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Sample records for element binding factor

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

    PubMed Central

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

    1992-01-01

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

  2. Determining the DNA sequence elements required for binding integration host factor to two different target sites.

    PubMed Central

    Hales, L M; Gumport, R I; Gardner, J F

    1994-01-01

    Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding. PMID:8188600

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

    PubMed

    Ghosh, R; Mitchell, D L

    1999-08-01

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

  4. GAGA factor binding to DNA via a single trinucleotide sequence element.

    PubMed Central

    Wilkins, R C; Lis, J T

    1998-01-01

    GAGA transcription factor (GAF) is an essential protein in Drosophila , important for the transcriptional regulation of numerous genes. GAF binds to GA repeats in the promoters of these genes via a DNA-binding domain containing a single zinc finger. While GAF binding sites are typically composed of 3.5 GA repeats, the Drosophila hsp70 gene contains much smaller elements, some of which are as little as three bases (GAG) in length. Interestingly, the binding of GAF to more distant trinucleotide elements is relatively strong and not appreciably affected by the removal of larger GA arrays in the promoter. Moreover, a simple synthetic GAG sequence is sufficient to bind GAF in vitro . Here we directly compare the affinity of GAF for different sequence elements by immunoprecipitation and gel mobility shift analysis. Furthermore, our measures of the concentration of GAF in vivo indicate that it is a highly abundant nuclear protein, prevalent enough to occupy a sizable fraction of correspondingly abundant trinucleotide sites. PMID:9592153

  5. Effects of binding factors on structural elements in F-actin.

    PubMed

    Scoville, Damon; Stamm, John D; Altenbach, Christian; Shvetsov, Alexander; Kokabi, Kaveh; Rubenstein, Peter A; Hubbell, Wayne L; Reisler, Emil

    2009-01-20

    Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

  6. Nonmyogenic factors bind nicotinic acetylcholine receptor promoter elements required for response to denervation.

    PubMed

    Bessereau, J L; Laudenbach, V; Le Poupon, C; Changeux, J P

    1998-05-22

    Nicotinic acetylcholine receptors (AChRs) belong to a class of muscle proteins whose expression is regulated by muscle electrical activity. In innervated muscle fiber, AChR genes are transcriptionally repressed outside of the synapse, while after denervation they become reexpressed throughout the fiber. The myogenic determination factors (MDFs) of the MyoD family have been shown to play a central role in this innervation-dependent regulation. In the chicken AChR alpha-subunit gene promoter, two E-boxes that bind MDFs are necessary to achieve the enhancement of transcription following muscle denervation. However, the deletion of promoter sequences located upstream to these E-boxes greatly impairs the response to denervation (Bessereau, J. L., Stratford- Perricaudet, L. D., Piette, J., Le Poupon, C. and Changeux, J. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1304-1308). Here we identified two additional cis-regulatory elements of the alpha-subunit gene promoter that cooperate with the E-boxes in the denervation response. One region binds the Sp1 and Sp3 zinc finger transcription factors. The second region binds at least three distinct factors, among which we identified an upstream stimulatory factor, a b-ZIP-HLH transcription factor. We propose that among MDF-responsive muscle promoters, a specific combination between myogenic and nonmyogenic factors specify innervation-dependent versus innervation-independent promoters.

  7. Database of repetitive elements in complete genomes and data mining using transcription factor binding sites.

    PubMed

    Horng, Jorng-Tzong; Lin, F M; Lin, J H; Huang, H D; Liu, B J

    2003-06-01

    Approximately 43% of the human genome is occupied by repetitive elements. Even more, around 51% of the rice genome is occupied by repetitive elements. The analysis presented here indicates that repetitive elements in complete genomes may have been very important in the evolutionary genomics. In this study, a database, called the Repeat Sequence Database, is first designed and implemented to store complete and comprehensive repetitive sequences. See http://rsdb.csie.ncu.edu.tw for more information. The database contains direct, inverted and palindromic repetitive sequences, and each repetitive sequence has a variable length ranging from seven to many hundred nucleotides. The repetitive sequences in the database are explored using a mathematical algorithm to mine rules on how combinations of individual binding sites are distributed among repetitive sequences in the database. Combinations of transcription factor binding sites in the repetitive sequences are obtained and then data mining techniques are applied to mine association rules from these combinations. The discovered associations are further pruned to remove insignificant associations and obtain a set of associations. The mined association rules facilitate efforts to identify gene classes regulated by similar mechanisms and accurately predict regulatory elements. Experiments are performed on several genomes including C. elegans, human chromosome 22, and yeast.

  8. Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

    PubMed Central

    Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

    2014-01-01

    The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

  9. Small yet effective: the ethylene responsive element binding factor-associated amphiphilic repression (EAR) motif.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2010-06-01

    The Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif is a small yet distinct regulatory motif that is conserved in many plant transcriptional regulator (TR) proteins associated with diverse biological functions. We have previously established a list of high-confidence Arabidopsis EAR repressors, the EAR repressome, comprising 219 TRs belonging to 21 different TR families. This class of proteins and the sequence context of the EAR motif exhibited a high degree of conservation across evolutionarily diverse plant species. Our comprehensive genome-wide analysis enabled refining EAR motifs as comprising either LxLxL or DLNxxP. Comparing the representation of these sequence signatures in TRs to that of other repressor motifs we show that the EAR motif is the one most frequently represented, detected in 10 to 25% of the TRs from diverse plant species. The mechanisms involved in regulation of EAR motif function and the cellular fates of EAR repressors are currently not well understood. Our earlier analysis had implicated amino acid residues flanking the EAR motifs in regulation of their functionality. Here, we present additional evidence supporting possible regulation of EAR motif function by phosphorylation of integral or adjacent Ser and/or Thr residues. Additionally, we discuss potential novel roles of EAR motifs in plant-pathogen interaction and processes other than transcriptional repression.

  10. PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing

    USDA-ARS?s Scientific Manuscript database

    Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

  11. Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish

    PubMed Central

    Ashikawa, Yoshifumi; Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish

  12. Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

    PubMed Central

    Frederick, Joshua P.; Liberati, Nicole T.; Waddell, David S.; Shi, Yigong; Wang, Xiao-Fan

    2004-01-01

    Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter. PMID:14993291

  13. Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis.

    PubMed

    L'honore, Aurore; Rana, Vanessa; Arsic, Nikola; Franckhauser, Celine; Lamb, Ned J; Fernandez, Anne

    2007-06-01

    MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.

  14. The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element

    PubMed Central

    Cichocki, Frank; Hanson, Rebecca J.; Lenvik, Todd; Pitt, Michelle; McCullar, Valarie; Li, Hongchuan; Anderson, Stephen K.

    2009-01-01

    The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5′ promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education. PMID:18987359

  15. Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis. Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication. Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region. A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter. This fragment contained both positively and negatively acting sequences. Four DNA-binding factors interacted with the RNR2 regulatory region. One factor was identified as the GRF1 protein, the product of the RAP1 gene. GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment. UAS2 activity was repressed by the 42-bp fragment. Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter. These factors are likely to mediate the response of RNR2 to DNA damage. Images PMID:2685561

  16. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  17. Molecular cloning and characterization of interferon. alpha. /. beta. response element binding factors of the murine (2 prime -5 prime )oligoadenylate synthetase ME-12 gene

    SciTech Connect

    Yan, Cong; Tamm, Igor )

    1991-01-01

    Seven clones encoding interferon response element binding factors have been isolated from a mouse fibroblast {lambda}gt11 cDNA library by using a {sup 32}P end-labeled tandem trimer of the mouse (2{prime}-5{prime})oligoadenylate synthetase gene interferon response element as a probe. Clone 16 shares strong similarity (95%) at both DNA and amino acid level with YB-1, a human major histocompatibility complex class II Y-box DNA-binding protein, and with dbpB, a human epidermal growth factor receptor gene enhancer region binding protein. The product of the gene represented by clone 16 may represent a factor that regulates multiple genes by binding to a variety of 5{prime} regulatory elements. Clone 25 is a 2407-base-pair-long cDNA and contains a putative 311-amino acid open reading frame corresponding to an estimated mass of 35.5 kDa. This putative protein, designated as interferon resonse element binding factor 1 (IREBF-1), contains an acidic domain, three heptad repeat leucine arrays, and a region that shares similarity with the yeast transcriptional factor CAL4 DNA-binding domain. Furthermore, the C terminus of IREBF-1 shows an unusual amphipathic property: within a 79-amino acid range, one side of the {alpha}-helical region contains a preponderance of hydrophobic amino acids and the other side contains hydrophilic amino acids. This type of structure provides a strong hydrophobic force for protein-protein interaction.

  18. Isolation and characterization of a buffalograss (Buchloe dactyloides) dehydration responsive element binding transcription factor, BdDREB2.

    PubMed

    Zhang, Pan; Yang, Peizhi; Zhang, Zhiqiang; Han, Bo; Wang, Weidong; Wang, Yafang; Cao, Yuman; Hu, Tianming

    2014-02-15

    Dehydration responsive element binding (DREB) transcription factors play an important role in the regulation of stress-related genes. These factors contribute to resistance to different abiotic stresses. In the present study, a novel DREB transcription factor, BdDREB2, isolated from Buchloe dactyloides, was cloned and characterized. The BdDREB2 protein was estimated to have a molecular weight of 28.36kDa, a pI of 5.53 and a typical AP2/ERF domain. The expression of BdDREB2 was involved in responses to drought and salt stresses. Overexpression of BdDREB2 in tobacco showed higher relative water and proline content, and was associated with lower MDA content under drought stress, suggesting that the transgenic tobacco may tolerate drought stress better. Results demonstrate that BdDREB2 may play an important role in the regulation of abiotic stress responses, and mediate many physiological pathways that enhance stress tolerance in plants. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder.

    PubMed

    Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

    2013-03-25

    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  20. Forkhead transcription factor 1 inhibits endometrial cancer cell proliferation via sterol regulatory element-binding protein 1

    PubMed Central

    Zhang, Yifang; Zhang, Lili; Sun, Hengzi; Lv, Qingtao; Qiu, Chunping; Che, Xiaoxia; Liu, Zhiming; Jiang, Jie

    2017-01-01

    The morbidity and mortality associated with endometrial cancer (EC) has increased in recent years. Regarded as a tumor suppressor, forkhead transcription factor 1 (FOXO1) has various biological activities and participates in cell cycle progression, apoptosis and differentiation. Notably, FOXO1 also functions in the regulation of lipogenesis and energy metabolism. Lipogenesis is a feature of cancer and is upregulated in EC. Sterol regulatory element-binding protein 1 (SREBP1) is a transcription factor that is also able to regulate lipogenesis. Increased expression of SREBP1 is directly correlated with malignant transformation of tumors. A previous study demonstrated that SREBP1 was highly expressed in EC and directly resulted in tumorigenesis. However, the association between FOXO1 and SREBP1 in EC is not clear. In the present study, lentiviruses overexpressing FOXO1 were used in cell transfection and transduction. Cell viability assays demonstrated that the overexpression of FOXO1 was able to suppress cell proliferation significantly in Ishikawa and AN3 CA cell lines. In addition, FOXO1 overexpression significantly inhibited cell migration and invasion ability in vitro. In xenograft models, overexpression of FOXO1 suppressed cell tumorigenesis, and western blot analysis demonstrated that SREBP1 expression was markedly reduced in the FOXO1-overexpressing cells. It may therefore be concluded that FOXO1 is able to inhibit the proliferative capacity of cells in vitro and in vivo, in addition to the migratory and invasive capacities in vitro by directly targeting SREBP1. PMID:28356952

  1. Constitutive and heat-inducible heat shock element binding activities of heat shock factor in a group of filamentous fungi

    PubMed Central

    Xavier, Ilungo J.; Khachatourians, George G.; Ovsenek, Nick

    1999-01-01

    This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation. PMID:10590835

  2. A DNA-binding factor specific for xenobiotic responsive elements of P-450c gene exists as a cryptic form in cytoplasm: Its possible translocation to nucleus

    SciTech Connect

    Fujisawa-Sehara, Atsuko; Yamane, Miyuki; Fujii-Kuriyama, Yoshiaki

    1988-08-01

    Transcription of the drug-metabolizing cytochrome P-450c gene is induced by 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Previously, the authors defined two xenobiotic responsive elements (XREs) of {approx}15 base pairs, both of which activate transcription in cis in response to these xenobiotics. Using a gel mobility shift assay, they have identified a factor that specifically binds to the XREs. This factor appears in nuclei of mouse hepatoma cell line Hepa-1 only when the cells are treated with the xenobiotics, while the factor is undetectable in the nuclei of a 3-methylcholanthrene-treated mutant of Hepa-1 with defective function of a xenobiotic receptor. In addition, the nuclear factor bound to the XRE in the gel was found to be associated with ({sup 3}H)TCDD when the cells were treated with it, suggesting that the xenobiotic receptor is at least a component of the DNA-binding factor. The cytoplasmic fraction from nontreated Hepa-1 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with 3-methylcholanthrene in vitro. These results not only suggest the involvement of the XRE-binding factor in transcriptional activation via XREs but also provide evidence that the binding of ligands to the preexisting factor in a cryptic form induces its XRE-binding activity, which is probably followed by its translocation from cytoplasm to nucleus.

  3. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter.

