventral spatial expression of COUP-TFI and -TFII. Through gain- and loss-of-function analyses, we found that both COUP-TFI and -TFII are required to suppress S-opsin expression in the dorsal retina but that only COUP-TFI plays an essential role in suppressing M-opsin expression in the ventral retina. Based on these findings, we propose a new molecular cascade involving BMP and COUP-TFs that conveys dorsoventral information to direct the expression of cone opsins during retinal development. PMID:19812316

Effect of circulating tissue factor on hypercoagulability in type 2 diabetes mellitus studied by rheometry and dielectric blood coagulometry.

PubMed

Uchimura, Isao; Kaibara, Makoto; Nagasawa, Masayuki; Hayashi, Yoshihito

2016-01-01

Hypercoagulability in type 2 diabetes mellitus (T2DM) patients increases their risk of cardiovascular diseases. The aim of this work was to investigate the hypercoagulation mechanism in T2DM patients in terms of circulating tissue factor (TF). Whole blood coagulation tests by damped oscillation rheometry and dielectric blood coagulometry (DBCM) were performed. The average coagulation time was significantly shorter for T2DM patients than for healthy controls. In vitro addition of either anti-TF or anti-activated factor VII (FVIIa) antibody to hypercoagulable blood samples prolonged coagulation times for one group of patients, while coagulation times remained short for another group. The levels of circulating TF were estimated in the former group by measuring the coagulation times for blood samples from healthy subjects with addition of various concentrations of TF and comparing them with the coagulation times for the group. The results indicated that the levels of circulating TF were on the order of subpicomolar at most. Circulating TF is at least partially responsible for a hypercoagulable group of T2DM patients, while an abnormality in the intrinsic coagulation pathway probably occurs in the other group.

Multipotent versus differentiated cell fate selection in the developing Drosophila airways

PubMed Central

Matsuda, Ryo; Hosono, Chie; Samakovlis, Christos; Saigo, Kaoru

2015-01-01

Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001 PMID:26633813

Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice.

PubMed

Smita, Shuchi; Katiyar, Amit; Chinnusamy, Viswanathan; Pandey, Dev M; Bansal, Kailash C

2015-01-01

MYB transcription factor (TF) is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by "top-down" and "guide-gene" approaches. More than 50% of OsMYBs were strongly correlated under 50 experimental conditions with 51 hub genes via "top-down" approach. Further, clusters were identified using Markov Clustering (MCL). To maximize the clustering performance, parameter evaluation of the MCL inflation score (I) was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by "guide-gene" approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought response in rice. Thus, the co-regulatory network analysis facilitated the identification of complex OsMYB regulatory networks, and candidate target regulon genes of selected guide MYB genes. The results contribute to the candidate gene screening, and experimentally testable hypotheses for potential regulatory MYB TFs, and their targets under stress conditions.

Iron Enhances Hepatic Fibrogenesis and Activates Transforming Growth Factor-β Signaling in Murine Hepatic Stellate Cells.

PubMed

Mehta, Kosha J; Coombes, Jason D; Briones-Orta, Marco; Manka, Paul P; Williams, Roger; Patel, Vinood B; Syn, Wing-Kin

2018-02-01

Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-β) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-β, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-β RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. HSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-β pathway with TGF-β messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-β RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-β (0.4-fold; P < 0.01) and reduced levels of TGF-β RII and phospho-Smad2 proteins. HSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-β pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this. Published by Elsevier Inc.

Selective tissue factor/factor VIIa Inhibitor, ER-410660, and its prodrug, E5539, have anti-venous and anti-arterial thrombotic effects with a low risk of bleeding.

PubMed

Nagakura, Tadashi; Tabata, Kimiyo; Kira, Kazunobu; Hirota, Shinsuke; Clark, Richard; Matsuura, Fumiyoshi; Hiyoshi, Hironobu

2013-08-01

Many anticoagulant drugs target factors common to both the intrinsic and extrinsic coagulation pathways, which may lead to bleeding complications. Since the tissue factor (TF)/factor VIIa complex is associated with thrombosis onset and specifically activates the extrinsic coagulation pathway, compounds that inhibit this complex may provide therapeutic and/or prophylactic benefits with a decreased risk of bleeding. The in vitro enzyme profile and anticoagulation selectivity of the TF/VIIa complex inhibitor, ER-410660, and its prodrug E5539 were assessed using enzyme inhibitory and plasma clotting assays. In vivo effects of ER-410660 and E5539 were determined using a TF-induced, thrombin generation rhesus monkey model; a stasis-induced, venous thrombosis rat model; a photochemically induced, arterial thrombosis rat model; and a rat tail-cut bleeding model. ER-410660 selectively prolonged prothrombin time, but had a less potent anticoagulant effect on the intrinsic pathway. It also exhibited a dose-dependent inhibitory effect on thrombin generation caused by TF-injection in the rhesus monkey model. ER-410660 also reduced venous thrombus weights in the TF-administered, stasis-induced, venous thrombosis rat model and prolonged the occlusion time induced by arterial thrombus formation after vascular injury. The compound was capable of doubling the total bleeding time in the rat tail-cut model, albeit with a considerably higher dose compared to the effective dose in the venous and arterial thrombosis models. Moreover, E5539, an orally available ER-410660 prodrug, reduced the thrombin-anti-thrombin complex levels, induced by TF-injection, in a dose-dependent manner. Selective TF/VIIa inhibitors have potential as novel anticoagulants with a lower propensity for enhancing bleeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ultra Fine Particles from Diesel Engines Induce Vascular Oxidative Stress via JNK Activation

PubMed Central

Li, Rongsong; Ning, Zhi; Cui, Jeffery; Khalsa, Bhavraj; Ai, Lisong; Takabe, Wakako; Beebe, Tyler; Majumdar, Rohit; Sioutas, Constantinos; Hsiai, Tzung

2011-01-01

Exposure of particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultra fine particles (UFP) from diesel vehicle engines have been shown to be pro-atherogenic in apoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induced vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intra-cellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O2·-) production in human aortic endothelial cells (HAEC). Flow cytometry (FACS) showed that UFP increased MitoSOX Red intensity specific for mitochondrial superoxide. Protein carbonyl content is increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated hemeoxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pre-treatment with antioxidant, N-acetyl cysteine (NAC), significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP stimulated O2·- production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation play an important role in UFP-induced oxidative stress and stress response gene expression. PMID:19154785

Tissue factor activity in women with preeclampsia or SGA: a potential explanation for the excessive thrombin generation in these syndromes.

PubMed

Erez, Offer; Romero, Roberto; Vaisbuch, Edi; Than, Nandor Gabor; Kusanovic, Juan Pedro; Mazaki-Tovi, Shali; Gotsch, Francesca; Mittal, Pooja; Dong, Zhong; Chaiworapongsa, Tinnakorn; Kim, Chong Jai; Nhan-Chang, Chia-Ling; Kim, Sun Kwon; Yeo, Lami; Mazor, Moshe; Hassan, Sonia S

2018-06-01

The aim of this study was to determine whether the activity of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the plasma of women with preeclampsia (PE) and small for gestational age (SGA) neonate differ from that of normal pregnant women and whether they are related to specific placental lesions. This cross-sectional study included the following groups: (1) normal pregnancy (n = 68); (2) PE (n= 128); and (3) SGA (n = 56). Maternal plasma TF and TFPI activity was determined with chromogenic assays. (1) The median maternal plasma TF activity, but not TFPI activity, differed among the study groups (p < .0001 and p = .4, respectively); (2) patients with PE had a higher median maternal plasma TF activity than women with normal pregnancies (p < .0001) and mothers with SGA fetuses (p = .002); (3) among patients with PE, those with distal villous hypoplasia had a higher median maternal TF activity than those without these placental lesions (p = .018); and (4) following adjustment for confounding variables, maternal plasma TF and TFPI activity were not associated with an SGA neonate. Plasma TF activity is higher in women with PE than in those with SGA or normal pregnancies. We propose that these changes may be responsible, at least in part, for the increased in-vivo thrombin generation observed in this obstetrical syndrome.

Improved in vivo gene transfer into tumor tissue by stabilization of pseudodendritic oligoethylenimine-based polyplexes.

PubMed

Russ, Verena; Fröhlich, Thomas; Li, Yunqiu; Halama, Anna; Ogris, Manfred; Wagner, Ernst

2010-02-01

HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations. HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP-linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by periodate oxidation of the glycosylation sites. Crosslinked DNA polyplexes showed an increased stability against exchange reaction by salt or heparin. Disulfide bond containing DSP-linked polyplexes were susceptible to reducing conditions. These polyplexes displayed the highest gene expression levels in vitro and in vivo (upon intratumoral application in mice), and these were significantly elevated and prolonged over standard or DSS-stabilized HD O formulations. DSP-stabilized HD O polyplexes with or without Tf coating were well-tolerated after intravenous application. High gene expression levels were found in tumor tissue, with negligible gene expression in any other organ. Lateral stabilization of HD O polyplexes with DSP crosslinker enhanced gene transfer efficacy and was essential for the incorporation of a ligand (Tf) into a stable particle formulation.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Conditioned media from (pre)adipocytes stimulate fibrinogen and PAI-1 production by HepG2 hepatoma cells

PubMed Central

Faber, D R; Kalkhoven, E; Westerink, J; Bouwman, J J; Monajemi, H M; Visseren, F L J

2012-01-01

Background: Obesity is associated with a prothrombotic state, which may contribute to the increased risk of thrombotic events. Objective: To assess the effects of (pre)adipocyte-derived adipokines on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) production by hepatocytes. Methods: HepG2 hepatocytes were incubated with conditioned media (CM) derived from preadipocytes and adipocytes, which had been untreated or prestimulated with tumor necrosis factor (TNF)-α, interleukin (IL)-1β or IL-6. After 24 h, supernatants and cell lysates were harvested for measurement of fibrinogen, PAI-1 and TF. Results: (Pre)adipocyte CM significantly enhanced the production of PAI-1 by HepG2 cells 2.5- to 4.4-fold. CM from cytokine-stimulated (pre)adipocytes significantly induced fibrinogen secretion 1.5- to 4.2-fold. TF production was not affected by the CM. After specific depletion of TNF-α, IL-1β or IL-6 from the CM, IL-6 was shown to be the most prominent stimulus of fibrinogen secretion and IL-1β of PAI-1 secretion. In addition, fibrinogen, PAI-1 and tissue factor production was evaluated by direct stimulation of HepG2 cells with TNF-α, IL-1β or IL-6. IL-6 enhanced fibrinogen synthesis 4.3-fold (P<0.01), whereas IL-1β induced PAI-1 production 5.0-fold (P<0.01). Gene expression analyses showed that TNF-α and IL-1β stimulate the adipocyte expression of TNF-α, IL-1β and IL-6. Cytokine stimulation of adipocytes may thus have induced an inflammatory response, which may have stimulated fibrinogen and PAI-1 production by HepG2 cells more potently. Conclusions: SGBS (pre)adipocytes release cytokines that increase the production of fibrinogen and PAI-1 by HepG2 cells. IL-6 and IL-1β produced by (pre)adipocytes were the strongest inducers of fibrinogen and PAI-1 secretion, respectively. PMID:23208413

A microfluidic method for synthesis of transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

PubMed Central

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2015-01-01

siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for targeted delivery of LOR-1284 siRNA to acute myeloid leukemia (AML) cells. In vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4–11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than free LOR-1284 in reducing the R2 mRNA and protein levels. Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific thereapeutic delivery of LOR-1284 to AML. PMID:25003978

DNA-bending properties of TF1.

PubMed

Schneider, G J; Sayre, M H; Geiduschek, E P

1991-10-05

Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of DNA-binding proteins that includes Escherichia coli HU and integration host factor, IHF. A gel electrophoretic retardation method has been used to show that a TF1 dimer binding to one of its preferred sites in (5-hydroxymethyl)uracil (hmUra)-containing DNA sharply bends the latter. In fact, the DNA-bending properties of TF1 and E. coli IHF are indistinguishable. Substitutions at amino acid 61 in the DNA-binding "arm" of TF1 are known to affect DNA-binding affinity and site selectivity. Experiments described here show that these substitutions also affect DNA bending. The selectivity of TF1 binding is very greatly diminished and the affinity is reduced when hmUra is replaced in DNA by thymine (T). An extension of the gel retardation method that permits an analysis of DNA bending by non-specifically bound TF1 is proposed. Under the assumptions of this analysis, the reduced affinity of TF1 for T-containing DNA is shown to be associated with bending that is still sharp. The analysis of the TF1-DNA interaction has also been extended by hydroxyl radical (.OH) and methylation interference footprinting at two DNA sites. At each of these sites, and on each strand, TF1 strongly protects three segments of DNA from attack by OH. Patches of protected DNA are centered approximately ten base-pairs apart and fall on one side of the B-helix. Methylation in either the major or minor groove in the central ten base-pairs of the two TF1 binding sites quantitatively diminishes, but does not abolish, TF1 binding. We propose that multiple protein contacts allow DNA to wrap around the relatively small TF1 dimer, considerably deforming the DNA B-helix in the process.

Prediction of TF target sites based on atomistic models of protein-DNA complexes

PubMed Central

Angarica, Vladimir Espinosa; Pérez, Abel González; Vasconcelos, Ana T; Collado-Vides, Julio; Contreras-Moreira, Bruno

2008-01-01

Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition. PMID:18922190

JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles.

PubMed

Mathelier, Anthony; Fornes, Oriol; Arenillas, David J; Chen, Chih-Yu; Denay, Grégoire; Lee, Jessica; Shi, Wenqiang; Shyr, Casper; Tan, Ge; Worsley-Hunt, Rebecca; Zhang, Allen W; Parcy, François; Lenhard, Boris; Sandelin, Albin; Wasserman, Wyeth W

2016-01-04

JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. We updated the structural annotation of the TF DNA binding domains (DBDs) following a published hierarchical structural classification. In addition, we introduced 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites. This new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, we provide the users with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. Finally, we provide the JASPAR2016 R/Bioconductor data package with the data of this release. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.

PubMed

Fang, Ying; Treffers, Emmely E; Li, Yanhua; Tas, Ali; Sun, Zhi; van der Meer, Yvonne; de Ru, Arnoud H; van Veelen, Peter A; Atkins, John F; Snijder, Eric J; Firth, Andrew E

2012-10-23

Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis[OPEN

PubMed Central

Penfold, Christopher A.; Jenkins, Dafyd J.; Legaie, Roxane; Lawson, Tracy; Vialet-Chabrand, Silvere R.M.; Subramaniam, Sunitha; Hickman, Richard; Feil, Regina; Bowden, Laura; Hill, Claire; Lunn, John E.; Finkenstädt, Bärbel; Buchanan-Wollaston, Vicky; Beynon, Jim; Wild, David L.; Ott, Sascha

2016-01-01

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses. PMID:26842464

PvTFDB: a Phaseolus vulgaris transcription factors database for expediting functional genomics in legumes.

PubMed

Bhawna; Bonthala, V S; Gajula, Mnv Prasad

2016-01-01

The common bean [Phaseolus vulgaris (L.)] is one of the essential proteinaceous vegetables grown in developing countries. However, its production is challenged by low yields caused by numerous biotic and abiotic stress conditions. Regulatory transcription factors (TFs) symbolize a key component of the genome and are the most significant targets for producing stress tolerant crop and hence functional genomic studies of these TFs are important. Therefore, here we have constructed a web-accessible TFs database for P. vulgaris, called PvTFDB, which contains 2370 putative TF gene models in 49 TF families. This database provides a comprehensive information for each of the identified TF that includes sequence data, functional annotation, SSRs with their primer sets, protein physical properties, chromosomal location, phylogeny, tissue-specific gene expression data, orthologues, cis-regulatory elements and gene ontology (GO) assignment. Altogether, this information would be used in expediting the functional genomic studies of a specific TF(s) of interest. The objectives of this database are to understand functional genomics study of common bean TFs and recognize the regulatory mechanisms underlying various stress responses to ease breeding strategy for variety production through a couple of search interfaces including gene ID, functional annotation and browsing interfaces including by family and by chromosome. This database will also serve as a promising central repository for researchers as well as breeders who are working towards crop improvement of legume crops. In addition, this database provide the user unrestricted public access and the user can download entire data present in the database freely.Database URL: http://www.multiomics.in/PvTFDB/. © The Author(s) 2016. Published by Oxford University Press.

Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

PubMed Central

Ma, Xiqing; Huang, Bingru

2016-01-01

Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

Over-expression of StZFP2 in Solanum tuberosum L. var. Kennebec (potato) inhibits growth of tobacco hornworm larvae (THW, Manduca sexta L.)

USDA-ARS?s Scientific Manuscript database

Tobacco hornworm (Manduca sexta, THW) is a voracious pest of Solanaceous plants such as tomato and potato. Finding new approaches to enhance protection against this pest in potato has led to investigating transcription factors (TF) that are induced upon insect infestation. StZFP2 is a Q-type C2H2 z...

Inhibitory Effects of the Four Main Theaflavin Derivatives Found in Black Tea on Ovarian Cancer Cells.

PubMed

Gao, Ying; Rankin, Gary O; Tu, Youying; Chen, Yi Charlie

2016-02-01

Some polyphenols induce apoptosis and inhibit angiogenesis. Consumption of black tea, rich in polyphenols, has been found to reduce ovarian cancer risk. Theaflavin (TF1), theaflavin-3-gallate (TF2a), theaflavin-3'-gallate (TF2b) and theaflavin-3, 3'-digallate (TF3) are four main theaflavin derivatives found in black tea. Cell proliferation assay, Hoechst 33342 staining assay, Caspase-Glo Assay, western blot, human umbilical vein endothelial cell tube formation assay and vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay were performed. All four theaflavin derivatives reduced viability of ovarian cancer cells at lower concentrations than with normal ovarian cells. TF1 mainly mediated apoptosis via the intrinsic pathway, while the others via the intrinsic and extrinsic pathways. TF1 inhibited tube formation via reducing VEGF secretion in a hypoxia-inducible factor 1α-independent manner, while the others in a HIF1α-dependent way. All four theaflavin derivatives inhibited ovarian cancer cells. Some of the effects and mechanisms of TF1 are different from those of the other three theaflavin derivatives. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development

PubMed Central

2013-01-01

Background Red coloration of fruit is an important trait in apple, and it is mainly attributed to the accumulation of anthocyanins, a class of plant flavonoid metabolites. Anthocyanin biosynthesis is genetically determined by structural and regulatory genes. Plant tissue pigmentation patterns are mainly controlled by expression profiles of regulatory genes. Among these regulatory genes are MYB transcription factors (TFs), wherein the class of two-repeats (R2R3) is deemed the largest, and these are associated with the anthocyanin biosynthesis pathway. Although three MdMYB genes, almost identical in nucleotide sequences, have been identified in apple, it is likely that there are other R2R3 MYB TFs that are present in the apple genome that are also involved in the regulation of coloration of red color pigmentation of the skin of apple fruits. Results In this study, a novel R2R3 MYB gene has been isolated and characterized in apple. This MYB gene is closely related to the Arabidopsis thaliana AtMYB3, and has been designated as MdMYB3. This TF belongs to the subgroup 4 R2R3 family of plant MYB transcription factors. This apple MdMYB3 gene is mapped onto linkage group 15 of the integrated apple genetic map. Transcripts of MdMYB3 are detected in all analyzed tissues including leaves, flowers, and fruits. However, transcripts of MdMYB3 are higher in excocarp of red-skinned apple cultivars than that in yellowish-green skinned apple cultivars. When this gene is ectopically expressed in Nicotiana tabacum cv. Petite Havana SR1, flowers of transgenic tobacco lines carrying MdMYB3 have exhibited increased pigmentation and accumulate higher levels of anthocyanins and flavonols than wild-type flowers. Overexpression of MdMYB3 has resulted in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS. Moreover, peduncles of flowers and styles of pistils of transgenic plants overexpressing MdMYB3 are longer than those of wild-type plants, thus suggesting that this TF is involved in regulation of flower development. Conclusions This study has identified a novel MYB transcription factor in the apple genome. This TF, designated as MdMYB3, is involved in transcriptional activation of several flavonoid pathway genes. Moreover, this TF not only regulates the accumulation of anthocyanin in the skin of apple fruits, but it is also involved in the regulation of flower development, particularly that of pistil development. PMID:24199943

An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development.

PubMed

Vimolmangkang, Sornkanok; Han, Yuepeng; Wei, Guochao; Korban, Schuyler S

2013-11-07

Red coloration of fruit is an important trait in apple, and it is mainly attributed to the accumulation of anthocyanins, a class of plant flavonoid metabolites. Anthocyanin biosynthesis is genetically determined by structural and regulatory genes. Plant tissue pigmentation patterns are mainly controlled by expression profiles of regulatory genes. Among these regulatory genes are MYB transcription factors (TFs), wherein the class of two-repeats (R2R3) is deemed the largest, and these are associated with the anthocyanin biosynthesis pathway. Although three MdMYB genes, almost identical in nucleotide sequences, have been identified in apple, it is likely that there are other R2R3 MYB TFs that are present in the apple genome that are also involved in the regulation of coloration of red color pigmentation of the skin of apple fruits. In this study, a novel R2R3 MYB gene has been isolated and characterized in apple. This MYB gene is closely related to the Arabidopsis thaliana AtMYB3, and has been designated as MdMYB3. This TF belongs to the subgroup 4 R2R3 family of plant MYB transcription factors. This apple MdMYB3 gene is mapped onto linkage group 15 of the integrated apple genetic map. Transcripts of MdMYB3 are detected in all analyzed tissues including leaves, flowers, and fruits. However, transcripts of MdMYB3 are higher in excocarp of red-skinned apple cultivars than that in yellowish-green skinned apple cultivars. When this gene is ectopically expressed in Nicotiana tabacum cv. Petite Havana SR1, flowers of transgenic tobacco lines carrying MdMYB3 have exhibited increased pigmentation and accumulate higher levels of anthocyanins and flavonols than wild-type flowers. Overexpression of MdMYB3 has resulted in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS. Moreover, peduncles of flowers and styles of pistils of transgenic plants overexpressing MdMYB3 are longer than those of wild-type plants, thus suggesting that this TF is involved in regulation of flower development. This study has identified a novel MYB transcription factor in the apple genome. This TF, designated as MdMYB3, is involved in transcriptional activation of several flavonoid pathway genes. Moreover, this TF not only regulates the accumulation of anthocyanin in the skin of apple fruits, but it is also involved in the regulation of flower development, particularly that of pistil development.

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat.

PubMed

Sheikh, Nadeem; Dudas, Jozsef; Ramadori, Giuliano

2007-07-01

In the present study, turpentine oil was injected in the hind limb muscle of the rat to stimulate an acute-phase response (APR). The changes in the gene expression of cytokines and proteins known to be involved in the iron regulatory pathway were then studied in the liver and in extra-hepatic tissue. In addition to the strong upregulation of interleukin-6 (IL-6) and IL-1 beta observed in the inflamed muscle, an upregulation of the genes for IL1-beta and tumor necrosis factor-alpha, but not IL-6, were detectable in the liver. Hepatic Hepc gene expression increased to a maximum at 6 h after the onset of APR. An upregulation of transferrin, transferrin receptor 1 (TfR1), TfR2, ferritin-H, iron responsive element binding protein-1 (IRP1), IRP2 and divalent metal transporter gene expression was also found. Hemojuvelin (Hjv)-, ferroportin 1-, Dcytb-, hemochromatosis-gene- and hephaestin gene expression was downregulated. Hepcidin (Hepc) gene expression was not only detectable in extra-hepatic tissues such as heart, small intestine, colon, spleen and kidney but it was also upregulated under acute-phase conditions, with the Hjv gene being regulated antagonistically. Fpn-1 gene expression was downregulated significantly in heart, colon and spleen. Most of the genes of the known proteins involved in iron metabolism are expressed not only in the liver but also in extra-hepatic tissues. Under acute-phase conditions, acute-phase cytokines (eg IL-6) may modulate the gene expression of such proteins not only in the liver but also in other organs.