    PubMed

    Sun, Chuanxin; Palmqvist, Sara; Olsson, Helena; Borén, Mats; Ahlandsberg, Staffan; Jansson, Christer

    2003-09-01

    SURE (sugar responsive) is a cis element in plant sugar signaling. The SURE element was reported first for potato, in which it confers sugar responsiveness to the patatin promoter. A SURE binding transcription factor has not been isolated. We have isolated a transcription factor cDNA from barley and purified the corresponding protein. The transcription factor, SUSIBA2 (sugar signaling in barley), belongs to the WRKY proteins and was shown to bind to SURE and W-box elements but not to the SP8a element in the iso1 promoter. Nuclear localization of SUSIBA2 was demonstrated in a transient assay system with a SUSIBA2:green fluorescent protein fusion protein. Exploiting the novel transcription factor oligodeoxynucleotide decoy strategy with transformed barley endosperm provided experimental evidence for the importance of the SURE elements in iso1 transcription. Antibodies against SUSIBA2 were produced, and the expression pattern for susiba2 was determined at the RNA and protein levels. It was found that susiba2 is expressed in endosperm but not in leaves. Transcription of susiba2 is sugar inducible, and ectopic susiba2 expression was obtained in sugar-treated leaves. Likewise, binding to SURE elements was observed for nuclear extracts from sugar-treated but not from control barley leaves. The temporal expression of susiba2 in barley endosperm followed that of iso1 and endogenous sucrose levels, with a peak at approximately 12 days after pollination. Our data indicate that SUSIBA2 binds to the SURE elements in the barley iso1 promoter as an activator. Furthermore, they show that SUSIBA2 is a regulatory transcription factor in starch synthesis and demonstrate the involvement of a WRKY protein in carbohydrate anabolism. Orthologs to SUSIBA2 were isolated from rice and wheat endosperm.

  4. Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α.

    PubMed

    Hanse, Eric A; Mashek, Douglas G; Becker, Jennifer R; Solmonson, Ashley D; Mullany, Lisa K; Mashek, Mara T; Towle, Howard C; Chau, Anhtung T; Albrecht, Jeffrey H

    2012-07-15

    Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

  5. BjMYB1, a transcription factor implicated in plant defence through activating BjCHI1 chitinase expression by binding to a W-box-like element

    PubMed Central

    Gao, Ying; Jia, Shuangwei; Wang, Chunlian; Wang, Fujun; Wang, Fajun; Zhao, Kaijun

    2016-01-01

    We previously identified the W-box-like-4 (Wbl-4) element (GTAGTGACTCAT), one of six Wbl elements in the BjC-P promoter of the unusual chitinase gene BjCHI1 from Brassica juncea, as the core element responsive to fungal infection. Here, we report the isolation and characterization of the cognate transcription factor interacting with the Wbl-4 element. Using Wbl-4 as a target, we performed yeast one-hybrid screening of a B. juncea cDNA library and isolated an R2R3-MYB transcription factor designated as BjMYB1. BjMYB1 was localized in the nucleus of plant cells. EMSA assays confirmed that BjMYB1 binds to the Wbl-4 element. Transiently expressed BjMYB1 up-regulated the activity of the BjC-P promoter through its binding to the Wbl-4 element in tobacco (Nicotiana benthamiana) leaves. In B. juncea, BjMYB1 displayed a similar induced expression pattern as that of BjCHI1 upon infection by the fungus Botrytis cinerea. Moreover, heterogeneous overexpression of BjMYB1 significantly elevated the resistance of transgenic Arabidopsis thaliana to the fungus B. cinerea. These results suggest that BjMYB1 is potentially involved in host defence against fungal attack through activating the expression of BjCHI1 by binding to the Wbl-4 element in the BjC-P promoter. This finding demonstrates a novel DNA target of plant MYB transcription factors. PMID:27353280

  6. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor. beta. 1 and by the retinoblastoma gene product

    SciTech Connect

    Pietenpol, J.A.; Stein, R.W.; Moses, H.L. ); Muenger, K.; Howley, P.M. )

    1991-11-15

    Previous studies have shown that transforming growth factor {beta}1 (TGF-{beta}1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter region was required for regulation by both TGF-{beta}1 and pRB. These sequences, termed the TGF-{beta} control element (TCE), lie between positions {minus}86 and {minus}63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-{beta}1 treatment of TGF-{beta}-sensitive but not TGF-{beta}-insensitive cells. These data indicate that pRB can suppress c-myc transcription and growth inhibition.

  7. The avian cardiac alpha-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive- and negative-acting factors.

    PubMed

    Moss, J B; McQuinn, T C; Schwartz, R J

    1994-04-29

    The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.

  8. A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

    PubMed Central

    Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

    1992-01-01

    Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

  9. Transposable element-mediated enhancement of gene expression in saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor

    SciTech Connect

    Goel, A.; Pearlman, R.E.

    1988-06-01

    In the authors' studies on the regulation of adjacent-gene expression by Ty sequences, they demonstrated that a single-base-pair change (T-A---C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of BETA-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility mobility assay, they showed that the same base pair transition is required for binding a trans-acting factor, TyBF, from crude cell extracts in vitro. The authors identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. They propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

  10. A binuclear zinc transcription factor binds the host isoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification.

    PubMed

    Khan, Rana; Tan, Reynold; Mariscal, Amanda Galvez; Straney, David

    2003-07-01

    The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450. Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus. Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus. Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea. The regulatory element within PDA1 that provides pisatin-responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis. The 40 bp pisatin-responsive element is located 635 bp upstream of the PDA1 transcription start site. This element was sufficient to provide strong pisatin-induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter. A gene encoding a DNA-binding protein specific to this 40 bp element was isolated from a N. haematococca cDNA library using the yeast one-hybrid screen. The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6-Zn 2) family of transcription factors unique to fungi. The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin-responsive regulation.

  11. HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements.

    PubMed

    Kabat, Jennifer L; Barberan-Soler, Sergio; Zahler, Alan M

    2009-10-16

    Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.

  12. Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

    PubMed

    Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

    2017-04-24

    Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  13. Content and activity of cAMP response element-binding protein regulate platelet-derived growth factor receptor-alpha content in vascular smooth muscles.

    PubMed

    Watson, Peter A; Vinson, Charles; Nesterova, Albina; Reusch, Jane E-B

    2002-08-01

    Experiments in vascular smooth muscle cells (SMCs) indicate that the transcription factor cAMP response element-binding protein (CREB), the cyclic nucleotide response element-binding protein, suppresses expression of the platelet-derived growth factor-alpha receptor gene (PDGFRalpha). Adenovirus-mediated expression of constitutively active CREB mutants decreases PDGFRalpha mRNA, PDGFRalpha protein, and PDGFRalpha promoter-luciferase reporter activity in cultured SMCs. Expression of dominant negative CREB protein, A-CREB, increases PDGFRalpha protein content and the PDGFRalpha-promoter activity in SMCs. Active CREB prevents activation of PDGFRalpha promoter-luciferase reporter activity by CCAAT/enhancer-binding protein-delta (C/EBPdelta), shown to mediate IL-1beta stimulation of PDGFRalpha expression. Exposure of cultured SMCs to high glucose or reactive oxidant stress, which decrease CREB protein content and activity, increases PDGFRalpha protein content and promoter activity. Expression of active CREB blunts reactive oxidant stress-induced PDGFRalpha accumulation in SMCs. Loss of CREB protein in aortic walls of rats with streptozotocin-induced diabetes is accompanied by an increase in PDGFRalpha content. In Ob/Ob mice (which demonstrate reduced aortic wall CREB content vs. Ob/- controls), treatment with the peroxisomal proliferator-activated receptor gamma rosiglitazone increases CREB content and decreases PDGFRalpha content in the aortic wall. Thus, both in vitro and in vivo loss of CREB content and activity and subsequent accumulation of PDGFRalpha may contribute to SMC activation during diabetes.

  14. Stimulation of the human intercellular adhesion molecule-1 promoter by interleukin-6 and interferon-gamma involves binding of distinct factors to a palindromic response element.

    PubMed

    Caldenhoven, E; Coffer, P; Yuan, J; Van de Stolpe, A; Horn, F; Kruijer, W; Van der Saag, P T

    1994-08-19

    Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is expressed on cells of many lineages and is induced by interleukin-6 (IL-6) and interferon-gamma (IFN-gamma). Functional analysis of ICAM-1 promoter-luciferase constructs in HepG2 cells enabled us to identify a region between -110 and -37 mediating IL-6 and IFN-gamma responsiveness and containing a palindromic IL-6/IFN-gamma response element (pIRE). Site-directed mutagenesis of key nucleotides in the ICAM-1 pIRE abolished the effect of both IL-6 and IFN-gamma stimulation, while this pIRE element was sufficient to confer IL-6 and IFN-gamma responsiveness to a heterologous promoter. We further show by gel retardation analysis that distinct nuclear factors induced by both IL-6 or IFN-gamma specifically bind to this pIRE. Furthermore, treatment with IL-6 results in the formation of multiple complexes while IFN-gamma induces a single binding complex, both in HepG2 and monocytic U937 cells. Differentiation of U937 cells by exposure to 12-O-tetradecanoyl phorbol-13-acetate abolishes response to IL-6 but not IFN-gamma. Supershift data utilizing the ICAM-1 pIRE revealed that IFN-gamma and IL-6 both induce a factor antigenically related to IFN-gamma activation factor. We further provide data suggesting that IL-6 additionally activates an ICAM-1 pIRE binding factor related to the previously described acute-phase response factor in disparate cell types. We therefore conclude that the activation of these related nuclear factors by IL-6 and IFN-gamma is important in the regulation of ICAM-1 gene expression.

  15. Phorbol ester-induced transcription of a fibroblast growth factor-binding protein is modulated by a complex interplay of positive and negative regulatory promoter elements.

    PubMed

    Harris, V K; Liaudet-Coopman, E D; Boyle, B J; Wellstein, A; Riegel, A T

    1998-07-24

    Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene

  16. Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response.

    PubMed Central

    Fawcett, T W; Martindale, J L; Guyton, K Z; Hai, T; Holbrook, N J

    1999-01-01

    Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site. PMID:10085237

  17. Identification of TIAR as a protein binding to the translational regulatory AU-rich element of tumor necrosis factor alpha mRNA.

    PubMed

    Gueydan, C; Droogmans, L; Chalon, P; Huez, G; Caput, D; Kruys, V

    1999-01-22

    In monocyte/macrophages, the translation of tumor necrosis factor alpha (TNF-alpha) mRNA is tightly regulated. In unstimulated cells, translation of TNF-alpha mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-alpha mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNA-binding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation.

  18. Transcription of angiogenin and ribonuclease 4 is regulated by RNA polymerase III elements and a CCCTC binding factor (CTCF)-dependent intragenic chromatin loop.

    PubMed

    Sheng, Jinghao; Luo, Chi; Jiang, Yuxiang; Hinds, Philip W; Xu, Zhengping; Hu, Guo-fu

    2014-05-02

    Angiogenin (ANG) and ribonuclease 4 (RNASE4), two members of the secreted and vertebrate-specific ribonuclease superfamily, play important roles in cancers and neurodegenerative diseases. The ANG and RNASE4 genes share genetic regions with promoter activities, but the structure and regulation of these putative promotes are unknown. We have characterized the promoter regions, defined the transcription start site, and identified a mechanism of transcription regulation that involves both RNA polymerase III (Pol III) elements and CCCTC binding factor (CTCF) sites. We found that two Pol III elements within the promoter region influence ANG and RNASE4 expression in a position- and orientation-dependent manner. We also provide evidence for the presence of an intragenic chromatin loop between the two CTCF binding sites located in two introns flanking the ANG coding exon. We found that formation of this intragenic loop preferentially enhances ANG transcription. These results suggest a multilayer transcriptional regulation of ANG and RNASE4 gene locus. These data also add more direct evidence to the notion that Pol III elements are able to directly influence Pol II gene transcription. Furthermore, our data indicate that a CTCF-dependent chromatin loop is able to differentially regulate transcription of genes that share the same promoters.

  19. Delta, a transcription factor that binds to downstream elements in several polymerase II promoters, is a functionally versatile zinc finger protein.

    PubMed Central

    Hariharan, N; Kelley, D E; Perry, R P

    1991-01-01

    The promoters of several eukaryotic genes transcribed by RNA polymerase II contain elements located downstream of the transcriptional start site. To gain insight into how these elements function in the formation of an active transcription complex, we have cloned and sequenced the cDNA that encodes delta, a protein that binds to critical downstream promoter elements in the mouse ribosomal protein rpL30 and rpL32 genes. Our results revealed that the delta protein contains four C-terminal zinc fingers, which are essential for its DNA binding capability and a very unusual N-terminal domain that includes stretches of 11 consecutive negatively charged amino acids and 12 consecutive histidines. The sequence of the delta protein was found to be essentially identical to a concurrently cloned human transcription factor that acts both positively and negatively in the context of immunoglobulin enhancers and a viral promoter. Our structural modeling of this protein indicates properties that could endow it with exquisite functional versatility. Images PMID:1946404

  20. Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1).

    PubMed Central

    Sully, Gareth; Dean, Jonathan L E; Wait, Robin; Rawlinson, Lesley; Santalucia, Tomas; Saklatvala, Jeremy; Clark, Andrew R

    2004-01-01

    COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present. PMID:14594446

  1. Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene.

    PubMed Central

    Saatcioglu, F; Perry, D J; Pasco, D S; Fagan, J B

    1990-01-01

    Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known

  2. The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

    PubMed

    Kuang, Jian-Fei; Chen, Jian-Ye; Liu, Xun-Cheng; Han, Yan-Chao; Xiao, Yun-Yi; Shan, Wei; Tang, Yang; Wu, Ke-Qiang; He, Jun-Xian; Lu, Wang-Jin

    2017-04-01

    Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  3. Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis.

    PubMed

    Kagale, Sateesh; Links, Matthew G; Rozwadowski, Kevin

    2010-03-01

    The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

  4. Protein binding elements in the human beta-polymerase promoter.

    PubMed Central

    Englander, E W; Wilson, S H

    1990-01-01

    The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein. Images PMID:2315044

  5. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  6. Antibody binding loop insertions as diversity elements

    PubMed Central

    Kiss, Csaba; Fisher, Hugh; Pesavento, Emanuele; Dai, Minghua; Valero, Rosa; Ovecka, Milan; Nolan, Rhiannon; Phipps, M. Lisa; Velappan, Nileena; Chasteen, Leslie; Martinez, Jennifer S.; Waldo, Geoffrey S.; Pavlik, Peter; Bradbury, Andrew R.M.

    2006-01-01

    In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments. PMID:17023486

  7. Post-transcriptional effects and interactions between chronic mild stress and acute sleep deprivation: regulation of translation factor and cytoplasmic polyadenylation element-binding protein phosphorylation.