Evaluation of speciated VOC emission factors for Air Force hush houses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, P.D.; Stevens, D.K.

1997-12-31

Data published in: ``Engine and Hush House Emissions from a TF30-P109 Jet Engine Tested at Cannon Air Force Base, NM`` by Radian Corporation and ``Aircraft Emissions. Characterization: TF41-A2, TF30-P103 , and TF30-P109 Engines`` by Battelle are reviewed and compared. Specifically CO, NO{sub x}, and VOC emission factors using EPA Method 19 are addressed, with comparisons between JP-4 and JP-8 jet fuels. CO and NO{sub x} emissions for JP-4 and JP-8 jet fuels were found to be essentially the same. VOC emission data exhibited high variability. Problems inherent in speciated VOC emission testing are discussed. A limiting of speciated VOC emissionmore » testing, with emission factor estimation based on fuel content is proposed.« less

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression.

PubMed

Coego, Alberto; Brizuela, Esther; Castillejo, Pablo; Ruíz, Sandra; Koncz, Csaba; del Pozo, Juan C; Piñeiro, Manuel; Jarillo, José A; Paz-Ares, Javier; León, José

2014-03-01

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β-estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β-estradiol treatment. As a proof of concept for the exploitation of this resource, β-estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl-root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

PubMed

Killisch, I; Steinlein, P; Römisch, K; Hollinshead, R; Beug, H; Griffiths, G

1992-09-01

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.

Gonad Transcriptome Analysis of High-Temperature-Treated Females and High-Temperature-Induced Sex-Reversed Neomales in Nile Tilapia

PubMed Central

Sun, Li Xue; Teng, Jian; Zhao, Yan; Li, Ning; Wang, Hui

2018-01-01

Background: Nowadays, the molecular mechanisms governing TSD (temperature-dependent sex determination) or GSD + TE (genotypic sex determination + temperature effects) remain a mystery in fish. Methods: We developed three all-female families of Nile tilapia (Oreochromis niloticus), and the family with the highest male ratio after high-temperature treatment was used for transcriptome analysis. Results: First, gonadal histology analysis indicated that the histological morphology of control females (CF) was not significantly different from that of high-temperature-treated females (TF) at various development stages. However, the high-temperature treatment caused a lag of spermatogenesis in high-temperature-induced neomales (IM). Next, we sequenced the transcriptome of CF, TF, and IM Nile tilapia. 79, 11,117, and 11,000 differentially expressed genes (DEGs) were detected in the CF–TF, CF–IM, and TF–IM comparisons, respectively, and 44 DEGs showed identical expression changes in the CF–TF and CF–IM comparisons. Principal component analysis (PCA) indicated that three individuals in CF and three individuals in TF formed a cluster, and three individuals in IM formed a distinct cluster, which confirmed that the gonad transcriptome profile of TF was similar to that of CF and different from that of IM. Finally, six sex-related genes were validated by qRT-PCR. Conclusions: This study identifies a number of genes that may be involved in GSD + TE, which will be useful for investigating the molecular mechanisms of TSD or GSD + TE in fish. PMID:29495590

"Hit-and-Run" transcription: de novo transcription initiated by a transient bZIP1 "hit" persists after the "run".

PubMed

Doidy, Joan; Li, Ying; Neymotin, Benjamin; Edwards, Molly B; Varala, Kranthi; Gresham, David; Coruzzi, Gloria M

2016-02-03

Dynamic transcriptional regulation is critical for an organism's response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named "hit-and-run". This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking. Here, we show that active transcription continues after transient TF-target interactions by tracking de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex ("hit"), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter ("run"). Our findings provide experimental proof for active transcription of transient TF-targets supporting a "hit-and-run" mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control.

Effect of circulating tissue factor on hypercoagulability in type 2 diabetes mellitus studied by rheometry and dielectric blood coagulometry

PubMed Central

Uchimura, Isao; Kaibara, Makoto; Nagasawa, Masayuki; Hayashi, Yoshihito

2016-01-01

Background: Hypercoagulability in type 2 diabetes mellitus (T2DM) patients increases their risk of cardiovascular diseases. Objective: The aim of this work was to investigate the hypercoagulation mechanism in T2DM patients in terms of circulating tissue factor (TF). Methods: Whole blood coagulation tests by damped oscillation rheometry and dielectric blood coagulometry (DBCM) were performed. Results: The average coagulation time was significantly shorter for T2DM patients than for healthy controls. In vitro addition of either anti-TF or anti-activated factor VII (FVIIa) antibody to hypercoagulable blood samples prolonged coagulation times for one group of patients, while coagulation times remained short for another group. The levels of circulating TF were estimated in the former group by measuring the coagulation times for blood samples from healthy subjects with addition of various concentrations of TF and comparing them with the coagulation times for the group. The results indicated that the levels of circulating TF were on the order of subpicomolar at most. Conclusions: Circulating TF is at least partially responsible for a hypercoagulable group of T2DM patients, while an abnormality in the intrinsic coagulation pathway probably occurs in the other group. PMID:27858671

In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia.

PubMed

Augustsson, Cecilia; Persson, Egon

2014-11-13

Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component. © 2014 by The American Society of Hematology.

RNA sequencing on Amomum villosum Lour. induced by MeJA identifies the genes of WRKY and terpene synthases involved in terpene biosynthesis.

PubMed

He, Xueying; Wang, Huan; Yang, Jinfen; Deng, Ke; Wang, Teng

2018-02-01

Amomum villosum Lour. is an important Chinese medicinal plant that has diverse medicinal functions, and mainly contains volatile terpenes. This study aims to explore the WRKY transcription factors (TFs) and terpene synthase (TPS) unigenes that might be involved in terpene biosynthesis in A. villosum, and thus providing some new information on the regulation of terpenes in plants. RNA sequencing of A. villosum induced by methyl jasmonate (MeJA) revealed that the WRKY family was the second largest TF family in the transcriptome. Thirty-six complete WRKY domain sequences were expressed in response to MeJA. Further, six WRKY unigenes were highly correlated with eight deduced TPS unigenes. Ultimately, we combined the terpene abundance with the expression of candidate WRKY TFs and TPS unigenes to presume a possible model wherein AvWRKY61, AvWRKY28, and AvWRKY40 might coordinately trans-activate the AvNeoD promoter. We propose an approach to further investigate TF unigenes that might be involved in terpenoid biosynthesis, and identified four unigenes for further analyses.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

PubMed

Yu, Olivia M; Benitez, Jorge A; Plouffe, Steven W; Ryback, Daniel; Klein, Andrea; Smith, Jeff; Greenbaum, Jason; Delatte, Benjamin; Rao, Anjana; Guan, Kun-Liang; Furnari, Frank B; Chaim, Olga Meiri; Miyamoto, Shigeki; Brown, Joan Heller

2018-06-11

The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

EXTRINSIC COAGULATION BLOCKADE ATTENUATES LUNG INJURY AND PROINFLAMMATORY CYTOKINE RELEASE AFTER INTRATRACHEAL LIPOPOLYSACCHARIDE

EPA Science Inventory

Initiation of coagulation by tissue factor (TF) is a potentially powerful regulator of local inflammatory responses. We hypothesized that blockade of TF-factor VIIa (FVIIa) complex would decrease lung inflammation and proinflammatory cytokine release after tracheal instillation o...

Genome-wide analysis reveals phytohormone action during cassava storage root initiation.

PubMed

Sojikul, Punchapat; Saithong, Treenut; Kalapanulak, Saowalak; Pisuttinusart, Nuttapat; Limsirichaikul, Siripan; Tanaka, Maho; Utsumi, Yoshinori; Sakurai, Tetsuya; Seki, Motoaki; Narangajavana, Jarunya

2015-08-01

Development of storage roots is a process associated with a phase change from cell division and elongation to radial growth and accumulation of massive amounts of reserve substances such as starch. Knowledge of the regulation of cassava storage root formation has accumulated over time; however, gene regulation during the initiation and early stage of storage root development is still poorly understood. In this study, transcription profiling of fibrous, intermediate and storage roots at eight weeks old were investigated using a 60-mer-oligo microarray. Transcription and gene expression were found to be the key regulating processes during the transition stage from fibrous to intermediate roots, while homeostasis and signal transduction influenced regulation from intermediate roots to storage roots. Clustering analysis of significant genes and transcription factors (TF) indicated that a number of phytohormone-related TF were differentially expressed; therefore, phytohormone-related genes were assembled into a network of correlative nodes. We propose a model showing the relationship between KNOX1 and phytohormones during storage root initiation. Exogeneous treatment of phytohormones N (6) -benzylaminopurine and 1-Naphthaleneacetic acid were used to induce the storage root initiation stage and to investigate expression patterns of the genes involved in storage root initiation. The results support the hypothesis that phytohormones are acting in concert to regulate the onset of cassava storage root development. Moreover, MeAGL20 is a factor that might play an important role at the onset of storage root initiation when the root tip becomes swollen.

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

PubMed Central

Reinhardt, Christoph; von Brühl, Marie-Luise; Manukyan, Davit; Grahl, Lenka; Lorenz, Michael; Altmann, Berid; Dlugai, Silke; Hess, Sonja; Konrad, Ildiko; Orschiedt, Lena; Mackman, Nigel; Ruddock, Lloyd; Massberg, Steffen; Engelmann, Bernd

2008-01-01

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury. PMID:18274674

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.

PubMed

Reinhardt, Christoph; von Brühl, Marie-Luise; Manukyan, Davit; Grahl, Lenka; Lorenz, Michael; Altmann, Berid; Dlugai, Silke; Hess, Sonja; Konrad, Ildiko; Orschiedt, Lena; Mackman, Nigel; Ruddock, Lloyd; Massberg, Steffen; Engelmann, Bernd

2008-03-01

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased - and, under conditions of decreased platelet adhesion, PDI inhibition reduced - fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet-secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.

FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139.

PubMed

Anas, Andrea Roxanne J; Mori, Akane; Tone, Mineka; Naruse, Chiaki; Nakajima, Anna; Asukabe, Hirohiko; Takaya, Yoshiaki; Imanishi, Susumu Y; Nishizawa, Tomoyasu; Shirai, Makoto; Harada, Ken-Ichi

2017-08-30

The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF) have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE) from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC 50 10.62 µM, which was more efficient than thrombin inhibition of EC 50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF) and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors.

Effects of mutations at amino acid 61 in the arm of TF1 on its DNA-binding properties.

PubMed

Sayre, M H; Geiduschek, E P

1990-12-20

Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of bacterial DNA-binding proteins that includes Escherichia coli HU and integration host factor (IHF). We have initiated a mutational analysis of the TF1 molecule to understand better its unique DNA-binding properties and to investigate its physiological function. We report here the consequences of mutating the putative DNA-binding "arms" of TF1. At position 61 in its primary sequence, TF1 contains a Phe residue in place of the Arg residue found in all other known members of the HU family. Substituting polar, uncharged residues for Phe61 substantially reduced the DNA-binding affinity and site-selectivity of TF1 in vitro, whereas the substitution of Tyr had no effect. Substituting Trp or Arg for Phe61 had little effect on the affinity of TF1 for SPO1 DNA, but altered the electrophoretic mobilities of protein-DNA complexes in non-denaturing gels. The Arg61 substitution increased the affinity of the protein for non-specific sites on thymine-containing DNA, thus reducing the natural preference of TF1 for (5-hydroxymethyluracil)-containing DNA. The Phe61-to-Arg mutation was also correlated with decreased phage yield and aberrant regulation of viral protein synthesis in vivo.

Thrombin generation and fibrin formation under flow on biomimetic tissue factor-rich surfaces.

PubMed

Onasoga-Jarvis, A A; Puls, T J; O'Brien, S K; Kuang, L; Liang, H J; Neeves, K B

2014-01-01

Blood flow regulates coagulation and fibrin assembly by controlling the rate of transport of zymogens, enzymes and plasma proteins to and from the site of an injury. The objective of this work was to define the hemodynamic conditions under which fibrin can form under flow on tissue factor (TF)-rich substrates. TF-coated silica beads (~ 800 nm) were patterned into 18-85-μm spots. Normal pooled plasma and factors VIII, IX and XI deficient plasmas were perfused over the beads coated with 0.08, 0.8 and 8 molecules-TF μm(-2) at shear rates of 50-1000 s(-1) . Fibrin deposition and thrombin generation were measured by fluorescence microscopy in a hydrodynamic focusing microfluidic device. Fibrin deposition was supported on patterned bead spots, but not planar TF substrates at the same surface TF concentration. There was a threshold spot size and a shear rate dependent TF concentration that was necessary to support fibrin polymerization. FVIII and FIX had minor effects on fibrin dynamics at 8 molecules-TF μm(-2) , but were essential at 0.8 molecules-TF μm(-2) . The absence of FXI influenced thrombin generation and fibrin deposition at both 0.8 and 8 molecules-TF μm(-2) . These results show that fibrin deposition requires perturbations in the flow field that protect reactions from dilution by flow under venous and arterial conditions. FVIII and FIX have a modest effect on fibrin deposition at high TF concentrations, but are necessary for fibrin deposition at low TF concentrations. FXI amplifies thrombin generation under flow at both low and high TF concentrations. © 2013 International Society on Thrombosis and Haemostasis.

“Hit‐and‐Run” leaves its mark: Catalyst transcription factors and chromatin modification

PubMed Central

Varala, Kranthi; Li, Ying; Marshall‐Colón, Amy; Para, Alessia

2015-01-01

Understanding how transcription factor (TF) binding is related to gene regulation is a moving target. We recently uncovered genome‐wide evidence for a “Hit‐and‐Run” model of transcription. In this model, a master TF “hits” a target promoter to initiate a rapid response to a signal. As the “hit” is transient, the model invokes recruitment of partner TFs to sustain transcription over time. Following the “run”, the master TF “hits” other targets to propagate the response genome‐wide. As such, a TF may act as a “catalyst” to mount a broad and acute response in cells that first sense the signal, while the recruited TF partners promote long‐term adaptive behavior in the whole organism. This “Hit‐and‐Run” model likely has broad relevance, as TF perturbation studies across eukaryotes show small overlaps between TF‐regulated and TF‐bound genes, implicating transient TF‐target binding. Here, we explore this “Hit‐and‐Run” model to suggest molecular mechanisms and its biological relevance. PMID:26108710

Effects of the Fruit Extract of Tribulus terrestris on Skin Inflammation in Mice with Oxazolone-Induced Atopic Dermatitis through Regulation of Calcium Channels, Orai-1 and TRPV3, and Mast Cell Activation

PubMed Central

Kang, Seok Yong; Jung, Hyo Won; Nam, Joo Hyun; Kim, Woo Kyung; Kang, Jong-Seong; Kim, Young-Ho; Cho, Cheong-Weon; Cho, Chong Woon

2017-01-01

Ethnopharmacological Relevance In this study, we investigated the effects of Tribulus terrestris fruit (Leguminosae, Tribuli Fructus, TF) extract on oxazolone-induced atopic dermatitis in mice. Materials and Methods TF extract was prepared with 30% ethanol as solvent. The 1% TF extract with or without 0.1% HC was applied to the back skin daily for 24 days. Results 1% TF extract with 0.1% HC improved AD symptoms and reduced TEWL and symptom scores in AD mice. 1% TF extract with 0.1% HC inhibited skin inflammation through decrease in inflammatory cells infiltration as well as inhibition of Orai-1 expression in skin tissues. TF extract inhibited Orai-1 activity in Orai-1-STIM1 cooverexpressing HEK293T cells but increased TRPV3 activity in TRPV3-overexpressing HEK293T cells. TF extract decreased β-hexosaminidase release in RBL-2H3 cells. Conclusions The present study demonstrates that the topical application of TF extract improves skin inflammation in AD mice, and the mechanism for this effect appears to be related to the modulation of calcium channels and mast cell activation. This outcome suggests that the combination of TF and steroids could be a more effective and safe approach for AD treatment. PMID:29348776

Tissue distribution and metabolism of triadimefon and triadimenol enantiomers in Chinese lizards (Eremias argus).

PubMed

Li, Jitong; Wang, Yinghuan; Li, Wei; Xu, Peng; Guo, Baoyuan; Li, Jianzhong; Wang, Huili

2017-08-01

Triadimefon (TF, S-(+)-TF, R-(-)-TF) and its metabolite triadimenol (TN, TN-A1, A2 and TN-B1, B2) are two systemic fungicides and both of them are chiral pharmaceuticals which are widely used in agricultural industry. Many researches focused on the toxicity effects of triadimefon on mammals, while the ecotoxicological data of tiradimefon on reptiles is limited. In order to understand the toxicity mechanism of triadimefon in reptiles, the current study administrated S-(+)-TF or R-(-)-TF traidimefon (50mg/kg bw ) to Chinese lizards (Eremias argus) respectively, the absorption, distribution of triadimefon and the formation of triadimenol were analysed at different sampling times. The metabolic pathways were demonstrated through relative gene expression using quantitative real-time PCR reaction. During the experiment time, triadimefon was quickly peaked to the maximum concentration within 12h in liver, brain, kidney, and plasma, eliminated slowly. The biotransformation in kidney was the lowest and fat possessed the worst degradation ability among others. The metabolite, triadimenol was detected in blood in 2h and reached to a plateau at about 12h in most organs (fat excepted), while the process of metabolism is stereoselective. The mainly metabolite in R-(-)-TF treated group was TN-B1, and TN-A2 in S-(+)-TF group which showed the selective metabolism to other species caused by environmental conditions, differences in the animal models and concentration of TF. The related gene expression of cyp1a1, cyp3a1 and hsd11β mRNA level in lizards showed different metabolic pathways in the liver and brain. Both P450s enzymes and 11β-hydroxysteroid dehydrogenase participated in metabolic reaction in liver, while no 11β-hydroxysteroid dehydrogenase pathway observed in brain. This diversity in liver and brain may cause different degradation rate and ecotoxicological effect in different organs. Copyright © 2017 Elsevier Inc. All rights reserved.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

PubMed

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

NASA Astrophysics Data System (ADS)

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2014-07-01

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

Functional and structural insights into novel DREB1A transcription factors in common wheat (Triticum aestivum L.): A molecular modeling approach.

PubMed

Kumar, Anuj; Kumar, Sanjay; Kumar, Upendra; Suravajhala, Prashanth; Gajula, M N V Prasad

2016-10-01

Triticum aestivum L. known as common wheat is one of the most important cereal crops feeding a large and growing population. Various environmental stress factors including drought, high salinity and heat etc. adversely affect wheat production in a significant manner. Dehydration-responsive element-binding (DREB1A) factors, a class of transcription factors (TF) play an important role in combating drought stress. It is known that DREB1A specifically interacts with the dehydration responsive elements (DRE/CRT) inducing expression of genes involved in environmental stress tolerance in plants. Despite its critical interplay in plants, the structural and functional aspects of DREB1A TF in wheat remain unresolved. Previous studies showed that wheat DREBs (DREB1 and DREB2) were isolated using various methods including yeast two-hybrid screens but no extensive structural models were reported. In this study, we made an extensive in silico study to gain insight into DREB1A TF and reported the location of novel DREB1A in wheat chromosomes. We inferred the three-dimensional structural model of DREB1A using homology modelling and further evaluated them using molecular dynamics(MD) simulations yielding refined modelled structures. Our biochemical function predictions suggested that the wheat DREB1A orthologs have similar biochemical functions and pathways to that of AtDREB1A. In conclusion, the current study presents a structural perspective of wheat DREB1A and helps in understanding the molecular basis for the mechanism of DREB1A in response to environmental stress. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

PubMed

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Research on the Sensing Performance of the Tuning Fork-Probe as a Micro Interaction Sensor

PubMed Central

Gao, Fengli; Li, Xide

2015-01-01

The shear force position system has been widely used in scanning near-field optical microscopy (SNOM) and recently extended into the force sensing area. The dynamic properties of a tuning fork (TF), the core component of this system, directly determine the sensing performance of the shear positioning system. Here, we combine experimental results and finite element method (FEM) analysis to investigate the dynamic behavior of the TF probe assembled structure (TF-probe). Results from experiments under varying atmospheric pressures illustrate that the oscillation amplitude of the TF-probe is linearly related to the quality factor, suggesting that decreasing the pressure will dramatically increase the quality factor. The results from FEM analysis reveal the influences of various parameters on the resonant performance of the TF-probe. We compared numerical results of the frequency spectrum with the experimental data collected by our recently developed laser Doppler vibrometer system. Then, we investigated the parameters affecting spatial resolution of the SNOM and the dynamic response of the TF-probe under longitudinal and transverse interactions. It is found that the interactions in transverse direction is much more sensitive than that in the longitudinal direction. Finally, the TF-probe was used to measure the friction coefficient of a silica–silica interface. PMID:26404310

The COUP-TFs compose a family of functionally related transcription factors

PubMed Central

Wang, Lee-Ho; Ing, Nancy H.; Tsai, Sophia Y.; O’Malley, Bert W.; Tsai, Ming-Jer

1991-01-01

The chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are members of the steroid/thyroid hormone receptor superfamily and function in transcriptional regulation of a wide variety of genes. The COUP-TFs purified from HeLa nuclear extract by COUP-affinity chromatography are composed of multiple Mr forms. The Low Mr COUP-TFs (43,000, 44,000, 46,000, and 47,000 Mr) produce a relatively fast migrating complex (Cl) with DNA in electrophoresis mobility shift assays, while the high Mr forms (66,000, 68,000, 72,000, and 74,000 Mr) produce a slower migrating (C2) complex. The high Mr COUP-TFs were purified by gel filtration chromatography and independently formed the C2 DNA complex, probably acting as dimers. The high Mr forms are indistinguishable from the low Mr COUP-TFs in DNA binding and in enhancement of in vitro transcription from the ovalbumin promoter. The finding of multiple COUP-TF forms led us to clone a second low Mr COUP-TF, “COUP-TF2.” The COUP-TF2 sequence has very strong homology with COUP-TF1. The N-termini of COUP-TF1 and COUP-TF2 are least similar, but both contain glutamine-rich and proline-rich motifs, putative activation domains. PMID:1820218

Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.

PubMed

Zhang, S; Zhang, H S; Reuter, V E; Slovin, S F; Scher, H I; Livingston, P O

1998-02-01

Defining the expression of tumor-associated antigens on primary and metastatic prostate cancer is the crucial first step in selecting appropriate targets for immune attack. In this study, the distribution of the tumor-associated antigens GM2, Tn, sTn, Thompson-Friedenreich antigen (TF), Globo H, Le(y), MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonic antigen, beta chain of human chorionic gonadotropin (hCG beta), HER2/neu, PSMA, and KSA on primary and metastatic prostate cancer and 16 types of normal tissues was compared by immunohistochemistry, using a panel of well-characterized monoclonal antibodies. Our results show that GM2, KSA, and MUC2 were strongly expressed on 8 or 9 of 9 metastatic prostate cancer biopsy specimens and, with PSMA, hCG beta, TF, Tn, and sTn, on 8 or more of 11 primary prostate cancer specimens. Tn, MUC1, and PSMA were expressed on 4-6 of 9 metastatic specimens. The remaining antigens were expressed on no more than three of nine metastatic specimens. Normal tissues were also tested with all antibodies. With regard to the eight antigens most widely expressed on prostate cancers, PSMA was not expressed significantly on any of the normal tissues except prostate epithelium. Tn, sTn, hCG beta, and MUC2 were detected on up to 3 of 10 types of normal epithelia. GM2, TF, MUC1, and KSA were more broadly distributed on normal epithelia, all primarily at the secretory borders. STn, KSA, and hCG beta were also detected in the testis, and GM2 was expressed on gray matter of brain. From the 30 antigens that we have screened, this study provides the basis for selecting GM2, TF, Tn, sTn, hCG beta, MUC1, MUC2, KSA, and PSMA as target antigens for specific immunotherapy of prostate cancer.

Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation

PubMed Central

Vischi Winck, Flavia; Arvidsson, Samuel; Riaño-Pachón, Diego Mauricio; Hempel, Sabrina; Koseska, Aneta; Nikoloski, Zoran; Urbina Gomez, David Alejandro; Rupprecht, Jens; Mueller-Roeber, Bernd

2013-01-01

The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1) and Lcr2 (Low-CO 2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in Chlamydomonas. PMID:24224019

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum (Hort.).

PubMed

Li, Chonghui; Qiu, Jian; Yang, Guangsui; Huang, Surong; Yin, Junmei

2016-10-01

A R2R3-MYB gene AaMYB2 was isolated from Anthurium andraeanum (Hort.) and was functionally characterized to be a positive transcriptional regulator for anthocyanin biosynthesis in the spathes and leaves. Spathe coloration is an important Anthurium andraeanum (Hort.) characteristic, which is mainly contributed by anthocyanins. R2R3-MYB transcription factors (TFs) are important regulators of anthocyanin biosynthesis in plants. Here we describe the identification and characterization of AaMYB2, a member in subgroup 6 of the R2R3-MYB TFs family, which correlated with anthocyanin biosynthesis in A. andraeanum. AaMYB2 was a nuclear-localization protein with positive transcriptional activity, and prominently expressed in the red spathes. Ectopic expression of AaMYB2 in tobacco led to anthocyanin accumulation and up-regulation of the early and late anthocyanin pathway genes, particularly NtDFR, NtANS, and NtUFGT, and the endogenous TF genes NtAn2 and NtAn1 in leaves. In the developing red spathes of 'Tropical' and 'Vitara', the expression of AaMYB2 was closely linked to anthocyanin accumulation, and co-expressed with AaCHS, AaF3H, and AaANS, the latter two of which were regarded as the potential targets of the R locus encoding a TF controlling spathe colors inheritance in anthurium. In addition, the transcription level of AaMYB2 in various cultivars with different color phenotypes showed that AaMYB2 was drastically expressed in the spathes from the red, pink, and purple cultivars, but hardly detected in the spathes from the white and green ones. Besides, AaMYB2 also showed higher expression in newly developmental leaves when anthocyanin was actively biosynthesized. Taken together, AaMYB2 positively related to anthocyanin biosynthesis in anthurium spathes and leaves, and appeared to regulate the expression of AaF3H, AaANS, and possibly AaCHS.

ETHYLENE RESPONSE FACTOR 96 positively regulates Arabidopsis resistance to necrotrophic pathogens by direct binding to GCC elements of jasmonate - and ethylene-responsive defence genes.

PubMed

Catinot, Jérémy; Huang, Jing-Bo; Huang, Pin-Yao; Tseng, Min-Yuan; Chen, Ying-Lan; Gu, Shin-Yuan; Lo, Wan-Sheng; Wang, Long-Chi; Chen, Yet-Ran; Zimmerli, Laurent

2015-12-01

The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens. © 2015 John Wiley & Sons Ltd.

Identification and positional distribution analysis of transcription factor binding sites for genes from the wheat fl-cDNA sequences.

PubMed

Chen, Zhen-Yong; Guo, Xiao-Jiang; Chen, Zhong-Xu; Chen, Wei-Ying; Wang, Ji-Rui

2017-06-01

The binding sites of transcription factors (TFs) in upstream DNA regions are called transcription factor binding sites (TFBSs). TFBSs are important elements for regulating gene expression. To date, there have been few studies on the profiles of TFBSs in plants. In total, 4,873 sequences with 5' upstream regions from 8530 wheat fl-cDNA sequences were used to predict TFBSs. We found 4572 TFBSs for the MADS TF family, which was twice as many as for bHLH (1951), B3 (1951), HB superfamily (1914), ERF (1820), and AP2/ERF (1725) TFs, and was approximately four times higher than the remaining TFBS types. The percentage of TFBSs and TF members showed a distinct distribution in different tissues. Overall, the distribution of TFBSs in the upstream regions of wheat fl-cDNA sequences had significant difference. Meanwhile, high frequencies of some types of TFBSs were found in specific regions in the upstream sequences. Both TFs and fl-cDNA with TFBSs predicted in the same tissues exhibited specific distribution preferences for regulating gene expression. The tissue-specific analysis of TFs and fl-cDNA with TFBSs provides useful information for functional research, and can be used to identify relationships between tissue-specific TFs and fl-cDNA with TFBSs. Moreover, the positional distribution of TFBSs indicates that some types of wheat TFBS have different positional distribution preferences in the upstream regions of genes.

Transcriptional network inference from functional similarity and expression data: a global supervised approach.

PubMed

Ambroise, Jérôme; Robert, Annie; Macq, Benoit; Gala, Jean-Luc

2012-01-06

An important challenge in system biology is the inference of biological networks from postgenomic data. Among these biological networks, a gene transcriptional regulatory network focuses on interactions existing between transcription factors (TFs) and and their corresponding target genes. A large number of reverse engineering algorithms were proposed to infer such networks from gene expression profiles, but most current methods have relatively low predictive performances. In this paper, we introduce the novel TNIFSED method (Transcriptional Network Inference from Functional Similarity and Expression Data), that infers a transcriptional network from the integration of correlations and partial correlations of gene expression profiles and gene functional similarities through a supervised classifier. In the current work, TNIFSED was applied to predict the transcriptional network in Escherichia coli and in Saccharomyces cerevisiae, using datasets of 445 and 170 affymetrix arrays, respectively. Using the area under the curve of the receiver operating characteristics and the F-measure as indicators, we showed the predictive performance of TNIFSED to be better than unsupervised state-of-the-art methods. TNIFSED performed slightly worse than the supervised SIRENE algorithm for the target genes identification of the TF having a wide range of yet identified target genes but better for TF having only few identified target genes. Our results indicate that TNIFSED is complementary to the SIRENE algorithm, and particularly suitable to discover target genes of "orphan" TFs.

Tissue factor and Toll-like receptor (TLR)4 in hyperglycaemia-hyperinsulinaemia. Effects in healthy subjects, and type 1 and type 2 diabetes mellitus.

PubMed

Singh, Anamika; Boden, Guenther; Rao, A Koneti

2015-04-01

Diabetes mellitus (DM) patients have an increased incidence of cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycaemia (HG), hyperinsulinaemia (HI) and combined HG+HI (induced using 24-hour infusion clamps) increases TF-PCA in healthy and type 2 DM (T2DM) subjects, but not in type 1 DM (T1DM) subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24-hour HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. In vivo, in healthy subjects, 24-hour HG + HI infusion increased TLR4 six-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hours). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects.

Tissue Factor and Toll Like Receptor (TLR)4 in Hyperglycemia-Hyperinsulinemia: Effects in Healthy Subjects, and Type 1 and Type 2 Diabetes Mellitus

PubMed Central

Singh, Anamika; Boden, Guenther; Rao, A. Koneti

2015-01-01

SUMMARY Background Diabetes mellitus (DM) patients have increased cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycemia (HG), hyperinsulinemia (HI) and combined HG+HI (induced using 24 hr infusion clamps) increases TF-PCA in healthy and T2DM subjects, but not in T1DM subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. Objectives and Methods We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24 hr HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. Results In vivo, in healthy subjects, 24 hr HG + HI infusion increased TLR4 6-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hr). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. Conclusions This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects. PMID:25653143

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Ramesh K.; Kern, Teresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch frommore » the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

How gene order is influenced by the biophysics of transcription regulation

PubMed Central

Kolesov, Grigory; Wunderlich, Zeba; Laikova, Olga N.; Gelfand, Mikhail S.; Mirny, Leonid A.

2007-01-01

What are the forces that shape the structure of prokaryotic genomes: the order of genes, their proximity, and their orientation? Coregulation and coordinated horizontal gene transfer are believed to promote the proximity of functionally related genes and the formation of operons. However, forces that influence the structure of the genome beyond the level of a single operon remain unknown. Here, we show that the biophysical mechanism by which regulatory proteins search for their sites on DNA can impose constraints on genome structure. Using simulations, we demonstrate that rapid and reliable gene regulation requires that the transcription factor (TF) gene be close to the site on DNA the TF has to bind, thus promoting the colocalization of TF genes and their targets on the genome. We use parameters that have been measured in recent experiments to estimate the relevant length and times scales of this process and demonstrate that the search for a cognate site may be prohibitively slow if a TF has a low copy number and is not colocalized. We also analyze TFs and their sites in a number of bacterial genomes, confirm that they are colocalized significantly more often than expected, and show that this observation cannot be attributed to the pressure for coregulation or formation of selfish gene clusters, thus supporting the role of the biophysical constraint in shaping the structure of prokaryotic genomes. Our results demonstrate how spatial organization can influence timing and noise in gene expression. PMID:17709750

Proteinase-Activated Receptor-1 and Immunomodulatory Effects of a PAR1-Activating Peptide in a Mouse Model of Prostatitis

PubMed Central

Stanton, M. Mark; Nelson, Lisa K.; Benediktsson, Hallgrimur; Hollenberg, Morley D.; Buret, Andre G.; Ceri, Howard

2013-01-01

Background. Nonbacterial prostatitis has no established etiology. We hypothesized that proteinase-activated receptor-1 (PAR1) can play a role in prostatitis. We therefore investigated the effects of PAR1 stimulation in the context of a new model of murine nonbacterial prostatitis. Methods. Using a hapten (ethanol-dinitrobenzene sulfonic acid- (DNBS-)) induced prostatitis model with both wild-type and PAR1-null mice, we examined (1) the location of PAR1 in the mouse prostate and (2) the impact of a PAR1-activating peptide (TFLLR-NH2: PAR1-TF) on ethanol-DNBS-induced inflammation. Results. Ethanol-DNBS-induced inflammation was maximal at 2 days. In the tissue, PAR1 was expressed predominantly along the apical acini of prostatic epithelium. Although PAR1-TF on its own did not cause inflammation, its coadministration with ethanol-DNBS reduced all indices of acute prostatitis. Further, PAR1-TF administration doubled the prostatic production of interleukin-10 (IL-10) compared with ethanol-DNBS treatment alone. This enhanced IL-10 was not observed in PAR1-null mice and was not caused by the reverse-sequence receptor-inactive peptide, RLLFT-NH2. Surprisingly, PAR1-TF, also diminished ethanol-DNBS-induced inflammation in PAR1-null mice. Conclusions. PAR1 is expressed in the mouse prostate and its activation by PAR1-TF elicits immunomodulatory effects during ethanol-DNBS-induced prostatitis. However, PAR1-TF also diminishes ethanol-DNBS-induced inflammation via a non-PAR1 mechanism by activating an as-yet unknown receptor. PMID:24459330

TF1, the bacteriophage SPO1-encoded type II DNA-binding protein, is essential for viral multiplication.

PubMed

Sayre, M H; Geiduschek, E P

1988-09-01

The lytic Bacillus subtilis bacteriophage SPO1 encodes an abundant, 99-amino-acid type II DNA-binding protein, transcription factor 1 (TF1). TF1 is special in this family of procaryotic chromatin-forming proteins in its preference for hydroxymethyluracil-containing DNA, such as SPO1 DNA, and in binding with high affinity to specific sites in the SPO1 chromosome. We constructed recessive null alleles of the TF1 gene and introduced them into SPO1 chromosomes. Segregation analysis with partially diploid phage heterozygous for TF1 showed that phage bearing only these null alleles was inviable. Deletion of the nine C-proximal amino acids of TF1 prohibited phage multiplication in vivo and abolished its site-specific DNA-binding activity in vitro.

Rapid screening of transferrin-binders in the flowers of Bauhinia blakeana Dunn by on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-transferrin-fluorescence detection system.