    PubMed

    Grønli, Janne; Dagestad, Grethe; Milde, Anne Marita; Murison, Robert; Bramham, Clive R

    2012-12-01

    Stress and restricted or disrupted sleep trigger adaptive responses in the brain at the level of gene transcription. We investigated the possible impact of chronic mild stress (CMS), acute sleep deprivation, and a combination of these in male rats on post-transcriptional mechanisms important for cognitive function and synaptic plasticity. Relationships between sleep architecture and translational regulators were also assessed. After four weeks of CMS, phosphorylation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor 2 (eEF2), was enhanced in the prefrontal cortex, but unchanged in the hippocampus and dentate gyrus. Sleep deprivation decreased phosphorylated eIF4E in the dentate gyrus. In contrast, eEF2 phosphorylation was elevated in all brain regions after sleep deprivation. Thus, CMS and sleep deprivation, when given alone, have distinct region-specific effects. Furthermore, the combined treatment revealed striking interactions with eEF2 phosphorylation in which sleep deprivation counteracts the effect of CMS cortically and CMS modulates the effects of sleep deprivation in the hippocampus proper. Although CMS exposure alone had no effect in the hippocampus, it inhibited the sleep deprivation-induced eIF4E phosphorylation, while inducing phosphorylation of a major regulatory RNA-binding protein, cytoplasmic polyadenylation element-binding protein (CPEB) in the combined treatment. CMS had no effect on plasma corticosterone, but led to disruption of sleep. Sleep quality and sleep quantity in non-stressed animals showed predictive changes in eIF4E and eEF2 phosphorylation cortically. Prior exposure to CMS abolishes this relationship. We conclude that CMS and acute sleep deprivation have interactive and brain region-specific effects on translational regulators of relevance to mechanisms of stress responsiveness and sleep homeostasis. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    SciTech Connect

    Wang, Feng-Wei; Wu, Xian-Rui; Liu, Wen-Ju; Liao, Yi-Ji; Lin, Sheng; Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin; Mai, Shi-Juan; Xie, Dan

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  9. The role of the glucose-sensing transcription factor carbohydrate-responsive element-binding protein pathway in termite queen fertility

    PubMed Central

    Sillam-Dussès, David; Hanus, Robert; Poulsen, Michael; Roy, Virginie; Favier, Maryline

    2016-01-01

    Termites are among the few animals that themselves can digest the most abundant organic polymer, cellulose, into glucose. In mice and Drosophila, glucose can activate genes via the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to induce glucose utilization and de novo lipogenesis. Here, we identify a termite orthologue of ChREBP and its downstream lipogenic targets, including acetyl-CoA carboxylase and fatty acid synthase. We show that all of these genes, including ChREBP, are upregulated in mature queens compared with kings, sterile workers and soldiers in eight different termite species. ChREBP is expressed in several tissues, including ovaries and fat bodies, and increases in expression in totipotent workers during their differentiation into neotenic mature queens. We further show that ChREBP is regulated by a carbohydrate diet in termite queens. Suppression of the lipogenic pathway by a pharmacological agent in queens elicits the same behavioural alterations in sterile workers as observed in queenless colonies, supporting that the ChREBP pathway partakes in the biosynthesis of semiochemicals that convey the signal of the presence of a fertile queen. Our results highlight ChREBP as a likely key factor for the regulation and signalling of queen fertility. PMID:27249798

  10. Isolation and characterization of dehydration-responsive element-binding factor 2C (MsDREB2C) from Malus sieversii Roem.

    PubMed

    Zhao, Kai; Shen, Xinjie; Yuan, Huazhao; Liu, Yun; Liao, Xiong; Wang, Qi; Liu, Linlin; Li, Fang; Li, Tianhong

    2013-09-01

    DREB2 (dehydration-responsive element-binding factor 2)-type transcription factors play a critical role in the stress-related regulation network in plants. In this study, we isolated and characterized a DREB2 homolog from Malus sieversii Roem., designated MsDREB2C (GenBank accession No. JQ790526). MsDREB2C localized to the nucleus and transactivated reporter genes in yeast strain YGR-2. Quantitative real-time PCR analysis demonstrated that MsDREB2C was constitutively expressed and significantly induced by drought, salt, cold, heat and ABA. Transgenic Arabidopsis plants overexpressing MsDREB2C exhibited increased root and leaf growth and proline levels, and reduced water loss and stomatal aperture. The transcriptional level of genes that function downstream of dehydration-responsive elements was greater in the transgenic Arabidopsis plants than in wild-type plants under control and abiotic stress conditions. Furthermore, constitutive expression of MsDREB2C repressed the expression of pathogenesis-related (PR) genes and the activity of peroxidase in transgenic plants under control and pathogenic conditions. As a result, transgenic plants were more tolerant to drought, heat and cold, but more sensitive to Pst DC3000 (Pseudomonas syringae pv . tomato DC3000) infection than control plants. β-Glucuronidase expression analysis of the MsDREB2C promoter in transgenic tobacco plants showed that MsDREB2C was mainly expressed in the vascular tissues and seeds. Deletion analysis identified the regulatory regions responsible for the plant's response to drought (-831 to -680), ABA (-831 to -680 and -335 to -148), salt (-831 to -335), cold (-1,317 to -831 and -335 to -148) and heat (-335 to -148).

  11. Chromatin landscape dictates HSF binding to target DNA elements.

    PubMed

    Guertin, Michael J; Lis, John T

    2010-09-09

    Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. We used the ChIP-seq assay to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in "active" chromatin is a primary determinant of whether HSF can bind following heat shock.

  12. The DNA replication-related element (DRE)/DRE-binding factor system is a transcriptional regulator of the Drosophila E2F gene.

    PubMed

    Sawado, T; Hirose, F; Takahashi, Y; Sasaki, T; Shinomiya, T; Sakaguchi, K; Matsukage, A; Yamaguchi, M

    1998-10-02

    Two mRNA species were observed for the Drosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. We found three tandemly aligned sequences, similar to the DNA replication-related element (DRE; 5'-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathione S-transferase fusion protein with the N-terminal fragment (1-125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. We have established transgenic lines of Drosophila in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity of dE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene.

  13. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species.

    PubMed

    Jaglo, K R; Kleff, S; Amundsen, K L; Zhang, X; Haake, V; Zhang, J Z; Deits, T; Thomashow, M F

    2001-11-01

    Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.

  14. Components of the Arabidopsis C-Repeat/Dehydration-Responsive Element Binding Factor Cold-Response Pathway Are Conserved in Brassica napus and Other Plant Species1

    PubMed Central

    Jaglo, Kirsten R.; Kleff, Susanne; Amundsen, Keenan L.; Zhang, Xin; Haake, Volker; Zhang, James Z.; Deits, Thomas; Thomashow, Michael F.

    2001-01-01

    Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate. PMID:11706173

  15. Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

    PubMed Central

    van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

    1992-01-01

    The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

  16. Association Study Between Metabolic Syndrome and rs8066560 Polymorphism in the Promoter Region of Sterol Regulatory Element-binding Transcription Factor 1 Gene in Iranian Children and Adolescents

    PubMed Central

    Miranzadeh-Mahabadi, Hajar; Emadi-Baygi, Modjtaba; Nikpour, Parvaneh; Kelishadi, Roya

    2016-01-01

    Background: Metabolic syndrome (MetS) is a prevalent disorder in pediatric age groups, described by a combination of genetic and environmental factors. Sterol regulatory element-binding transcription factor 1 (SREBF-1) induces the expression of a family of genes involved in fatty acid synthesis. Moreover, dysregulation of miR-33b, which is located within the intron 17 of the SREBF-1 gene, disrupts fatty acid oxidation and insulin signaling, thus leading to MetS. The aim of the present study was to investigate the association between SREBF-1 rs8066560 polymorphism and MetS in Iranian children and adolescents. Methods: This study includes 100 MetS and 100 normal individuals aged 9–19 years. Anthropological and biochemical indexes were measured. The -1099G > A polymorphism was genotyped by TaqMan real-time polymerase chain reaction. Results: Significant differences were observed in anthropometric measurements and lipid profiles between MetS and normal children. There were no differences in the genotype frequencies or allele distribution for -1099G > A polymorphism between MetS and control groups. High-density lipoprotein cholesterol levels were significantly higher in the MetS GG group than in the A allele carrier group. The genotype AA controls had significantly increased cholesterol and low-density lipoprotein cholesterol levels than AG genotypes. By logistic regression using different genetic models, no significant association was observed between SREBF-1 rs8066560 polymorphism and the risk of MetS. Conclusions: We conclude that the -1099G > A variant on SREBF-1 gene associated with serum lipid profiles, however, it may not be a major risk factor for the MetS in Iranian children and adolescents. PMID:27076879

  17. High glucose induces platelet-derived growth factor-C via carbohydrate response element-binding protein in glomerular mesangial cells.

    PubMed

    Kitsunai, Hiroya; Makino, Yuichi; Sakagami, Hidemitsu; Mizumoto, Katsutoshi; Yanagimachi, Tsuyoshi; Atageldiyeva, Kuralay; Takeda, Yasutaka; Fujita, Yukihiro; Abiko, Atsuko; Takiyama, Yumi; Haneda, Masakazu

    2016-03-01

    Persistent high concentration of glucose causes cellular stress and damage in diabetes via derangement of gene expressions. We previously reported high glucose activates hypoxia-inducible factor-1αand downstream gene expression in mesangial cells, leading to an extracellular matrix expansion in the glomeruli. A glucose-responsive transcription factor carbohydrate response element-binding protein (ChREBP) is a key mediator for such perturbation of gene regulation. To provide insight into glucose-mediated gene regulation in mesangial cells, we performed chromatin immunoprecipitation followed byDNAmicroarray analysis and identified platelet-derived growth factor-C (PDGF-C) as a novel target gene of ChREBP In streptozotocin-induced diabetic mice, glomerular cells showed a significant increase inPDGF-C expression; the ratio ofPDGF-C-positive cells to the total number glomerular cells demonstrated more than threefold increase when compared with control animals. In cultured human mesangial cells, high glucose enhanced expression ofPDGF-C protein by 1.9-fold. Knock-down of ChREBPabrogated this induction response. UpregulatedPDGF-C contributed to the production of typeIVand typeVIcollagen, possibly via an autocrine mechanism. Interestingly, urinaryPDGF-C levels in diabetic model mice were significantly elevated in a fashion similar to urinary albumin. Taken together, we hypothesize that a high glucose-mediated induction ofPDGF-C via ChREBPin mesangial cells contributes to the development of glomerular mesangial expansion in diabetes, which may provide a platform for novel predictive and therapeutic strategies for diabetic nephropathy.

  18. SlDREB2, a tomato dehydration-responsive element-binding 2 transcription factor, mediates salt stress tolerance in tomato and Arabidopsis.

    PubMed

    Hichri, Imène; Muhovski, Yordan; Clippe, André; Žižková, Eva; Dobrev, Petre I; Motyka, Vaclav; Lutts, Stanley

    2016-01-01

    To counter environmental cues, cultivated tomato (Solanum lycopersicum L.) has evolved adaptive mechanisms requiring regulation of downstream genes. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors regulate abiotic stresses responses in plants. Herein, we isolated a novel DREB2-type regulator involved in salinity response, named SlDREB2. Spatio-temporal expression profile together with investigation of its promoter activity indicated that SlDREB2 is expressed during early stages of seedling establishment and in various vegetative and reproductive organs of adult plants. SlDREB2 is up-regulated in roots and young leaves following exposure to NaCl, but is also induced by KCl and drought. Its overexpression in WT Arabidopsis and atdreb2a mutants improved seed germination and plant growth in presence of different osmotica. In tomato, SlDREB2 affected vegetative and reproductive organs development and the intronic sequence present in the 5' UTR drives its expression. Physiological, biochemical and transcriptomic analyses showed that SlDREB2 enhanced plant tolerance to salinity by improvement of K(+) /Na(+) ratio, and proline and polyamines biosynthesis. Exogenous hormonal treatments (abscisic acid, auxin and cytokinins) and analysis of WT and 35S::SlDREB2 tomatoes hormonal contents highlighted SlDREB2 involvement in abscisic acid biosynthesis/signalling. Altogether, our results provide an overview of SlDREB2 mode of action during early salt stress response. © 2015 John Wiley & Sons Ltd.

  19. Molecular characterization of cotton C-repeat/dehydration-responsive element binding factor genes that are involved in response to cold stress.

    PubMed

    Ma, Liu-Feng; Zhang, Jian-Min; Huang, Geng-Qing; Li, Yang; Li, Xue-Bao; Zheng, Yong

    2014-07-01

    Low temperature, drought and salinity are major abiotic stresses that influence survival, productivity and geographical distribution of many important crops across the globe. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important proteins involved in response to abiotic stresses in plants. In this study, twenty-one CBF genes were identified in cotton (Gossypium hirsutum) by bioinformatic approach. The twenty-one CBF genes (named as GhCBF1--GhCBF21) were characterized to encode proteins that share high similarity with those plant cold stress-related CBF proteins, which contain the classic AP2 domain of 58 amino acid residues. Phylogenetic analysis revealed that the isolated cotton CBF genes can be classified into 4 groups: GhCBF I, GhCBF II, GhCBF III and GhCBF IV. RT-PCR analysis indicated that GhCBF genes were up-regulated in cotton plants under cold stress. Furthermore, four GhCBF genes were up-regulated in cotton under salinity and drought treatments. Our data provided valuable information for further exploring the roles of the CBF genes in cotton development and in response to cold stress.

  20. A dehydration-responsive element binding (DREB) transcription factor from the succulent halophyte Salicornia brachiata enhances abiotic stress tolerance in transgenic tobacco.

    PubMed

    Gupta, Kapil; Jha, Bhavanath; Agarwal, Pradeep K

    2014-12-01

    Dehydration-responsive element binding (DREB) transcription factor (TF) plays a key role for abiotic stress tolerance in plants. Earlier, we have published the isolation and characterisation of an A-2-type SbDREB2A TF from an extreme halophyte Salicornia brachiata. The SbDREB2A protein lacks potential proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST) sequence which is known to act as signal peptide for protein degradation. In this study, SbDREB2A TF was over-expressed in tobacco plants without any modification in polypeptide sequence. Transgenic plants showed better seed germination and growth characteristics in both hyperionic and hyperosmotic stresses. Transgenic plants exhibited higher water content, membrane stability and less electrolyte leakage in stress conditions. The transgenic plants accumulated less Na(+) and higher K(+) than wildtype (WT) plants. The transgenic plants revealed higher chlorophyll content, water use efficiency (WUE) and net photosynthesis rate. Transgenics exhibited higher level of proline and low amount of MDA and H2O2 under stress conditions. The real-time PCR of transgenics showed higher expression of downstream heat shock genes (Hsp18, Hsp26 and Hsp70), TFs (AP2 domain containing TF, HSF2 and ZFP), signalling components (PLC3 and Ca (2+) /calmodulin) and dehydrins (ERD10B, ERD10D and LEA5) under different abiotic stress treatments.

  1. The gene transcription factor cyclic AMP-responsive element binding protein: role in positive and negative affective states of alcohol addiction.

    PubMed

    Pandey, Subhash C

    2004-10-01

    The gene transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding (CREB) protein is a nuclear protein that regulates synaptic plasticity via modulating the expression of several (cAMP)-inducible genes. Alcohol addiction is a complex psychiatric disorder and is characterized by a compulsive and uncontrolled pattern of alcohol drinking by an individual in spite of the adverse consequences of its abuse. Ethanol produces both euphoric (reward and reinforcing) and dysphoric (negative withdrawal reactions) effects and these are most likely involved in the initiation and maintenance of alcohol use and abuse. Several neurotransmitter systems in the brain might be involved in the effects of alcohol but the exact molecular mechanisms of both the positive and negative affective states of alcohol abuse are still unclear. Recent research in molecular neurosciences using animal models have identified the role of extended amygdaloid (shell structures of nucleus accumbens [NAc] and central and medial amygdaloid nuclei) CREB signaling in positive and negative affective states of alcohol drinking behaviors. This review article highlights the current findings on the role of nucleus accumbal and amygdaloid CREB signaling in behavioral consequences of alcohol use and abuse.

  2. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs.

    PubMed

    Chen, J; Yang, X J; Xia, D; Chen, J; Wegner, J; Jiang, Z; Zhao, R Q

    2008-01-01

    Two experiments were performed to elucidate the role of sterol regulatory element binding transcription factor 1 (SREBF1) in i.m. fat (IMF) deposition in pigs. In Exp. 1, LM samples were removed from 4 male and 4 female Erhualian piglets at 3, 20, and 45 d of age, and SREBF1 mRNA expression level and IMF content were measured. Intramuscular fat content and expression of SREBF1 mRNA was greater (P < 0.05) in females than males at all 3 stages of age, providing initial evidence that the level of SREBF1 mRNA expression is related to IMF deposition in muscle of suckling pigs. Additionally, in Exp. 2 there was a positive correlation between the SREBF1 mRNA level and IMF content (r = 0.67, P < 0.01) in 100 Sutai finishing pigs, a synthetic line produced by crossing Erhualian and Duroc pigs. Single-strand conformation polymorphism (SSCP) analysis of the reverse transcription PCR products of the SREBF1 gene revealed 3 genotypes in Sutai pigs with frequencies of 50% for AA, 36% for AB, and 14% for BB, respectively. Both SREBF1 mRNA level and IMF content in muscle were greater (P < 0.05) in AB and BB animals than in AA animals, whereas no difference in backfat thickness was observed among the 3 genotypes. Sequencing analysis identified 2 SNP at T1006C and C1033T within the open reading frame of the SREBF1 gene (NM_214157). Although both are silent mutations, they affected the secondary structure of SREBF1 mRNA. These results suggest that SREBF1 might play an important role in regulation of muscle fat deposition during postnatal growth of pigs. The SNP identified in the SREBF1 gene suggest that it could be used as a genetic marker to improve IMF content in pigs.

  3. Heterology Expression of the Arabidopsis C-Repeat/Dehydration Response Element Binding Factor 1 Gene Confers Elevated Tolerance to Chilling and Oxidative Stresses in Transgenic Tomato1

    PubMed Central

    Hsieh, Tsai-Hung; Lee, Jent-Turn; Yang, Pei-Tzu; Chiu, Li-Hui; Charng, Yee-yung; Wang, Yu-Chie; Chan, Ming-Tsair

    2002-01-01

    In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T1 and T2 plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA3 (gibberellic acid) treatment. More importantly, GA3-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H2O2 in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress. PMID:12114563

  4. FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c.

    PubMed

    Deng, Xiong; Zhang, Wenwei; O-Sullivan, InSug; Williams, J Bradley; Dong, Qingming; Park, Edwards A; Raghow, Rajendra; Unterman, Terry G; Elam, Marshall B

    2012-06-08

    Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

  5. Anoxic induction of a sarcoma virus-related VL30 retrotransposon is mediated by a cis-acting element which binds hypoxia-inducible factor 1 and an anoxia-inducible factor.

    PubMed Central

    Estes, S D; Stoler, D L; Anderson, G R

    1995-01-01

    Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with metabolic adaptation and increasing oxygen delivery. In contrast, responses distinct from those elicited by hypoxia are induced in anoxic fibroblasts; this includes activation of a set of VL30 elements. The responses seen in anoxically cultured fibroblasts are expressed physiologically in vivo during the anaerobic phase of wound healing. A fundamental question is whether transcriptional regulatory pathways utilized during anoxia are distinct from those already characterized for hypoxic cells. We report here the isolation of a 14-bp sequence within a VL30 retrotransposon promoter which mediates its anoxia responsiveness. Analyses of the protein complexes binding to this sequence demonstrated the presence of two distinct inducible DNA binding activities. The first is present in both hypoxic and anoxic fibroblasts and is indistinguishable from hypoxia-inducible factor 1. The second activity, which is present only in anoxic fibroblasts, is a previously uncharacterized heterodimeric DNA binding activity that appears to arise via posttranslational modification of an existing complex found in aerobic cells. These results indicate that the strong VL30 transcriptional induction seen with anoxia occurs through a mechanism specific to anoxia. PMID:7666534

  6. Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) replication and transcription factor activates the K9 (vIRF) gene through two distinct cis elements by a non-DNA-binding mechanism.

    PubMed

    Ueda, Keiji; Ishikawa, Kayo; Nishimura, Ken; Sakakibara, Shuhei; Do, Eunju; Yamanishi, Koichi

    2002-12-01

    The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent cis-acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5'-TCTGGGACAGTC-3' in responsive element (RE) I-2B and the sequence 5'-GTACTTAAAATA-3' in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in Escherichia coli, however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique cis elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.

  7. Minocycline upregulates cyclic AMP response element binding protein and brain-derived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits.

    PubMed

    Zhao, Yu; Xiao, Ming; He, Wenbo; Cai, Zhiyou

    2015-01-01

    The cAMP response element binding protein (CREB) plays an important role in the mechanism of cognitive impairment and is also pivotal in the switch from short-term to long-term memory. Brain-derived neurotrophic factor (BDNF) seems a promising avenue in the treatment of cerebral ischemia injury since this neurotrophin could stimulate structural plasticity and repair cognitive impairment. Several findings have displayed that the dysregulation of the CREB-BDNF cascade has been involved in cognitive impairment. The aim of this study was to investigate the effect of cerebral ischemia on learning and memory as well as on the levels of CREB, phosphorylated CREB (pCREB), and BDNF, and to determine the effect of minocycline on CREB, pCREB, BDNF, and behavioral functional recovery after cerebral ischemia. The animal model was established by permanent bilateral occlusion of both common carotid arteries. Behavior was evaluated 5 days before decapitation with Morris water maze and open-field task. Four days after permanent bilateral occlusion of both common carotid arteries, minocycline was administered by douche via the stomach for 4 weeks. CREB and pCREB were examined by Western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry. BDNF was measured by immunohistochemistry and Western blotting. The model rats after minocycline treatment swam shorter distances than control rats before finding the platform (P=0.0007). The number of times the platform position was crossed for sham-operation rats was more than that of the model groups in the corresponding platform location (P=0.0021). The number of times the platform position was crossed for minocycline treatment animals was significantly increased compared to the model groups in the corresponding platform position (P=0.0016). CREB, pCREB, and BDNF were downregulated after permanent bilateral occlusion of both common carotid arteries in the model group. Minocycline increased the expression of CREB

  8. Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast.

    PubMed Central

    Dorsman, J C; van Heeswijk, W C; Grivell, L A

    1988-01-01

    Two abundant factors, GFI and GFII which interact with the 5' flanking regions of nuclear genes coding for proteins of the mitochondrial respiratory chain have been identified. In one case (subunit VIII of QH2: cytochrome c oxidoreductase) the binding sites for both factors overlap completely and their binding is mutually exclusive. For the other 5' regions tested the GFI and GFII binding sites do not coincide. Interestingly, binding sites for GFI and GFII are also present in or at the 3' ends of the coding regions of two genes of the PHO gene family and in DNA elements important for optimal ARS and CEN function respectively. The sites recognized by GFI conform to the consensus RTCRNNNNNNACGNR, while those recognized by GFII contain the element RTCACGTG. We speculate that GFI and GFII may play a role in different cellular processes, dependent on the context of their binding sites and that one of these processes may be the coordination of the expression of genes involved in mitochondrial biogenesis with the progress of the cell cycle. Images PMID:3045755

  9. Soy isoflavones increase quinone reductase in hepa-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element.

    PubMed

    Froyen, Erik B; Steinberg, Francene M

    2011-09-01

    Soy protein and isoflavones (genistein and daidzein) have been demonstrated to increase quinone reductase (QR) activity, protein, and mRNA in animal and cell culture models. However, their mechanism of action has not been completely characterized. Additionally, it has not been determined if equol, a daidzein metabolite, can modulate QR activity and expression. Estrogen receptor beta (ERβ) is thought to be involved in stimulating QR gene transcription by anti-estrogens and phytoestrogens, along with nuclear factor erythroid 2-related factor 2 (Nrf2). This study tested the hypothesis that genistein, daidzein and equol increase quinone reductase activity, protein and mRNA via ERβ and Nrf2 binding to the QR antioxidant response element (ARE). QR expression and activity were determined using TaqMan polymerase chain reaction, protein immunoblots and activity assays. Molecular events were investigated using luciferase reporter gene assays and chromatin immunoprecipitation (ChIP). Hepa-1c1c7 cells were treated with control [0.1% (v:v) dimethyl sulfoxide (DMSO)]; 1 μmol/L β-naphthoflavone (positive control); 5 μmol/L resveratrol (ChIP positive control for ERβ binding) and 1, 5 and 25 μmol/L genistein, daidzein or equol. Treatment durations were 1 h (ChIP), 24 h (mRNA and luciferase assays) and 24 and 48 h (protein and activity). Genistein, daidzein and equol increased QR activity, protein and mRNA, with daidzein and equol having more of an impact at physiologic concentrations (1 and 5 μmol/L) compared to genistein. Furthermore, the study results demonstrate that genistein, daidzein and equol interact with the QR ARE and that daidzein and equol act via both ERβ and Nrf2 binding strongly to the QR ARE. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. 170 SUPPLEMENT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE DURING IN VITRO MATURATION ACTIVATES SMAD2 AND cAMP RESPONSIVE ELEMENT BINDING PROTEIN SIGNALING.

    PubMed

    Yoon, J D; Lee, E; Hyun, S-H

    2016-01-01

    -related genes HAS2, PTX3, and TNFAIP6 mRNA expression levels after IVM (4 times). To determine effect of GDF8 treatment during IVM, GDF8 downstream effector and oocyte ovulation-related protein expression and activation levels were analysed in CC after IVM by Western blotting. The 1 and 10ngmL(-1) treatment groups showed significantly increased phosphorylated (P)-SMAD2 (1.25 and 1.23 times increased compared with the control) and cyclic adenosine monophosphate responsive element binding protein (CREB; 1.31 and 1.32 times increased compared with the control) activation levels (4 times). In conclusion, supplementation of 10ngmL(-1) of GDF8 during IVM effectively increased the oocytes cytoplasmic maturation by reducing of intracellular ROS, and it seems correlated with significantly increased P-SMAD2, which is possibly related with induction of the cumulus cell expansion related genes expression and P-CREB while process of IVM.

  11. The long non-coding RNA GAS5 regulates transforming growth factor β (TGF-β)-induced smooth muscle cell differentiation via RNA Smad-binding elements.

    PubMed

    Tang, Rui; Zhang, Gui; Wang, Yung-Chun; Mei, Xiaohan; Chen, Shi-You

    2017-08-25

    Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-β signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-β/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-β-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-β/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-β/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-β-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-β/Smad3 signaling during SMC differentiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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.