PubMed

Liu, Meixian; Dong, Jing; Lin, Zongtao; Niu, Yanyan; Zhang, Xiaotian; Jiang, Haixiu; Guo, Ning; Li, Wei; Wang, Hong; Chen, Shizhong

2016-06-10

Transferrin (Transferrin, TRF, TF) has drawn increasing attention in cancer therapy due to its potential applications in drug delivery. TF receptor, highly expressed in tumor cells, recognizes and transports Fe(3+)-TF into cells to release iron into cytoplasm. Thus, discovering TF-binding compounds has become an active research area and is of great importance for target therapy. In this study, an on-line analysis method was established for screening TF-binding compounds from the flowers of Bauhinia blakeana Dunn using a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-transferrin-fluorescence detector (HPLC-DAD-MS(n)-TF-FLD) method. As a result, 33 of 80 identified or tentatively characterized compounds in the sample were TF-binding active. Twenty-five flavonol glycosides and eight phenolic acids were identified as TF-binders. Twelve of these active compounds together with six standard compounds were used to study the dose-response effects and structure-activity relationships of flavonoids and phenolic acids. The method was validated by vitexin with a good linearity in the range of concentrations used in the study. The limit of detection for vitexin was 0.1596 nmol. Our study indicated that the established method is simple, rapid and sensitive for screening TF-binding active compounds in the extract of Bauhinia blakeana Dunn, and therefore is important for discovering potential anti-cancer ingredients from complex samples for TF related drug delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1.

PubMed

Sayre, M H; Geiduschek, E P

1990-08-01

The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.

Disentangling the many layers of eukaryotic transcriptional regulation.

PubMed

Lelli, Katherine M; Slattery, Matthew; Mann, Richard S

2012-01-01

Regulation of gene expression in eukaryotes is an extremely complex process. In this review, we break down several critical steps, emphasizing new data and techniques that have expanded current gene regulatory models. We begin at the level of DNA sequence where cis-regulatory modules (CRMs) provide important regulatory information in the form of transcription factor (TF) binding sites. In this respect, CRMs function as instructional platforms for the assembly of gene regulatory complexes. We discuss multiple mechanisms controlling complex assembly, including cooperative DNA binding, combinatorial codes, and CRM architecture. The second section of this review places CRM assembly in the context of nucleosomes and condensed chromatin. We discuss how DNA accessibility and histone modifications contribute to TF function. Lastly, new advances in chromosomal mapping techniques have provided increased understanding of intra- and interchromosomal interactions. We discuss how these topological maps influence gene regulatory models.

Expression and in vitro functional analyses of recombinant Gam1 protein

PubMed Central

Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2014-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

Expression and in vitro functional analyses of recombinant Gam1 protein.

PubMed

Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2015-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

PubMed

Han, Xinxin; Yin, Linlin; Xue, Hongwei

2012-07-01

Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

Conservation of transcription factor binding events predicts gene expression across species

PubMed Central

Hemberg, Martin; Kreiman, Gabriel

2011-01-01

Recent technological advances have made it possible to determine the genome-wide binding sites of transcription factors (TFs). Comparisons across species have suggested a relatively low degree of evolutionary conservation of experimentally defined TF binding events (TFBEs). Using binding data for six different TFs in hepatocytes and embryonic stem cells from human and mouse, we demonstrate that evolutionary conservation of TFBEs within orthologous proximal promoters is closely linked to function, defined as expression of the target genes. We show that (i) there is a significantly higher degree of conservation of TFBEs when the target gene is expressed in both species; (ii) there is increased conservation of binding events for groups of TFs compared to individual TFs; and (iii) conserved TFBEs have a greater impact on the expression of their target genes than non-conserved ones. These results link conservation of structural elements (TFBEs) to conservation of function (gene expression) and suggest a higher degree of functional conservation than implied by previous studies. PMID:21622661

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line

PubMed Central

Lizio, Marina; Ishizu, Yuri; Itoh, Masayoshi; Lassmann, Timo; Hasegawa, Akira; Kubosaki, Atsutaka; Severin, Jessica; Kawaji, Hideya; Nakamura, Yukio; Suzuki, Harukazu; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.

2015-01-01

Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment. PMID:26635867

Plasmin-dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia.

PubMed

Lupu, C; Herlea, O; Tang, H; Lijnen, R H; Lupu, F

2013-01-01

The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications. © 2012 International Society on Thrombosis and Haemostasis.

Porcine Tissue-Specific Regulatory Networks Derived from Meta-Analysis of the Transcriptome

PubMed Central

Pérez-Montarelo, Dafne; Hudson, Nicholas J.; Fernández, Ana I.; Ramayo-Caldas, Yuliaxis; Dalrymple, Brian P.; Reverter, Antonio

2012-01-01

The processes that drive tissue identity and differentiation remain unclear for most tissue types. So are the gene networks and transcription factors (TF) responsible for the differential structure and function of each particular tissue, and this is particularly true for non model species with incomplete genomic resources. To better understand the regulation of genes responsible for tissue identity in pigs, we have inferred regulatory networks from a meta-analysis of 20 gene expression studies spanning 480 Porcine Affymetrix chips for 134 experimental conditions on 27 distinct tissues. We developed a mixed-model normalization approach with a covariance structure that accommodated the disparity in the origin of the individual studies, and obtained the normalized expression of 12,320 genes across the 27 tissues. Using this resource, we constructed a network, based on the co-expression patterns of 1,072 TF and 1,232 tissue specific genes. The resulting network is consistent with the known biology of tissue development. Within the network, genes clustered by tissue and tissues clustered by site of embryonic origin. These clusters were significantly enriched for genes annotated in key relevant biological processes and confirm gene functions and interactions from the literature. We implemented a Regulatory Impact Factor (RIF) metric to identify the key regulators in skeletal muscle and tissues from the central nervous systems. The normalization of the meta-analysis, the inference of the gene co-expression network and the RIF metric, operated synergistically towards a successful search for tissue-specific regulators. Novel among these findings are evidence suggesting a novel key role of ERCC3 as a muscle regulator. Together, our results recapitulate the known biology behind tissue specificity and provide new valuable insights in a less studied but valuable model species. PMID:23049964

Genome wide predictions of miRNA regulation by transcription factors.

PubMed

Ruffalo, Matthew; Bar-Joseph, Ziv

2016-09-01

Reconstructing regulatory networks from expression and interaction data is a major goal of systems biology. While much work has focused on trying to experimentally and computationally determine the set of transcription-factors (TFs) and microRNAs (miRNAs) that regulate genes in these networks, relatively little work has focused on inferring the regulation of miRNAs by TFs. Such regulation can play an important role in several biological processes including development and disease. The main challenge for predicting such interactions is the very small positive training set currently available. Another challenge is the fact that a large fraction of miRNAs are encoded within genes making it hard to determine the specific way in which they are regulated. To enable genome wide predictions of TF-miRNA interactions, we extended semi-supervised machine-learning approaches to integrate a large set of different types of data including sequence, expression, ChIP-seq and epigenetic data. As we show, the methods we develop achieve good performance on both a labeled test set, and when analyzing general co-expression networks. We next analyze mRNA and miRNA cancer expression data, demonstrating the advantage of using the predicted set of interactions for identifying more coherent and relevant modules, genes, and miRNAs. The complete set of predictions is available on the supporting website and can be used by any method that combines miRNAs, genes, and TFs. Code and full set of predictions are available from the supporting website: http://cs.cmu.edu/~mruffalo/tf-mirna/ zivbj@cs.cmu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella.

PubMed

Zhao, Qian; Ma, Dongna; Huang, Yuping; He, Weiyi; Li, Yiying; Vasseur, Liette; You, Minsheng

2018-04-01

Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P. xylostella or Lepidoptera. RNA-seq analysis showed that some of the TF families, such as zinc fingers, homeobox, bZIP, bHLH, and MADF_DNA_bdg genes, were highly expressed in certain tissues including midgut, salivary glands, fat body, and hemocytes, with an obvious sex-biased expression pattern. In addition, a number of TFs showed significant differences in expression between insecticide susceptible and resistant strains, suggesting that these TFs play a role in regulating genes related to insecticide resistance. Finally, we identified an expansion of the HOX cluster in Lepidoptera, which might be related to Lepidoptera-specific evolution. Knockout of this cluster using CRISPR/Cas9 showed that the egg cannot hatch, indicating that this cluster may be related to egg development and maturation. This is the first comprehensive study on identifying and characterizing TFs in P. xylostella. Our results suggest that some TF families are expanded in the P. xylostella genome, and these TFs may have important biological roles in growth, development, sexual dimorphism, and resistance to insecticides. The present work provides a solid foundation for understanding regulation via TFs in P. xylostella and insights into the evolution of the P. xylostella genome.

Inferring genome-wide functional modulatory network: a case study on NF-κB/RelA transcription factor.

PubMed

Li, Xueling; Zhu, Min; Brasier, Allan R; Kudlicki, Andrzej S

2015-04-01

How different pathways lead to the activation of a specific transcription factor (TF) with specific effects is not fully understood. We model context-specific transcriptional regulation as a modulatory network: triplets composed of a TF, target gene, and modulator. Modulators usually affect the activity of a specific TF at the posttranscriptional level in a target gene-specific action mode. This action may be classified as enhancement, attenuation, or inversion of either activation or inhibition. As a case study, we inferred, from a large collection of expression profiles, all potential modulations of NF-κB/RelA. The predicted modulators include many proteins previously not reported as physically binding to RelA but with relevant functions, such as RNA processing, cell cycle, mitochondrion, ubiquitin-dependent proteolysis, and chromatin modification. Modulators from different processes exert specific prevalent action modes on distinct pathways. Modulators from noncoding RNA, RNA-binding proteins, TFs, and kinases modulate the NF-κB/RelA activity with specific action modes consistent with their molecular functions and modulation level. The modulatory networks of NF-κB/RelA in the context epithelial-mesenchymal transition (EMT) and burn injury have different modulators, including those involved in extracellular matrix (FBN1), cytoskeletal regulation (ACTN1), and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long intergenic nonprotein coding RNA, and tumor suppression (FOXP1) for EMT, and TXNIP, GAPDH, PKM2, IFIT5, LDHA, NID1, and TPP1 for burn injury.

Conformational dynamics of bacterial trigger factor in apo and ribosome-bound states.

PubMed

Can, Mehmet Tarik; Kurkcuoglu, Zeynep; Ezeroglu, Gokce; Uyar, Arzu; Kurkcuoglu, Ozge; Doruker, Pemra

2017-01-01

The chaperone trigger factor (TF) binds to the ribosome exit tunnel and helps cotranslational folding of nascent chains (NC) in bacterial cells and chloroplasts. In this study, we aim to investigate the functional dynamics of fully-atomistic apo TF and its complex with 50S. As TF accomodates a high percentage of charged residues on its surface, the effect of ionic strength on TF dynamics is assessed here by performing five independent molecular dynamics (MD) simulations (total of 1.3 micro-second duration) at 29 mM and 150 mM ionic strengths. At both concentrations, TF exhibits high inter- and intra-domain flexibility related to its binding (BD), core (CD), and head (HD) domains. Even though large oscillations in gyration radius exist during each run, we do not detect the so-called 'fully collapsed' state with both HD and BD collapsed upon the core. In fact, the extended conformers with relatively open HD and BD are highly populated at the physiological concentration of 150 mM. More importantly, extended TF snapshots stand out in terms of favorable docking onto the 50S subunit. Elastic network modeling (ENM) indicates significant changes in TF's functional dynamics and domain decomposition depending on its conformation and positioning on the 50S. The most dominant slow motions are the lateral sweeping and vertical opening/closing of HD relative to 50S. Finally, our ENM-based efficient technique -ClustENM- is used to sample atomistic conformers starting with an extended TF-50S complex. Specifically, BD and CD motions are restricted near the tunnel exit, while HD is highly mobile. The atomistic conformers generated without an NC are in agreement with the cryo-EM maps available for TF-ribosome-NC complex.

Heterodimer formation by Oct4 and Smad3 differentially regulates epithelial-to-mesenchymal transition-associated factors in breast cancer progression.