  13. The Basic Leucine Zipper Transcription Factor ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 Is an Important Transcriptional Regulator of Abscisic Acid-Dependent Grape Berry Ripening Processes1[W][OPEN

    PubMed Central

    Nicolas, Philippe; Lecourieux, David; Kappel, Christian; Cluzet, Stéphanie; Cramer, Grant; Delrot, Serge; Lecourieux, Fatma

    2014-01-01

    In grape (Vitis vinifera), abscisic acid (ABA) accumulates during fruit ripening and is thought to play a pivotal role in this process, but the molecular basis of this control is poorly understood. This work characterizes ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTOR2 (VvABF2), a grape basic leucine zipper transcription factor belonging to a phylogenetic subgroup previously shown to be involved in ABA and abiotic stress signaling in other plant species. VvABF2 transcripts mainly accumulated in the berry, from the onset of ripening to the harvesting stage, and were up-regulated by ABA. Microarray analysis of transgenic grape cells overexpressing VvABF2 showed that this transcription factor up-regulates and/or modifies existing networks related to ABA responses. In addition, grape cells overexpressing VvABF2 exhibited enhanced responses to ABA treatment compared with control cells. Among the VvABF2-mediated responses highlighted in this study, the synthesis of phenolic compounds and cell wall softening were the most strongly affected. VvABF2 overexpression strongly increased the accumulation of stilbenes that play a role in plant defense and human health (resveratrol and piceid). In addition, the firmness of fruits from tomato (Solanum lycopersicum) plants overexpressing VvABF2 was strongly reduced. These data indicate that VvABF2 is an important transcriptional regulator of ABA-dependent grape berry ripening. PMID:24276949

  14. Functional Analysis of Regulatory Elements in the Gene Promoter for an Abscission-Specific Cellulase from Bean and Isolation, Expression, and Binding Affinity of Three TGA-Type Basic Leucine Zipper Transcription Factors

    PubMed Central

    Tucker, Mark L.; Whitelaw, Catherine A.; Lyssenko, Nicholas N.; Nath, Pravendra

    2002-01-01

    Site-directed mutagenesis was used to identify cis-acting elements that control hormonal and abscission-specific expression of the bean (Phaseolus vulgaris) abscission cellulase (BAC) promoter. Auxin inhibition of BAC promoter expression is at least in part controlled by a negatively regulated element and ethylene induction by a positively regulated element. One of a series of 15 different 10-bp mutations created in a 2.9-kb BAC promoter reduced reporter gene expression by 60%. The native sequence for this 10-bp mutation includes a TGA-type basic leucine zipper (bZIP) motif. Tandem ligation of three 18-bp BAC elements (Z-BAC), which includes the bZIP motif to a minimal −50 35S cauliflower mosaic virus promoter, enhanced expression in abscission zones (AZs) 13-fold over that of the minimal promoter alone. The native forward orientation of the Z-BAC elements was essential for high expression levels. Expression of the Z-BAC minimal construct was 3-fold greater in AZ than stems when compared with the expression levels of an internal control with an enhanced 35S cauliflower mosaic virus promoter. Polymerase chain reaction was used to identify three TGA-type bZIP transcription factors in an AZ cDNA library. One of these factors was of the class I type and two of the class II type. RNA-blot analysis was completed for these genes and electrophoretic mobility shift assays used to confirm their binding to the Z-BAC element. Electrophoretic mobility shift assay-binding affinity was greatest for the class I TGA-type bZIP factor. The results indicate a complex interaction of negative and positive regulating transcription factors that control BAC gene expression. PMID:12428013

  15. A nuclear factor that binds purine-rich, single-stranded oligonucleotides derived from S1-sensitive elements upstream of the CFTR gene and the MUC1 gene.

    PubMed Central

    Hollingsworth, M A; Closken, C; Harris, A; McDonald, C D; Pahwa, G S; Maher, L J

    1994-01-01

    We have identified two regions of non-random purine/pyrimidine strand asymmetry that were nearly identical in sequence in the 5' flanking (promoter) regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene and the human MUC1 gene. These regions contain perfect mirror repeat elements, a sequence motif previously found to be associated with the formation of H-DNA conformations. In this report we demonstrate that a single-stranded non-B DNA conformation exists at low pH in supercoiled plasmids containing the similar mirror repeat elements, and that S1 nuclease digestion maps the single-stranded region to the position of the mirror repeats. In addition, we identify a nuclear protein of approximately 27 kD that binds to single-stranded oligonucleotides corresponding to the purine-rich strand of this region, but not to the pyrimidine-rich strands or to double-stranded oligonucleotides with corresponding purine/pyrimidine strand asymmetry. Images PMID:7513081

  16. Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.

    PubMed

    Balmer, L A; Beveridge, D J; Jazayeri, J A; Thomson, A M; Walker, C E; Leedman, P J

    2001-03-01

    The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU

  17. Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor.

    PubMed

    Desjardins, Geneviève; Meeker, Charles A; Bhachech, Niraja; Currie, Simon L; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2014-07-29

    The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.

  18. Suppression of granulocyte-macrophage colony-stimulating factor expression by glucocorticoids involves inhibition of enhancer function by the glucocorticoid receptor binding to composite NF-AT/activator protein-1 elements.

    PubMed

    Smith, P J; Cousins, D J; Jee, Y K; Staynov, D Z; Lee, T H; Lavender, P

    2001-09-01

    Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.

  19. Synthetic heparin-binding growth factor analogs

    DOEpatents

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

    2007-01-23

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

  20. Induction by interleukin-6 of interferon regulatory factor 1 (IRF-1) gene expression through the palindromic interferon response element pIRE and cell type-dependent control of IRF-1 binding to DNA.

    PubMed

    Harroch, S; Revel, M; Chebath, J

    1994-04-15

    The effects of interleukin-6 (IL-6) on interferon regulatory factor 1 (IRF-1) gene expression were studied in B-hybridoma B9 cells which are growth-stimulated by IL-6 and breast carcinoma T47D cells which are growth-inhibited. IL-6 induced the production of IRF-1 mRNA and protein in both cell types, but IRF-1 binding activity to its target DNA sequence was induced only in T47D cells. With B9 cells, there was no IRF-1 binding but instead strong constitutive binding of the IRF-2 repressor, indicating that binding of IRF-1 to DNA is an important regulatory step. The IRF-1 gene promoter element, palindromic IFN-response element (pIRE), was found to respond to IL-6 with high efficiency as compared with IFN-gamma or IFN-beta. On this palindromic TTC...GAA sequence, two protein complexes (pIRE-a and pIRE-b) were induced within minutes by IL-6. pIRE-b is similar to the main complex induced by IFN-gamma and contains the Stat91 protein. pIRE-a predominantly induced by IL-6 is a slowly migrating complex which does not contain Stat91 and has low affinity for IFN-gamma activated sequence (GAS)-type sequences. Comparison of the relative effects of IL-6 and IFN-gamma shows that pIRE enhancers are differently regulated than GAS elements. Distinct transcription complexes, forming in ratios dependent on the inducer, help explain how various cytokines sharing effects through Stat91 on related enhancers can produce specific patterns of gene expression. Activation of the pIRE-a factors defines a novel transcriptional activity of IL-6 in epithelial and lymphoid cells.

  1. Induction by interleukin-6 of interferon regulatory factor 1 (IRF-1) gene expression through the palindromic interferon response element pIRE and cell type-dependent control of IRF-1 binding to DNA.

    PubMed Central

    Harroch, S; Revel, M; Chebath, J

    1994-01-01

    The effects of interleukin-6 (IL-6) on interferon regulatory factor 1 (IRF-1) gene expression were studied in B-hybridoma B9 cells which are growth-stimulated by IL-6 and breast carcinoma T47D cells which are growth-inhibited. IL-6 induced the production of IRF-1 mRNA and protein in both cell types, but IRF-1 binding activity to its target DNA sequence was induced only in T47D cells. With B9 cells, there was no IRF-1 binding but instead strong constitutive binding of the IRF-2 repressor, indicating that binding of IRF-1 to DNA is an important regulatory step. The IRF-1 gene promoter element, palindromic IFN-response element (pIRE), was found to respond to IL-6 with high efficiency as compared with IFN-gamma or IFN-beta. On this palindromic TTC...GAA sequence, two protein complexes (pIRE-a and pIRE-b) were induced within minutes by IL-6. pIRE-b is similar to the main complex induced by IFN-gamma and contains the Stat91 protein. pIRE-a predominantly induced by IL-6 is a slowly migrating complex which does not contain Stat91 and has low affinity for IFN-gamma activated sequence (GAS)-type sequences. Comparison of the relative effects of IL-6 and IFN-gamma shows that pIRE enhancers are differently regulated than GAS elements. Distinct transcription complexes, forming in ratios dependent on the inducer, help explain how various cytokines sharing effects through Stat91 on related enhancers can produce specific patterns of gene expression. Activation of the pIRE-a factors defines a novel transcriptional activity of IL-6 in epithelial and lymphoid cells. Images PMID:8168491

  2. Mutant copper-zinc superoxide dismutase associated with amyotrophic lateral sclerosis binds to adenine/uridine-rich stability elements in the vascular endothelial growth factor 3′-untranslated region

    PubMed Central

    Li, Xuelin; Lu, Liang; Bush, Donald J.; Zhang, Xiaowen; Zheng, Lei; Suswam, Esther A.; King, Peter H.

    2009-01-01

    Vascular endothelial growth factor (VEGF) is a neurotrophic factor essential for maintenance of motor neurons. Loss of this factor produces a phenotype similar to amyotrophic lateral sclerosis (ALS). We recently showed that ALS-producing mutations of Cu/Zn-superoxide dismutase (SOD1) disrupt post-transcriptional regulation of VEGF mRNA, leading to significant loss of expression. Mutant SOD1 was present in the ribonucleoprotein complex associated with adenine/uridine-rich elements (ARE) of the VEGF 3′-untranslated region (UTR). Here, we show by electrophoretic mobility shift assay that mutant SOD1 bound directly to the VEGF 3′-UTR with a predilection for AREs similar to the RNA stabilizer HuR. SOD1 mutants A4V and G37R showed higher affinity for the ARE than L38V or G93A. Wild-type SOD1 bound very weakly with an apparent Kd 11- to 72-fold higher than mutant forms. Mutant SOD1 showed an additional lower shift with VEGF ARE that was accentuated in the metal-free state. A similar pattern of binding was observed with AREs of tumor necrosis factor-α and interleukin-8, except only a single shift predominated. Using an ELISA-based assay, we demonstrated that mutant SOD1 competes with HuR and neuronal HuC for VEGF 3′-UTR binding. To define potential RNA-binding domains, we truncated G37R, G93A and wild-type SOD1 and found that peptides from the N-terminal portion of the protein that included amino acids 32-49 could recapitulate the binding pattern of full-length protein. Thus, the strong RNA-binding affinity conferred by ALS-associated mutations of SOD1 may contribute to the post-transcriptional dysregulation of VEGF mRNA. PMID:19196430

  3. Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins1[W

    PubMed Central

    Vom Endt, Débora; Soares e Silva, Marina; Kijne, Jan W.; Pasquali, Giancarlo; Memelink, Johan

    2007-01-01

    Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate. PMID:17496112

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

    PubMed

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

    2012-01-01

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

  5. Synthetic heparin-binding factor analogs

    DOEpatents

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

    2010-04-20

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

  6. c-Jun enhancement of cyclic adenosine 3',5'-monophosphate response element-dependent transcription induced by transforming growth factor-beta is independent of c-Jun binding to DNA.

    PubMed

    Hu, P P; Harvat, B L; Hook, S S; Shen, X; Wang, X F; Means, A R

    1999-12-01

    Transforming growth factor-beta (TGFbeta) enhances transcription from reporter genes regulated by a single consensus cAMP-response element (CRE) upon transfection into the immortalized human keratinocyte cell line, HaCaT. Whereas both CRE-binding protein (CREB) and c-Jun present in extracts of unstimulated cells can complex with a CRE in gel-shift experiments, TGFbeta treatment increases the amount of c-Jun found in the complex. Overexpression of c-Jun is sufficient to increase CRE and GAL4-CREB-dependent transcription and mimics the stimulatory effects of TGFbeta on transcription from either reporter gene. Surprisingly, although a portion of CREB in unstimulated cells is phosphorylated on the activating serine residue, Ser-133, this level of phospho-CREB is not altered by TGFbeta treatment. In fact, the CREB-dependent transcriptional effects of TGFbeta or c-Jun do not require phosphorylation of Ser-133, although CREB-binding protein (CBP) is required as evidenced by the observation that the adenoviral oncoprotein E1A can block the effects of both agents. c-Jun enhancement of CRE or GAL4-CREB-dependent transcription neither requires the DNA-binding nor N-terminal domains of c-Jun. Collectively, these results are consistent with a model in which signaling pathways initiated by TGFbeta can stimulate CREB-dependent transcription by increasing the cellular concentration of c-Jun, which participates in activation of the CBP-containing transcription complex.

  7. Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

    PubMed

    Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

    2014-01-01

    This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

  8. The Arabidopsis ETHYLENE RESPONSE FACTOR1 Regulates Abiotic Stress-Responsive Gene Expression by Binding to Different cis-Acting Elements in Response to Different Stress Signals1[W][OA

    PubMed Central

    Cheng, Mei-Chun; Liao, Po-Ming; Kuo, Wei-Wen; Lin, Tsan-Piao

    2013-01-01

    ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals. PMID:23719892

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

    PubMed Central

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

    2014-01-01

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

  10. Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism.

    PubMed

    Ferat-Osorio, Eduardo; Sánchez-Anaya, Aldair; Gutiérrez-Mendoza, Mireille; Boscó-Gárate, Ilka; Wong-Baeza, Isabel; Pastelin-Palacios, Rodolfo; Pedraza-Alva, Gustavo; Bonifaz, Laura C; Cortés-Reynosa, Pedro; Pérez-Salazar, Eduardo; Arriaga-Pizano, Lourdes; López-Macías, Constantino; Rosenstein, Yvonne; Isibasi, Armando

    2014-01-01

    Heat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70. Human peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data. The addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter. Extracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response.

  11. Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism

    PubMed Central

    2014-01-01

    Background Heat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70. Methods Human peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data. Results The addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter. Conclusion Extracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response. PMID:25053922

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

    Kwon, H; Green, M R

    1994-12-02

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

  14. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    SciTech Connect

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  15. Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein 1 in hot pepper (Capsicum annuum L. cv. Pukang).

    PubMed

    Hong, Jong-Pil; Kim, Woo Taek

    2005-04-01

    Through the use of subtractive hybridization analysis, we have identified 14 partial cDNA clones (pCa-DSRs) that are rapidly induced by dehydration in hot pepper (Capsicum annuum L.) roots. The predicted proteins encoded by Ca-DSRs are putatively involved in processes as diverse as primary and secondary metabolism, protein degradation, and stress responses, indicating the complexity of cellular responses to water deficit in hot pepper roots. Particularly, we investigated the detailed structural properties and expression profiles of Ca-DSR2 (Ca-DREBLP1: dehydration-responsive element binding-factor-like protein 1) encoding a protein that contains a single ERF/AP2 DNA-binding domain. Based on the conserved 14th valine and 19th glutamic acid residues in the ERF/AP2 domain, a basic amino acid stretch (PKKPAGRKKFR) near its N-terminal region, and DSAW signature sequence at the end of its ERF/AP2 domain, Ca-DREBLP1 was classified as a member of a DREB1-type subfamily. Gel retardation assays revealed that Ca-DREBLP1 was able to form a specific complex with the DRE/CRT motif, but not with the GCC box. When fused to the GAL4 DNA-binding domain, the Ca-DREBLP1(190-215) mutant could effectively function as a trans-activator in yeast. This suggests that the extreme C-terminal region plays an essential role in transcription activation. In hot pepper plants, Ca-DREBLP1 was rapidly induced by dehydration, high salinity and, to a lesser extent, mechanical wounding, but not by cold stress. Thus, although the structural features of Ca-DREBLP1 resemble those of the DREB1-type proteins of Arabidopsis thaliana and rice plants, its induction patterns are reminiscent of the DREB2-type proteins, indicating that Ca-DREBLP1 is a novel class DREB subfamily in hot pepper.

  16. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

    USDA-ARS?s Scientific Manuscript database

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  17. Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

    PubMed

    Dhar, M; Mascareno, E M; Siddiqui, M A

    1997-07-18

    The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

  18. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  19. Role of Hypoxia-Inducible Factor 1, α Subunit and cAMP-Response Element Binding Protein 1 in Synergistic Release of Interleukin 8 by Prostaglandin E2 and Nickel in Lung Fibroblasts

    PubMed Central

    Fabisiak, James P.

    2013-01-01

    Numerous epidemiological studies have linked exposure to particulate matter (PM) air pollution with acute respiratory infection and chronic respiratory and cardiovascular diseases. We have previously shown that soluble nickel (Ni), a common component of PM, alters the release of CXC chemokines from cultured human lung fibroblasts (HLF) in response to microbial stimuli via a pathway dependent on disrupted prostaglandin (PG)E2 signaling. The current study sought to identify the molecular events underlying Ni-induced alterations in PGE2 signaling and its effects on IL-8 production. PGE2 synergistically enhances Ni-induced IL-8 release from HLF in a concentration-dependent manner. The effects of PGE2 were mimicked by butaprost and PGE1-alcohol and inhibited with antagonists AH6809 and L-161,982, indicating PGE2 signals via PGE2 receptors 2 and 4. PGE2 and forskolin stimulated cAMP, but it was only in the presence of Ni-induced hypoxia-inducible factor 1, α subunit (HIF1A) that these agents stimulated IL-8 release. The Ni-induced HIF1A DNA binding was enhanced by PGE2 and mediated, in part, by activation of p38 MAPK. Negation of cAMP-response element binding protein 1 or HIF1A using short interfering RNA blocked the synergistic interactions between Ni and PGE2. The results of the current study provide novel information on the ability of atmospheric hypoxia-mimetic metals to disrupt the release of immune-modulating chemokines by HLF in response to PGE2. Moreover, in the presence of HIF1A, cAMP-mediated signaling pathways may be altered to exacerbate inflammatory-like processes in lung tissue, imparting a susceptibility of PM-exposed populations to adverse respiratory health effects. PMID:23526216

  20. Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus.

    PubMed

    Hattiangady, Bharathi; Rao, Muddanna S; Shetty, Geetha A; Shetty, Ashok K

    2005-10-01

    The hippocampus is very susceptible to aging. Severely diminished dentate neurogenesis at middle age is one of the most conspicuous early changes in the aging hippocampus, which is likely linked to an early decline in the concentration of neurotrophic factors and signaling proteins that influence neurogenesis. We analyzed three proteins that are well-known to promote dentate neurogenesis and learning and memory function in the dentate gyrus and the hippocampal CA1 and CA3 subfields of young, middle-aged and aged F344 rats. These include the brain-derived neurotrophic factor (BDNF), the transcription factor phosphorylated cyclic AMP response element binding protein (p-CREB) and the neuropeptide neuropeptide Y (NPY). The BDNF was analyzed via ELISA and BDNF immunohistochemistry, the p-CREB through densitometric analysis of p-CREB immunopositive cells, and the NPY via stereological counting of NPY-immunopositive interneurons. We provide new evidence that the BDNF concentration, the p-CREB immunoreactivity and the number of NPY immunopositive interneurons decline considerably by middle age in both dentate gyrus and CA1 and CA3 subfields of the hippocampus. However, both BDNF concentration and NPY immunopositive interneuron numbers exhibit no significant decrease between middle age and old age. In contrast, the p-CREB immunoreactivity diminishes further during this period, which is also associated with reduced BDNF immunoreaction within the soma of dentate granule cells and hippocampal pyramidal neurons. Collectively, these results suggest that severely dampened dentate neurogenesis observed at middle age is linked at least partially to reduced concentrations of BDNF, p-CREB and NPY, as each of these proteins is a positive regulator of dentate neurogenesis. Dramatically diminished CREB phosphorylation (and persistently reduced levels of BDNF and NPY) at old age may underlie the learning and memory impairments observed during senescence.

  1. Salicylic-Acid-Induced Chilling- and Oxidative-Stress Tolerance in Relation to Gibberellin Homeostasis, C-Repeat/Dehydration-Responsive Element Binding Factor Pathway, and Antioxidant Enzyme Systems in Cold-Stored Tomato Fruit.

    PubMed

    Ding, Yang; Zhao, Jinhong; Nie, Ying; Fan, Bei; Wu, Shujuan; Zhang, Yu; Sheng, Jiping; Shen, Lin; Zhao, Ruirui; Tang, Xuanming

    2016-11-02

    Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, C-repeat/dehydration-responsive element binding factor (CBF) pathway, and antioxidant enzyme systems linked to chilling- and oxidative-stress tolerance in tomato fruit were investigated. Mature green tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1 mM SA solution for 15 min before storage at 4 °C for 28 days. In comparison to 0 or 0.5 mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1) expression was followed by gibberellic acid (GA3) surge and DELLA protein degradation. CBF1 participated in the SA-modulated tolerance and stimulated the expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced activities of antioxidant enzymes and, thus, reduced reactive oxygen species accumulation. Our findings suggest that SA might protect tomato fruit from CI and oxidative damage through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme activities.

  2. The Transacting Factor CBF-A/Hnrnpab Binds to the A2RE/RTS Element of Protamine 2 mRNA and Contributes to Its Translational Regulation during Mouse Spermatogenesis

    PubMed Central

    Fukuda, Nanaho; Fukuda, Tomoyuki; Sinnamon, John; Hernandez-Hernandez, Abrahan; Izadi, Manizheh; Raju, Chandrasekhar S.; Czaplinski, Kevin; Percipalle, Piergiorgio

    2013-01-01

    During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3′ UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5′cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs. PMID:24146628

  3. Transcription factor binding energy vs. biological function

    NASA Astrophysics Data System (ADS)

    Djordjevic, M.; Grotewold, E.

    2007-03-01

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

  4. [Blockade of NMDA receptor enhances corticosterone-induced downregulation of brain-derived neurotrophic factor gene expression in the rat hippocampus through cAMP response element binding protein pathway].

    PubMed

    Feng, Hao; Lu, Li-Min; Huang, Ying; Zhu, Yi-Chun; Yao, Tai

    2005-10-25

    High concentration of corticosterone leads to morphological and functional impairments in hippocampus, ranging from a reversible atrophy of pyramidal CA3 apical dendrites to the impairment of long-term potentiation (LTP) and hippocampus-dependent learning and memory. Glutamate and N-methyl-D-aspartate (NMDA) receptor play an important role in this effect. Because of the importance of brain-derived neurotrophic factor (BDNF) in the functions of the hippocampal neurons, alteration of the expression of BDNF is thought to be involved in the corticosterone effect on the hippocampus. To determine whether change in BDNF in the hippocampus is involved in the corticosterone effect, we injected corticosterone (2 mg/kg, s.c.) to Sprague-Dawley rats and measured the mRNA, proBDNF and mature BDNF protein levels in the hippocampus. We also measured the phosphorylation level of the transcription factor cAMP response element binding protein (CREB). Furthermore, we intraperitoneally injected NMDA receptor antagonist MK801 (0.1 mg/kg) 30 min before corticosterone administration to investigate whether and how MK801 affected the regulation of BDNF gene expression by corticosterone. Our results showed that 3 h after single s.c. injection of corticsterone, the expression of BDNF mRNA, proBDNF and mature BDNF protein decreased significantly (P<0.01). MK801 promoted the downregulation of BDNF gene expression in the rat hippocampus by corticosterone. We also found that either applying corticosterone or co-applying corticosterone with MK801 downregulated the phosphoration level of CREB, the latter (corticosterone plus MK801) being more effective (P<0.05). Taken together, our results indicate that corticosterone downregulates BDNF gene expression in the rat hippocampus through CREB pathway and that blockade of NMDA receptor enhances this effect of corticosterone in reducing BDNF expression.

  5. Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts.

    PubMed Central

    Imai, S; Fujino, T; Nishibayashi, S; Manabe, T; Takano, T

    1994-01-01

    Dramatic changes occur in expression of the type I collagenase gene during the process of immortalization in simian virus 40 large T antigen-transformed human fibroblasts (S. Imai and T. Takano, Biochem. Biophys. Res. Commun. 189:148-153, 1992). From transient transfection assays, it was determined that these changes involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The profiles of binding of an activator, Proserpine, to the enhancer ISE1 were similar in the extracts of young, senescent preimmortalized and immortalized cells. ISE2 contained both negative and positive regulatory elements located adjacent to each other. The positive regulatory element consisted of a tandem array of putative Ets family- and AP-1-binding sites. An activator, Pluto, interacted with this positive regulatory element and had an AP-1-related component as a complex. The binding activity of Pluto was predominantly detected only in the extract from senescent preimmortalized cells. In contrast, a repressor, Orpheus, which bound to the ATG-rich negative regulatory element of ISE2, was prominently detected in extracts from both young preimmortalized and immortalized cells and appeared to suppress transcription in an orientation-dependent manner. Thus, the interplay of Pluto and Orpheus was suggested to be crucial for regulation of the collagenase gene accompanying in vitro aging and immortalization. Proserpine seemed to interact with Pluto to mediate strong expression of the collagenase gene in cellular senescence. On the basis of these results, we propose a model for regulation of the collagenase gene during in vitro aging and immortalization. Images PMID:7935433

  6. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  7. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    PubMed

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  8. Effects of estrogen treatment on expression of brain-derived neurotrophic factor and cAMP response element-binding protein expression and phosphorylation in rat amygdaloid and hippocampal structures.

    PubMed

    Zhou, Jin; Zhang, Huaibo; Cohen, Rochelle S; Pandey, Subhash C

    2005-01-01

    Clinical studies indicate an effect of estrogen (E2) on affect and cognition, which may be mediated by the cAMP response element-binding protein (CREB) pathway and CREB-related gene target brain-derived neurotrophic factor (BDNF). We investigated the effect of E2 on CREB expression and phosphorylation and BDNF expression in the amygdala and hippocampus, areas involved in emotional processing. Ovariectomized rats were given 10 microg 17beta-estradiol or vehicle for 14 days and expression of components of the CREB signaling pathway, i.e., CREB, phosphorylated CREB (pCREB), and BDNF in amygdala and hippocampus were investigated using immunogold labeling. Levels of BDNF mRNA were determined by in situ reverse-transcriptase polymerase chain reaction. We also examined the effect of E2 on calcium/calmodulin kinase (CaMK IV) immunolabeling in the hippocampus. E2 increased immunolabeling and mRNA levels of BDNF in the medial and basomedial amygdala and CA1 and CA3 regions of the hippocampus, but not in any other amygdaloid or hippocampal regions examined. E2 increased immunolabeling of CREB and pCREB in the medial and basomedial, but not central or basolateral amygdala. E2 also increased CaMK IV and pCREB immunolabeling in the CA1 and CA3 regions, but not CA2 region or dentate gyrus, of the hippocampus. There was no change in immunolabeling of CREB in any hippocampal region. These data identify a signaling pathway through which E2 increases BDNF expression that may underlie some actions of E2 on affective behavior and indicate neuroanatomical heterogeneity in the E2 effect within the amygdala and hippocampus.

  9. Caloric restriction-associated remodeling of rat white adipose tissue: effects on the growth hormone/insulin-like growth factor-1 axis, sterol regulatory element binding protein-1, and macrophage infiltration.

    PubMed

    Chujo, Yoshikazu; Fujii, Namiki; Okita, Naoyuki; Konishi, Tomokazu; Narita, Takumi; Yamada, Atsushi; Haruyama, Yushi; Tashiro, Kosuke; Chiba, Takuya; Shimokawa, Isao; Higami, Yoshikazu

    2013-08-01

    The role of the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis in the lifelong caloric restriction (CR)-associated remodeling of white adipose tissue (WAT), adipocyte size, and gene expression profiles was explored in this study. We analyzed the WAT morphology of 6-7-month-old wild-type Wistar rats fed ad libitum (WdAL) or subjected to CR (WdCR), and of heterozygous transgenic dwarf rats bearing an anti-sense GH transgene fed ad libitum (TgAL) or subjected to CR (TgCR). Although less effective in TgAL, the adipocyte size was significantly reduced in WdCR compared with WdAL. This CR effect was blunted in Tg rats. We also used high-density oligonucleotide microarrays to examine the gene expression profile of WAT of WdAL, WdCR, and TgAL rats. The gene expression profile of WdCR, but not TgAL, differed greatly from that of WdAL. The gene clusters with the largest changes induced by CR but not by Tg were genes involved in lipid biosynthesis and inflammation, particularly sterol regulatory element binding proteins (SREBPs)-regulated and macrophage-related genes, respectively. Real-time reverse-transcription polymerase chain reaction analysis confirmed that the expression of SREBP-1 and its downstream targets was upregulated, whereas the macrophage-related genes were downregulated in WdCR, but not in TgAL. In addition, CR affected the gene expression profile of Tg rats similarly to wild-type rats. Our findings suggest that CR-associated remodeling of WAT, which involves SREBP-1-mediated transcriptional activation and suppression of macrophage infiltration, is regulated in a GH-IGF-1-independent manner.