PubMed

Mandal, Gunjan; Biswas, Subir; Roy Chowdhury, Sougata; Chatterjee, Annesha; Purohit, Suman; Khamaru, Poulomi; Chakraborty, Sayan; Mandal, Palash Kumar; Gupta, Arnab; de la Mare, Jo-Anne; Edkins, Adrienne Lesley; Bhattacharyya, Arindam

2018-06-01

The multifunctional cytokine TGF-β crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-β and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-β in late stages compared to early stages of the disease. In vitro studies showed that TGF-β elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-β-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-β, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-β signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of platelet-derived β-thromboglobulins on coagulation.

PubMed

Egan, Karl; van Geffen, Johanna P; Ma, Hui; Kevane, Barry; Lennon, Aine; Allen, Seamus; Neary, Elaine; Parsons, Martin; Maguire, Patricia; Wynne, Kieran; O' Kennedy, Richard; Heemskerk, Johan W M; Áinle, Fionnuala Ní

2017-06-01

β-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived β-thromboglobulins (βTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of βTG on coagulation is unknown. Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified βTG on coagulation. In normal pooled plasma, βTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, βTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when βTG was incubated with factor X, suggesting a direct interaction between βTG and factor X. Using SPR, βTG were found to bind to immobilised factor X in a dose dependent manner. βTG modulate coagulation in vitro via an interaction with factor X. Copyright © 2017 Elsevier Ltd. All rights reserved.

An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1.

PubMed

Andera, L; Schneider, G J; Geiduschek, E P

1994-01-01

TF1 is the Bacillus subtilis bacteriophage-encoded dimeric type II DNA-binding protein. This relative of the eubacterial HU proteins and of the Escherichia coli integration host factor binds preferentially to 5-(hydroxymethyluracil)-containing DNA. We have examined the dynamics of exchange of monomer subunits between molecules of dimeric TF1. The analysis takes advantage of the fact that replacement of phenylalanine with arginine at amino acid 61 in the beta-loop 'arm' of TF1 alters DNA-bending and -binding properties, generating DNA complexes with distinctively different mobilities in gel electrophoresis. New species of DNA-protein complexes were formed by mixtures of wild type and mutant TF1, reflecting the formation of heterodimeric TF1, and making the dynamics of monomer exchange between TF1 dimers accessible to a simple gel retardation analysis. Exchange was rapid at high protein concentrations, even at 0 degrees C, and is proposed to be capable of proceeding through an interaction of molecules of TF1 dimer rather than exclusively through dissociation into monomer subunits. Evidence suggesting that DNA-bound TF1 dimers do not exchange subunits readily is also presented.

oPOSSUM-3: Advanced Analysis of Regulatory Motif Over-Representation Across Genes or ChIP-Seq Datasets

PubMed Central

Kwon, Andrew T.; Arenillas, David J.; Hunt, Rebecca Worsley; Wasserman, Wyeth W.

2012-01-01

oPOSSUM-3 is a web-accessible software system for identification of over-represented transcription factor binding sites (TFBS) and TFBS families in either DNA sequences of co-expressed genes or sequences generated from high-throughput methods, such as ChIP-Seq. Validation of the system with known sets of co-regulated genes and published ChIP-Seq data demonstrates the capacity for oPOSSUM-3 to identify mediating transcription factors (TF) for co-regulated genes or co-recovered sequences. oPOSSUM-3 is available at http://opossum.cisreg.ca. PMID:22973536

oPOSSUM-3: advanced analysis of regulatory motif over-representation across genes or ChIP-Seq datasets.

PubMed

Kwon, Andrew T; Arenillas, David J; Worsley Hunt, Rebecca; Wasserman, Wyeth W

2012-09-01

oPOSSUM-3 is a web-accessible software system for identification of over-represented transcription factor binding sites (TFBS) and TFBS families in either DNA sequences of co-expressed genes or sequences generated from high-throughput methods, such as ChIP-Seq. Validation of the system with known sets of co-regulated genes and published ChIP-Seq data demonstrates the capacity for oPOSSUM-3 to identify mediating transcription factors (TF) for co-regulated genes or co-recovered sequences. oPOSSUM-3 is available at http://opossum.cisreg.ca.

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia.

PubMed

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-07-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre‑processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)‑gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein‑DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid‑repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF‑pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF‑gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA‑box binding protein associated factor 1 and CCCTC‑binding factor, which may be potential therapeutic targets of AML.

Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis

PubMed Central

2014-01-01

Background Increased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains. Results Surface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages. Conclusion Inhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys. PMID:24383984

[The clinical significance of the measurement of plasma transferrin as a growth factor. II. The changes in various endocrine status].

PubMed

Matsubara, M; Odagaki, E; Morioka, T; Nakagawa, K

1987-05-20

The clinical significance of the measurement of plasma transferrin (Tf) in patients with hypophysial disorders was reported in our previous papers. In the present study, we determined plasma Tf levels in 55 patients with various endocrine states and considered their clinical significance compared with plasma somatomedin-C (SM-C) levels. Plasma Tf levels decreased significantly in patients with anorexia nervosa (p less than 0.02), hyperthyroidism (p less than 0.05), primary hypothyroidism (p less than 0.05) and Cushing's syndrome (p less than 0.05), while they were elevated significantly in pregnancy (p less than 0.01) or females using estrogens (p less than 0.05). The former two declines were considered a reflection of the malnutritional state of the patients since a significant negative correlation was observed between plasma Tf levels and the percentile deficit from the ideal body weight in patients with anorexia nervosa (p less than 0.01), or between plasma Tf levels and elevated T3 levels which induce hypermetabolism in patients with hyperthyroidism (p less than 0.01). A significant correlation was observed between the SM-C and Tf levels in these subjects (including normal controls and patients with hypophysial disorders) as a whole (r = 0.79, p less than 0.001). These data indicate that plasma Tf is changeable according to the endocrinological and nutritional conditions with good correlation to the SM-C, and it is suggested that Tf also operates as a growth factor in vivo.

Antigen targeting to antigen-presenting cells enhances presentation to class II-restricted T lymphocytes.

PubMed Central

Scardino, A; Paroli, M; De Petrillo, G; Michel, M L; Barnaba, V

1994-01-01

Receptor-mediated uptake increases by several orders of magnitude the efficiency of APC to internalize Ag, and is stringently required for the Ag-presenting function of T lymphocytes due to their inability to take up Ag non-specifically. We have previously reported that hepatitis B envelope antigen (HBenvAg) can be internalized by T cells via transferrin receptor (TfR). To evaluate if Ag targeting to receptors expressed on APC could be an effective tool for promoting Ag uptake and presentation, we tested the capacity of activated T cells not expressing TfR to induce HBenvAg-specific T-cell responses when pulsed with a hybrid particle containing HBenvAg coupled to gp120 of human immunodeficiency virus (HIV), exploiting the ability of gp120 to bind to CD4 receptor. We found that CD4+/TfR- T cells pulsed either with the hybrid particle or peptide (S193-207) but not with S, L Ag, a recombinant form of HBenvAg, induced a specific proliferative response of a T-cell clone recognizing peptide (S193-207) of HBenvAg. The finding that the addition of anti-CD4 monoclonal antibody (mAb) before the pulsing of CD4+/TfR- T cells with the hybrid particle drastically blocked the specific T-cell response, together with the finding that CD8+/TfR- T cells were unable to serve as APC even if pulsed with this molecule, demonstrated that CD4 receptor was crucial for the HBenvAg internalization. On the other hand, HBenvAg presentation by CD4+/TfR+ T cells pulsed with the hybrid particle was inhibited only when both anti-CD4 and anti-TfR were added before the pulsing. These results suggest that Ag targeting to APC receptors may be usefully exploited to improve Ag-presentation efficiency in potential immunotherapeutic approaches. PMID:7907575

p53 determines prognostic significance of the carbohydrate stem cell marker TF1 (CD176) in ovarian cancer.

PubMed

Heublein, Sabine; Page, Sabina K; Mayr, Doris; Ditsch, Nina; Jeschke, Udo

2016-06-01

The oncofoetal Thomsen-Friedenreich (TF1, CD176) epitope is a carbohydrate cancer stem cell (CSC) antigen, and TF1-mediated cancer progression can be widely reversed by anti-TF1 antibodies. Particularly, CSC-like cells are regarded to be tumorigenic and chemoresistant. Aberrant p53 is probably the factor most closely associated with chemoresistance and tumour aggressiveness in ovarian tumours. We thus questioned whether TF1 in combination with p53 or as a single marker may be related to clinico-pathological features and survival of ovarian cancer patients. Both markers were quantified in ovarian cancer tissue (n = 151) by immunohistochemistry. p53 staining was subdivided into three subgroups [n (completely negative) = 57, n (moderately stained) = 28, n (overexpressing) = 66]. TF1 was scored as positive (n = 30) versus negative (n = 121). Only in those cancers classified with moderate p53 staining-and thus most likely displaying with wild-type TP53-TF1 positivity turned out to be a predictor for shortened overall survival (univariate: p < 0.001, multivariate: p = 0.001). By screening 17 different protein markers for correlation with TF1, only mucin-1 emerged as a potential TF1 carrier protein. It is hypothesized that TF1 may confer tumour-promoting features, especially in a TP53 wild-type genetic background. In addition, TF1 is an attractive immunotherapeutic target. Whether those cases classified as TF1 positive and at the same time as moderately stained for p53 might particularly benefit from a future anti-TF1 antibody treatment or from TF1 vaccination therapy remains to be determined.

Ectopic Expression of JcWRKY Transcription Factor Confers Salinity Tolerance via Salicylic Acid Signaling.

PubMed

Agarwal, Parinita; Dabi, Mitali; Sapara, Komal K; Joshi, Priyanka S; Agarwal, Pradeep K

2016-01-01

Plants, being sessile, have developed intricate signaling network to specifically respond to the diverse environmental stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signaling through auto and cross regulation with different genes and TFs. Here, we report the functional characterization of a salicylic acid -inducible JcWRKY TF. The JcWRKY overexpression confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H 2 O 2 and [Formula: see text]) as compared to the wild type. Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K + /Na + ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes ( CAT and SOD ) showed upregulation during stress. Thus, the present study reflects that JcWRKY is working in co-ordination with SA signaling to orchestrate the different biochemical and molecular pathways to maneuvre salt stress tolerance of the transgenic plants.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

PubMed

Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

2007-08-01

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Binding of galectin-1 to breast cancer cells MCF7 induces apoptosis and inhibition of proliferation in vitro in a 2D- and 3D- cell culture model.

PubMed

Geiger, Pamina; Mayer, Barbara; Wiest, Irmi; Schulze, Sandra; Jeschke, Udo; Weissenbacher, Tobias

2016-11-08

Galectin-1 (gal-1) belongs to the family of β-galactoside-binding proteins which primarily recognizes the Galβ1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. The lectin recognizes correspondent glycoepitopes on human breast cancer cells. Galectin-1 is expressed both in normal and malignant tissues. Lymphatic organs naturally possessing high rates of apoptotic cells, express high levels of Galectin-1. Furthermore galectin-1 can initiate T cell apoptosis. Binding of galectin-1 to trophoblast tumor cells presenting the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen inhibits tumor cell proliferation. In this study we examined the impact galectin-1 has in vitro on cell proliferation, apoptotic potential and metabolic activity of MCF-7 and T-47D breast cancer cells in dependence to their expression of the Thomsen-Friedenreich (TF) tumor antigen. For proliferation and apoptosis assays cells were grown in presence of 10, 30 and 60 μg gal-1/ml medium. Cell proliferation was determined by a BrdU uptake ELISA. Detection of apoptotic cells was done by M30 cyto death staining, in situ nick translation and by a nucleosome ELISA method. Furthermore we studied the impact galectin-1 has on the metabolic activity of MCF-7 and T-47D cells in a homotypic three-dimensional spheroid cell culture model mimicking a micro tumour environment. Gal-1 inhibited proliferation of MCF-7 cells (strong expression of the TF epitope) but did not significantly change proliferation of T-47D cells (weak expression of the TF epitope). The incubation of MCF-7 cells with gal-1 raised number of apoptotic cells significantly. Treating the spheroids with 30 μg/ml galectin-1 in addition to standard chemotherapeutic regimes (FEC, TAC) resulted in further suppression of the metabolic activity in MCF-7 cells whereas T-47D cells were not affected. Our results demonstrate that galectin-1 can inhibit proliferation und metabolic cell activity and induce apoptosis in breast tumor cell lines with high expression levels of the Thomsen-Friedenreich (TF) antigen in monolayer and spheroid cell culture models.

Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames.

PubMed

Behrens, R; Hayles, J; Nurse, P

2000-12-01

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Fission yeast retrotransposon Tf1 integration is targeted to 5′ ends of open reading frames

PubMed Central

Behrens, Ralf; Hayles, Jacky; Nurse, Paul

2000-01-01

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100–420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed. PMID:11095681

Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.

PubMed

Tampo, Yoshiko; Kotamraju, Srigiridhar; Chitambar, Christopher R; Kalivendi, Shasi V; Keszler, Agnes; Joseph, Joy; Kalyanaraman, B

2003-01-10

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.

Suppression of lipopolysaccharide-induced nuclear factor-kappaB activity by theaflavin-3,3'-digallate from black tea and other polyphenols through down-regulation of IkappaB kinase activity in macrophages.

PubMed

Pan, M H; Lin-Shiau, S Y; Ho, C T; Lin, J H; Lin, J K

2000-02-15

We investigated the inhibition of IkappaB kinase (IKK) activity in lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 cell line) by various polyphenols including (-)-epigallocatechin-3-gallate, theaflavin, a mixture of theaflavin-3 gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate (TF-3), pyrocyanidin B-3, casuarinin, geraniin, and penta-O-galloyl-beta-D-glucose (5GG). TF-3 inhibited IKK activity in activated macrophages more strongly than did the other polyphenols. TF-3 strongly inhibited both IKK1 and IKK2 activity and prevented the degradation of IkappaBalpha and IkappaBbeta in activated macrophage cells. The results suggested that the inhibition of IKK activity by TF-3 could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. Furthermore, geraniin, 5GG, and TF-3 all blocked phosphorylation of IKB from the cytosolic fraction, inhibited nuclear factor-kappaB (NFkappaB) activity, and inhibited increases in inducible nitric oxide synthase levels in activated macrophages. These results suggest that TF-3 may exert its anti-inflammatory and cancer chemopreventive actions by suppressing the activation of NFkappaB through inhibition of IKK activity.

Characterization of iron uptake from transferrin by murine endothelial cells.

PubMed

Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

2000-01-01

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

PubMed

Atwood, A; Lin, J H; Levin, H L

1996-01-01

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Novel Cytochrome P45OlBl as a Mammary Cancer Risk Factor.

DTIC Science & Technology

1998-07-01

removed, weighed, and processed for isolation of microsomal protein . In the studies measuring the effect of ß-NF on CYP1B1 expression in the mammary...presented represent the mean and range of results from two experiments. 22 Figure 3. Effect of DEX on TCDD induction of CYP1B1 protein . RMF were isolated ...tumor (TF) or skin (SKF) of two patients (G-163 & G-149). Total cellular proteins were isolated from Trizole® lysates of these cells after they were

A global view of regulatory networks in lung cancer: An approach to understand homogeneity and heterogeneity.

PubMed

Lu, Jiapei; Wang, William; Xu, Menglin; Li, Yuping; Chen, Chengshui; Wang, Xiangdong

2017-02-01

A number of new biotechnologies are used to identify potential biomarkers for the early detection of lung cancer, enabling a personalized therapy to be developed in response. The combinatorial cross-regulation of hundreds of biological function-specific transcription factors (TFs) is defined as the understanding of regulatory networks of molecules within the cell. Here we integrated global databases with 537 patients with lung adenocarcinoma (ADC), 140 with lung squamous carcinoma (SCC), 9 with lung large-cell carcinoma (LCC), 56 with small-cell lung cancer (SCLC), and 590 without cancer with the understanding of TF functions. The present review aims at the homogeneity or heterogeneity of gene expression profiles among subtypes of lung cancer. About 5, 136, 52, or 16 up-regulated or 19, 24, 122, or 97down-regulated type-special TF genes were identified in ADC, SCC, LCC or SCLC, respectively. DNA-binding and transcription regulator activity associated genes play a dominant role in the differentiation of subtypes in lung cancer. Subtype-specific TF gene regulatory networks with elements should be an alternative for diagnostic and therapeutic targets for early identification of lung cancer and can provide insightful clues to etiology and pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

PubMed

Salmon, D; Hanocq-Quertier, J; Paturiaux-Hanocq, F; Pays, A; Tebabi, P; Nolan, D P; Michel, A; Pays, E

1997-12-15

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

A stele-enriched gene regulatory network in the Arabidopsis root

PubMed Central

Brady, Siobhan M; Zhang, Lifang; Megraw, Molly; Martinez, Natalia J; Jiang, Eric; Yi, Charles S; Liu, Weilin; Zeng, Anna; Taylor-Teeples, Mallorie; Kim, Dahae; Ahnert, Sebastian; Ohler, Uwe; Ware, Doreen; Walhout, Albertha J M; Benfey, Philip N

2011-01-01

Tightly controlled gene expression is a hallmark of multicellular development and is accomplished by transcription factors (TFs) and microRNAs (miRNAs). Although many studies have focused on identifying downstream targets of these molecules, less is known about the factors that regulate their differential expression. We used data from high spatial resolution gene expression experiments and yeast one-hybrid (Y1H) and two-hybrid (Y2H) assays to delineate a subset of interactions occurring within a gene regulatory network (GRN) that determines tissue-specific TF and miRNA expression in plants. We find that upstream TFs are expressed in more diverse cell types than their targets and that promoters that are bound by a relatively large number of TFs correspond to key developmental regulators. The regulatory consequence of many TFs for their target was experimentally determined using genetic analysis. Remarkably, molecular phenotypes were identified for 65% of the TFs, but morphological phenotypes were associated with only 16%. This indicates that the GRN is robust, and that gene expression changes may be canalized or buffered. PMID:21245844

Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals.

PubMed

Li, Chonghui; Qiu, Jian; Ding, Ling; Huang, Mingzhong; Huang, Surong; Yang, Guangsui; Yin, Junmei

2017-03-01

Dendrobium hybrids orchid are popular throughout the world. They have various floral color and pigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyanin biosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in most plants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from a Dendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobium petals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1 resulted in anthocyanin production in white petals. The interaction between the two TFs was observed in vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 were co-expressed with DhDFR and DhANS, which are regarded as potential regulatory targets of the two TFs. In flowers with distinct purple lips but white or yellow petals/sepals, the expression of DhbHLH1 was only related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for the distinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 and DhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

PubMed

Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

2005-04-26

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear.

PubMed

Govindarajan, Vijay; Zhu, Shu; Li, Ruizhi; Lu, Yichen; Diamond, Scott L; Reifman, Jaques; Mitrophanov, Alexander Y

2018-02-27

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ∼0.1 and ∼2 molecules/μm 2 . Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot's structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot's occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow. Copyright © 2018 Biophysical Society. All rights reserved.

Genome-wide identification and comparative expression analysis reveal a rapid expansion and functional divergence of duplicated genes in the WRKY gene family of cabbage, Brassica oleracea var. capitata.

PubMed

Yao, Qiu-Yang; Xia, En-Hua; Liu, Fei-Hu; Gao, Li-Zhi

2015-02-15

WRKY transcription factors (TFs), one of the ten largest TF families in higher plants, play important roles in regulating plant development and resistance. To date, little is known about the WRKY TF family in Brassica oleracea. Recently, the completed genome sequence of cabbage (B. oleracea var. capitata) allows us to systematically analyze WRKY genes in this species. A total of 148 WRKY genes were characterized and classified into seven subgroups that belong to three major groups. Phylogenetic and synteny analyses revealed that the repertoire of cabbage WRKY genes was derived from a common ancestor shared with Arabidopsis thaliana. The B. oleracea WRKY genes were found to be preferentially retained after the whole-genome triplication (WGT) event in its recent ancestor, suggesting that the WGT event had largely contributed to a rapid expansion of the WRKY gene family in B. oleracea. The analysis of RNA-Seq data from various tissues (i.e., roots, stems, leaves, buds, flowers and siliques) revealed that most of the identified WRKY genes were positively expressed in cabbage, and a large portion of them exhibited patterns of differential and tissue-specific expression, demonstrating that these gene members might play essential roles in plant developmental processes. Comparative analysis of the expression level among duplicated genes showed that gene expression divergence was evidently presented among cabbage WRKY paralogs, indicating functional divergence of these duplicated WRKY genes. Copyright © 2014 Elsevier B.V. All rights reserved.

A Novel Method to Predict Highly Expressed Genes Based on Radius Clustering and Relative Synonymous Codon Usage.

PubMed

Tran, Tuan-Anh; Vo, Nam Tri; Nguyen, Hoang Duc; Pham, Bao The

2015-12-01

Recombinant proteins play an important role in many aspects of life and have generated a huge income, notably in the industrial enzyme business. A gene is introduced into a vector and expressed in a host organism-for example, E. coli-to obtain a high productivity of target protein. However, transferred genes from particular organisms are not usually compatible with the host's expression system because of various reasons, for example, codon usage bias, GC content, repetitive sequences, and secondary structure. The solution is developing programs to optimize for designing a nucleotide sequence whose origin is from peptide sequences using properties of highly expressed genes (HEGs) of the host organism. Existing data of HEGs determined by practical and computer-based methods do not satisfy for qualifying and quantifying. Therefore, the demand for developing a new HEG prediction method is critical. We proposed a new method for predicting HEGs and criteria to evaluate gene optimization. Codon usage bias was weighted by amplifying the difference between HEGs and non-highly expressed genes (non-HEGs). The number of predicted HEGs is 5% of the genome. In comparison with Puigbò's method, the result is twice as good as Puigbò's one, in kernel ratio and kernel sensitivity. Concerning transcription/translation factor proteins (TF), the proposed method gives low TF sensitivity, while Puigbò's method gives moderate one. In summary, the results indicated that the proposed method can be a good optional applying method to predict optimized genes for particular organisms, and we generated an HEG database for further researches in gene design.

Differential Gene Expression between Leaf and Rhizome in Atractylodes lancea: A Comparative Transcriptome Analysis

PubMed Central

Huang, Qianqian; Huang, Xiao; Deng, Juan; Liu, Hegang; Liu, Yanwen; Yu, Kun; Huang, Bisheng

2016-01-01

The rhizome of Atractylodes lancea is extensively used in the practice of Traditional Chinese Medicine because of its broad pharmacological activities. This study was designed to characterize the transcriptome profiling of the rhizome and leaf of Atractylodes lancea in an attempt to uncover the molecular mechanisms regulating rhizome formation and growth. Over 270 million clean reads were assembled into 92,366 unigenes, 58% of which are homologous with sequences in public protein databases (NR, Swiss-Prot, GO, and KEGG). Analysis of expression levels showed that genes involved in photosynthesis, stress response, and translation were the most abundant transcripts in the leaf, while transcripts involved in stress response, transcription regulation, translation, and metabolism were dominant in the rhizome. Tissue-specific gene analysis identified distinct gene families active in the leaf and rhizome. Differential gene expression analysis revealed a clear difference in gene expression pattern, identifying 1518 up-regulated genes and 3464 down-regulated genes in the rhizome compared with the leaf, including a series of genes related to signal transduction, primary and secondary metabolism. Transcription factor (TF) analysis identified 42 TF families, with 67 and 60 TFs up-regulated in the rhizome and leaf, respectively. A total of 104 unigenes were identified as candidates for regulating rhizome formation and development. These data offer an overview of the gene expression pattern of the rhizome and leaf and provide essential information for future studies on the molecular mechanisms of controlling rhizome formation and growth. The extensive transcriptome data generated in this study will be a valuable resource for further functional genomics studies of A. lancea. PMID:27066021