  10. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

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

  11. Neuron-restrictive Silencer Factor (NRSF) Represses Cocaine- and Amphetamine-regulated Transcript (CART) Transcription and Antagonizes cAMP-response Element-binding Protein Signaling through a Dual NRSE Mechanism*

    PubMed Central

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao

    2012-01-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. PMID:23086924

  12. Streptococcus pneumoniae Genome-wide Identification and Characterization of BOX Element-binding Domains.

    PubMed

    Zhang, Qiao; Wang, Changzheng; Wan, Min; Wu, Yin; Ma, Qianli

    2015-11-01

    The BOX elements are short repetitive DNA sequences that distribute randomly in intergenic regions of the Streptococcus pneumoniae genome. The function and origin of such elements are still unknown, but they were found to modulate expression of neighboring genes. Evidences suggested that the modulation's mechanism can be fulfilled by sequence-specific interaction of BOX elements with transcription factor family proteins. However, the type and function of these BOX-binding proteins still remain largely unexplored to date. In the current study we described a synthetic protocol to investigate the recognition and interaction between a highly conserved site of BOX elements and the DNA-binding domains of a variety of putative transcription factors in the pneumococcal genome. With the protocol we were able to predict those high-affinity domain binders of the conserved BOX DNA site (BOX DNA) in a high-throughput manner, and analyzed sequence-specific interaction in the domainDNA recognition at molecular level. Consequently, a number of putative transcription factor domains with both high affinity and specificity for the BOX DNA were identified, from which the helix-turn-helix (HTH) motif of a small heat shock factor was selected as a case study and tested for its binding capability toward the double-stranded BOX DNA using fluorescence anisotropy analysis. As might be expected, a relatively high affinity was detected for the interaction of HTH motif with BOX DNA with dissociation constant at nanomolar level. Molecular dynamics simulation, atomic structure examination and binding energy analysis revealed a complicated network of intensive nonbonded interactions across the complex interface, which confers both stability and specificity for the complex architecture.

  13. Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter.

    PubMed

    Casolaro, V; Keane-Myers, A M; Swendeman, S L; Steindler, C; Zhong, F; Sheffery, M; Georas, S N; Ono, S J

    2000-11-24

    Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

  14. Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements.

    PubMed

    Telorac, Jonas; Prykhozhij, Sergey V; Schöne, Stefanie; Meierhofer, David; Sauer, Sascha; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-07-27

    Out of the myriad of potential DNA binding sites of the glucocorticoid receptor (GR) found in the human genome, only a cell-type specific minority is actually bound, indicating that the presence of a recognition sequence alone is insufficient to specify where GR binds. Cooperative interactions with other transcription factors (TFs) are known to contribute to binding specificity. Here, we reasoned that sequence signals preventing GR recruitment to certain loci provide an alternative means to confer specificity. Motif analyses uncovered candidate Negative Regulatory Sequences (NRSs) that interfere with genomic GR binding. Subsequent functional analyses demonstrated that NRSs indeed prevent GR binding to nearby response elements. We show that NRS activity is conserved across species, found in most tissues and that they also interfere with the genomic binding of other TFs. Interestingly, the effects of NRSs appear not to be a simple consequence of changes in chromatin accessibility. Instead, we find that NRSs interact with proteins found at sub-nuclear structures called paraspeckles and that these proteins might mediate the repressive effects of NRSs. Together, our studies suggest that the joint influence of positive and negative sequence signals partition the genome into regions where GR can bind and those where it cannot.

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

    PubMed Central

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

    1994-01-01

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

  16. Effects of rare earth elements and REE-binding proteins on physiological responses in plants.

    PubMed

    Liu, Dongwu; Wang, Xue; Chen, Zhiwei

    2012-02-01

    Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

  17. Eukaryotic translation initiation factor 4G (eIF4G) coordinates interactions with eIF4A, eIF4B, and eIF4E in binding and translation of the barley yellow dwarf virus 3' cap-independent translation element (BTE).

    PubMed

    Zhao, Pei; Liu, Qiao; Miller, W Allen; Goss, Dixie J

    2017-04-07

    Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

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

    1994-01-01

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

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

    MedlinePlus

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

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

    PubMed Central

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

    1991-01-01

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

  1. Far Upstream Element-Binding Protein 1 Binds the 3' Untranslated Region of PKD2 and Suppresses Its Translation.

    PubMed

    Zheng, Wang; Shen, Fan; Hu, Ruikun; Roy, Birbickram; Yang, JungWoo; Wang, Qian; Zhang, Fan; King, Jennifer C; Sergi, Consolato; Liu, Song-Mei; Cordat, Emmanuelle; Tang, Jingfeng; Cao, Ying; Ali, Declan; Chen, Xing-Zhen

    2016-09-01

    Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD. Copyright © 2016 by the American Society of Nephrology.

  2. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

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

    2015-01-01

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

  3. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  4. Physical factors affecting chloroquine binding to melanin.

    PubMed

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

    2015-10-01

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

  5. Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

    PubMed

    Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

    2000-11-10

    In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells.

  6. Mouse models for core binding factor leukemia.

    PubMed

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

    2015-10-01

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

  7. A zinc-dependent DNA-binding activity co-operates with cAMP-responsive-element-binding protein to activate the human thyroglobulin enhancer.

    PubMed Central

    Berg, V; Vassart, G; Christophe, D

    1997-01-01

    Footprinting experiments involving the human thyroglobulin gene enhancer and thyroid nuclear extracts revealed a protected region called X2, containing an incomplete cAMP-responsive element (CRE). Band-shift experiments identified two binding activities recognizing the X2 element: a CRE-binding protein (CREB)/activating transcription factor (ATF) relative that binds the half CRE motif and a second factor that interacts with a G-rich motif located just upstream from the CRE. The first factor appears to be CREB itself, as indicated by the supershifting when using an antibody directed against CREB, and the second DNA-binding activity involved was shown to be zinc-dependent and exhibited an apparent molecular mass of 42-44 kDa in South-Western blotting experiments. This factor may represent a novel entity, which we named CAF, for 'CREB Associated Factor'. Three copies of X2 sequence conferred a strong cAMP-dependent transcriptional activation to a heterologous promoter in transient transfection assay in cAMP-stimulated primary thyrocytes and HeLa cells. Transfection experiments of constructs containing the X2 element mutated in either the CRE or the G-rich site showed that both motifs were required for this transcription activating function. Moreover, the combination of several individual X2 elements mutated in either the CRE or the G-rich motif did not exhibit full transcriptional activity. This suggests that, in the context of the X2 element, CREB requires a close interaction with CAF to achieve both basal and cAMP-dependent transcriptional activation. PMID:9163323

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

    SciTech Connect

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

    2002-03-08

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

  9. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  10. Element-by-element factorization algorithms for heat conduction

    NASA Technical Reports Server (NTRS)

    Hughes, T. J. R.; Winget, J. M.; Park, K. C.

    1983-01-01

    Element-by-element solution strategies are developed for transient heat conduction problems. Results of numerical tests indicate the effectiveness of the procedures proposed. The small database requirements and attractive architectural features of the algorithms suggest considerable potential for solving large scale problems.

  11. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

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

    2012-04-24

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

  12. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

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

    2009-10-06

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

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

    PubMed Central

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

    2010-01-01

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

  14. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    SciTech Connect

    Mao, Grace; Brody, James P.

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s{sup -1}. We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

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

    PubMed

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

    2013-07-01

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

  16. Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.

    PubMed

    Ajuria, Leiore; Nieva, Claudia; Winkler, Clint; Kuo, Dennis; Samper, Núria; Andreu, María José; Helman, Aharon; González-Crespo, Sergio; Paroush, Ze'ev; Courey, Albert J; Jiménez, Gerardo

    2011-03-01

    RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

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

    PubMed

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

    2016-12-07

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

  18. A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes

    SciTech Connect

    Shannon, M.F.; Pell, L.M.; Kuczek, E.S.; Occhiodoro, F.S.; Dunn, S.M.; Vadas, M.A. ); Lenardo, M.J. )

    1990-06-01

    A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. The authors show that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor {alpha} (TNF-{alpha}) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-{alpha}-responsive enhancer in these cells.

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

    PubMed Central

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

    2003-01-01

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

  20. Sequential coagulation factor VIIa domain binding to tissue factor

    SciTech Connect

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

    2005-12-02

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

  1. Why Transcription Factor Binding Sites Are Ten Nucleotides Long

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-14

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

  4. RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements

    PubMed Central

    Chatagnon, Amandine; Veber, Philippe; Morin, Valérie; Bedo, Justin; Triqueneaux, Gérard; Sémon, Marie; Laudet, Vincent; d'Alché-Buc, Florence; Benoit, Gérard

    2015-01-01

    In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status. PMID:25897113

  5. RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

    PubMed

    Chatagnon, Amandine; Veber, Philippe; Morin, Valérie; Bedo, Justin; Triqueneaux, Gérard; Sémon, Marie; Laudet, Vincent; d'Alché-Buc, Florence; Benoit, Gérard

    2015-05-26

    In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

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

    2013-07-01

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

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

    PubMed

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

    1996-12-15

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

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

    PubMed

    Schleif, Robert F

    2013-10-01

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

  9. Differentiation-specific element binding protein (DSEB) binds to a defined element in the promoter of the angiotensinogen gene required for the irreversible induction of gene expression during differentiation of 3T3-L1 adipoblasts to adipocytes.

    PubMed

    McGehee, R E; Habener, J F

    1995-04-01

    The differentiation-specific element (DSE) is a cis-acting transcriptional element located at nucleotide--1000 in the 5'-flanking promoter of the angiotensinogen gene. It is required for the irreversible and sustained increase in transcription of the angiotensinogen gene that occurs during differentiation of 3T3-L1 adipoblasts into adipocytes induced by a 3-day hormonal pulse. We report here the cloning of 3T3-L1 adipocyte cDNA encoding a 150 kilodalton protein designated Differentiation Specific Element Binding Protein (DSEB) that exhibits sequence-specific binding to a DSE oligonucleotide. Two DSEB mRNAs (3.6 and 4.2 kilobases) are observed in adipose, brain, kidney, testis, liver, and lung. Both DSEB mRNA and protein are induced during, and remain elevated after, 3T3-L1 cell adipogenesis. Analysis of adipoblasts by immunocytochemistry with an antiserum directed to bacterial expressed DSEB reveals that DSEB is localized to the nucleus and is induced during differentiation. DNA-binding assays show that binding is specific and exhibits high affinity and specificity for the DSE. Deletional analyses of bacterial expressed recombinant DSEB identifies a DNA-binding domain of 120 amino acids that contains two predicted helical regions. A sequence of 72 amino acids within the DNA-binding domain of DSEB is 60% identical to domains found in the sequences of several bacterial ligases. Further, DSEB is homologous to several proteins reported recently that are proposed to be a component(s) of the DNA replication-C complex raising the possibility that DSEB may be both a transcription factor and a DNA-replication factor.

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

    PubMed

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

    2003-07-01

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

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

    PubMed

    Litovchenko, Maria; Laurent, Stefan

    2016-11-01

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

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

    2000-01-01

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

  14. New alternating direction procedures in finite element analysis based upon EBE approximate factorizations. [element-by-element

    NASA Technical Reports Server (NTRS)

    Hughes, T. J. R.; Winget, J.; Levit, I.; Tezduyar, T. E.

    1983-01-01

    Element-by-element approximate factorization procedures are proposed for solving the large finite element equation systems which arise in computational mechanics. A variety of techniques are compared on problems of structural mechanics, heat conduction and fluid mechanics. The results obtained suggest considerable potential for the methods described.

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

    PubMed

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

    2012-06-06

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

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

    PubMed Central

    2012-01-01

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

  17. A nucleolar localizing Rev binding element inhibits HIV replication

    PubMed Central

    Michienzi, Alessandro; De Angelis, Fernanda G; Bozzoni, Irene; Rossi, John J

    2006-01-01

    The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment. PMID:16712721

  18. Formation of STAT5-containing DNA binding complexes in response to colony-stimulating factor-1 and platelet-derived growth factor.

    PubMed

    Novak, U; Mui, A; Miyajima, A; Paradiso, L

    1996-08-02

    Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

  19. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

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

    1986-05-01

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

  20. Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

    PubMed

    Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin; Urban, Sylvia; Osz, Judit; Chatagnon, Amandine; Delacroix, Laurence; Langer, Diana; Rochel, Natacha; Moras, Dino; Benoit, Gerard; Davidson, Irwin

    2012-07-27

    Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

  1. Retinoic Acid Receptors Recognize the Mouse Genome through Binding Elements with Diverse Spacing and Topology*

    PubMed Central

    Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin; Urban, Sylvia; Osz, Judit; Chatagnon, Amandine; Delacroix, Laurence; Langer, Diana; Rochel, Natacha; Moras, Dino; Benoit, Gerard; Davidson, Irwin

    2012-01-01

    Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function. PMID:22661711

  2. A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter.

    PubMed Central

    Merchant, J L; Demediuk, B; Brand, S J

    1991-01-01

    Epidermal growth factor (EGF) and transforming growth factor alpha are important determinants of mucosal integrity in the gastrointestinal tract, and they act both directly and indirectly to prevent ulceration in the stomach. Consistent with this physiological role, EGF stimulates transcription of gastrin, a peptide hormone which regulates gastric acid secretion and mucosal growth. EGF stimulation of gastrin transcription is mediated by a GC-rich gastrin EGF response element (gERE) (GGGGCGGGGTGGGGGG) which lies between -54 and -68 in the human gastrin promoter. The gERE sequence also confers weaker responsiveness to phorbol ester stimulation. The gERE sequence differs from previously described EGF response elements. The gERE DNA sequence specifically interacts with a GH4 DNA-binding protein distinct from previously described transcription factors (Egr-1 and AP2) which bind GC-rich sequences and mediate transcriptional activation by growth factors. Furthermore, the gERE element does not bind the Sp1 transcription factor even though the gERE sequence contains a high-affinity Sp1-binding site (GGCGGG). Images PMID:2017173

  3. TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus.

    PubMed

    Braun, I C; Rohrbach, E; Schmitt, C; Izaurralde, E

    1999-04-01

    The constitutive transport element (CTE) of the simian type D retroviruses overcomes nuclear retention and allows nuclear export of unspliced viral RNAs by recruiting TAP, a host factor which is thought to be required for export of cellular mRNAs. In this report, we show that the first 372 amino acid residues of TAP, comprising a stretch of leucine-rich repeats, are both necessary and sufficient for binding to the CTE RNA and promoting its export to the cytoplasm. Moreover, like the full-length protein, this domain migrates to the cytoplasm upon nuclear co-injection with the CTE RNA. Together, these results indicate that the CTE-binding domain includes the signals for nuclear export. We also describe a derivative of TAP that bears a triple amino acid substitution within the CTE-binding domain and substantially reduces the export of mRNAs from the nucleus. This provides further evidence for a role for TAP in this process. Thus, the CTE-binding domain of TAP defines a novel RNA-binding motif which has dual functions, both recognizing the CTE RNA and interacting with other components of the nuclear transport machinery.

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

    SciTech Connect

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

    1983-01-01

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

  5. Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements.

    PubMed

    Gustin, Kortney M; Guan, Bo-Jhih; Dziduszko, Agnieszka; Brian, David A

    2009-06-01

    Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

  6. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.

    PubMed

    Webb, Christopher J; Zakian, Virginia A

    2015-09-08

    The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

  7. Transcription Factor Binding Sites Prediction Based on Modified Nucleosomes

    PubMed Central

    Talebzadeh, Mohammad; Zare-Mirakabad, Fatemeh

    2014-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  9. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter.

    PubMed

    Wang, Zhi; Zhu, Yan; Wang, Lili; Liu, Xia; Liu, Yongxiu; Phillips, Jonathan; Deng, Xin

    2009-11-01

    Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-plant species; however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene (BhGolS1) that was dehydration and ABA-inducible in the resurrection plant Boea hygrometrica and conferred dehydration tolerance in a transgenic tobacco system. Four W-box cis-elements were identified in the BhGolS1 promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (BhWRKY1). These data suggest a mechanism where BhWRKY1 is likely to function in an ABA-dependent signal pathway to regulate BhGolS1 expression, which leads to the accumulation of RFOs in desiccation-tolerant B. hygrometrica leaves.

  10. Transcription factor binding dynamics during human ESC differentiation

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1999-01-01

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

  12. Thermodynamic and structural determinants of differential Pdx1 binding to elements from the insulin and IAPP promoters.

    PubMed

    Bastidas, Monique; Showalter, Scott A

    2013-09-23

    In adult mammals, the production of insulin and other peptide hormones, such as the islet amyloid polypeptide (IAPP), is limited to β-cells due to tissue-specific expression of a set of transcription factors, the best known of which is pancreatic duodenal homeobox protein 1 (Pdx1). Like many homeodomain transcription factors, Pdx1 binds to a core DNA recognition sequence containing the tetranucleotide 5'-TAAT-3'; its consensus recognition element is 5'-CTCTAAT(T/G)AG-3'. Currently, a complete thermodynamic profile of Pdx1 binding to near-consensus and native promoter sequences has not been established, obscuring the mechanism of target site selection by this critical transcription factor. Strikingly, while Pdx1 responsive elements in the human insulin promoter conform to the pentanucleotide 5'-CTAAT-3' sequence, the Pdx1 responsive elements in the human iapp promoter all contain a substitution to 5'-TTAAT-3'. The crystal structure of Pdx1 bound to the consensus nucleotide sequence does not explain how Pdx1 identifies this natural variation, if it does at all. Here we report a combination of isothermal calorimetric titrations, NMR spectroscopy, and extensive multi-microsecond molecular dynamics calculations of Pdx1 that define its interactions with a panel of natural promoter elements and consensus-derived sequences. Our results show a small preference of Pdx1 for a C base 5' relative to the core TAAT promoter element. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. Taken together, our results suggest a molecular mechanism for differential Pdx1 affinity to elements from the insulin and iapp promoter sequences. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    SciTech Connect

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil Lee, Zang Hee

    2010-07-23

    Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

  14. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism.

    PubMed

    Iizuka, Katsumi

    2017-02-22

    Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp(-/-) mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  15. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    PubMed Central

    Iizuka, Katsumi

    2017-01-01

    Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome. PMID:28241431

  16. SeqGL Identifies Context-Dependent Binding Signals in Genome-Wide Regulatory Element Maps.

    PubMed

    Setty, Manu; Leslie, Christina S

    2015-05-01

    Genome-wide maps of transcription factor (TF) occupancy and regions of open chromatin implicitly contain DNA sequence signals for multiple factors. We present SeqGL, a novel de novo motif discovery algorithm to identify multiple TF sequence signals from ChIP-, DNase-, and ATAC-seq profiles. SeqGL trains a discriminative model using a k-mer feature representation together with group lasso regularization to extract a collection of sequence signals that distinguish peak sequences from flanking regions. Benchmarked on over 100 ChIP-seq experiments, SeqGL outperformed traditional motif discovery tools in discriminative accuracy. Furthermore, SeqGL can be naturally used with multitask learning to identify genomic and cell-type context determinants of TF binding. SeqGL successfully scales to the large multiplicity of sequence signals in DNase- or ATAC-seq maps. In particular, SeqGL was able to identify a number of ChIP-seq validated sequence signals that were not found by traditional motif discovery algorithms. Thus compared to widely used motif discovery algorithms, SeqGL demonstrates both greater discriminative accuracy and higher sensitivity for detecting the DNA sequence signals underlying regulatory element maps. SeqGL is available at http://cbio.mskcc.org/public/Leslie/SeqGL/.

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

    PubMed Central

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

    1987-01-01

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

  18. Stress-Mediated cis-Element Transcription Factor Interactions Interconnecting Primary and Specialized Metabolism in planta

    PubMed Central

    Sheshadri, S. A.; Nishanth, M. J.; Simon, Bindu

    2016-01-01

    Plant specialized metabolites are being used worldwide as therapeutic agents against several diseases. Since the precursors for specialized metabolites come through primary metabolism, extensive investigations have been carried out to understand the detailed connection between primary and specialized metabolism at various levels. Stress regulates the expression of primary and specialized metabolism genes at the transcriptional level via transcription factors binding to specific cis-elements. The presence of varied cis-element signatures upstream to different stress-responsive genes and their transcription factor binding patterns provide a prospective molecular link among diverse metabolic pathways. The pattern of occurrence of these cis-elements (overrepresentation/common) decipher the mechanism of stress-responsive upregulation of downstream genes, simultaneously forming a molecular bridge between primary and specialized metabolisms. Though many studies have been conducted on the transcriptional regulation of stress-mediated primary or specialized metabolism genes, but not much data is available with regard to cis-element signatures and transcription factors that simultaneously modulate both pathway genes. Hence, our major focus would be to present a comprehensive analysis of the stress-mediated interconnection between primary and specialized metabolism genes via the interaction between different transcription factors and their corresponding cis-elements. In future, this study could be further utilized for the overexpression of the specific transcription factors that upregulate both primary and specialized metabolism, thereby simultaneously improving the yield and therapeutic content of plants. PMID:27933071

  19. Cloning of a protein binding to the most proximal Pit-1 binding element of prolactin gene from human pituitary cDNA library.

    PubMed

    Fumoto, Mariko; Okimura, Yasuhiko; Sakagami, Yoshio; Iguchi, Genzo; Kishimoto, Masahiko; Takahashi, Yutaka; Kaji, Hidesuke; Chihara, Kazuo

    2003-09-30

    A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1. Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family. Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors. Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P. mPOU activated the expression of 0.6 k PRL and 7x1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7x3P reporter gene. These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1.

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

    PubMed

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

    2015-01-01

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

  1. Identifying differential transcription factor binding in ChIP-seq

    PubMed Central

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

    2015-01-01

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

  2. Dietary and nutritional manipulation of the nuclear transcription factors peroxisome proliferator-activated receptor and sterol regulatory element-binding proteins as a tool for reversing the primary diseases of premature death and delaying aging.

    PubMed

    Kurtak, Karen A

    2014-04-01

    Evolution over 2.1 billion years has equipped us with a biochemical pathway that has the power to literally reverse the primary disease etiologies that have become the leading causes of death and aging in the developed world. Activation of the peroxisome proliferator-activated receptor (PPAR) pathway arrests inflammatory signaling throughout the body, reverses damage to tissues, reverses insulin resistance, and can even dissolve beta-amyloid plaque in the brain. It has played a critical role in the evolution of the metazoans and the successful migration of humans to all corners of the Earth. For two decades, various pharmaceuticals have been designed to activate the PPAR pathway but have consistently fallen short of expectations. There is nothing wrong with these drugs. The problem has been the standard "healthy" diet creating mixed signals that render the drugs ineffective. This article explores the ongoing dance between the two primary nuclear receptors that mediate gene regulation of fatty acids. It discusses their interaction with sirtuins and telomerase, optimization of their obligate heterodimers, and why manipulation of dietary and nutritional factors, like the ketogenic diet, is the most effective means of activation. These are effective tools that we can start implementing now to slow, and in some cases reverse, the diseases of aging.

  3. Binding among Select Episodic Elements Is Altered via Active Short-Term Retrieval

    ERIC Educational Resources Information Center

    Bridge, Donna J.; Voss, Joel L.

    2015-01-01

    Of the many elements that comprise an episode, are any disproportionately bound to the others? We tested whether active short-term retrieval selectively increases binding. Individual objects from multiobject displays were retrieved after brief delays. Memory was later tested for the other objects. Cueing with actively retrieved objects facilitated…

  4. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

    PubMed Central

    Rajendra, S; Vandenberg, R J; Pierce, K D; Cunningham, A M; French, P W; Barry, P H; Schofield, P R

    1995-01-01

    A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily. Images PMID:7621814

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

    PubMed Central

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

    2016-01-01

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

  6. A Potential Structural Switch for Regulating DNA-Binding by TEAD Transcription Factors.

    PubMed

    Lee, Dong-Sun; Vonrhein, Clemens; Albarado, Diana; Raman, C S; Veeraraghavan, Sudha

    2016-06-19

    TEA domain (TEAD) transcription factors are essential for the normal development of eukaryotes and are the downstream effectors of the Hippo tumor suppressor pathway. Whereas our earlier work established the three-dimensional structure of the highly conserved DNA-binding domain using solution NMR spectroscopy, the structural basis for regulating the DNA-binding activity remains unknown. Here, we present the X-ray crystallographic structure and activity of a TEAD mutant containing a truncated L1 loop, ΔL1 TEAD DBD. Unexpectedly, the three-dimensional structure of the ΔL1 TEAD DBD reveals a helix-swapped homodimer wherein helix 1 is swapped between monomers. Furthermore, each three-helix bundle in the domain-swapped dimer is a structural homolog of MYB-like domains. Our investigations of the DNA-binding activity reveal that although the formation of the three-helix bundle by the ΔL1 TEAD DBD is sufficient for binding to an isolated M-CAT-like DNA element, multimeric forms are deficient for cooperative binding to tandemly duplicated elements, indicating that the L1 loop contributes to the DNA-binding activity of TEAD. These results suggest that switching between monomeric and domain-swapped forms may regulate DNA selectivity of TEAD proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

    SciTech Connect

    Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R.

    2010-02-08

    The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

  8. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    PubMed Central

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  9. DNA-binding specificities of human transcription factors.

    PubMed

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

    2013-01-17

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

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

    PubMed Central

    Pajon, Rolando; Beernink, Peter T.

    2012-01-01

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

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

    PubMed

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

    2012-08-01

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

  12. Metal and DNA binding properties of a two-domain fragment of neural zinc finger factor 1, a CCHC-type zinc binding protein.

    PubMed

    Berkovits, H J; Berg, J M

    1999-12-21

    Neural zinc finger factor 1 (NZF-1) is a member of a family of neural-specific transcription factors that contain multiple copies of a relatively uncharacterized zinc binding motif. We have studied the metal binding and DNA binding properties of a fragment of NZF-1 containing two adjacent zinc binding domains. Partial proteolysis with endoproteinase Lys-C identified metal-stabilized fragments containing either one or both of the zinc binding domains. Both domains were required for specific DNA binding to the beta-retinoic acid receptor element, producing a DNase I footprint covering predominantly one strand. The metal binding site was probed via cobalt(II) substitution. The visible absorption spectrum of the cobalt(II) complex is consistent with Cys-Cys-His-Cys coordination of the metal. The two domains appear to have similar affinities for metal and bind cobalt(II) and zinc(II) with dissociation constants of 4 (+/- 2) x 10(-)(7) M and 1.4 (+/- 0.8) x 10(-)(10) M, respectively. The domains fold upon the addition of zinc, as observed by (1)H NMR. However, an additional weak binding site causes line broadening in the presence of excess zinc, presumably due to aggregation.

  13. cAMP-responsive element binding protein: a vital link in embryonic hormonal adaptation.

    PubMed

    Schindler, Maria; Fischer, Sünje; Thieme, René; Fischer, Bernd; Santos, Anne Navarrete

    2013-06-01

    The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    1997-12-18

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

  16. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  17. The function of circadian RNA-binding proteins and their cis-acting elements in microalgae.

    PubMed

    Mittag, Maria

    2003-07-01

    An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin-binding protein (LBP) is controlled at the translational level. Thereby, a clock-controlled RNA-binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG-repeat, which is situated in the lbp 3' UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3' UTRs of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG-repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO2 metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.

  18. Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

    PubMed Central

    Quitschke, W W; DePonti-Zilli, L; Lin, Z Y; Paterson, B M

    1989-01-01

    The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis. Images PMID:2552286

  19. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway.

    PubMed

    Bhattacharya, Bonhi S; Sweby, Peter K; Minihane, Anne-Marie; Jackson, Kim G; Tindall, Marcus J

    2014-05-21

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

  20. Aralia elata prevents neuronal death by downregulating tonicity response element binding protein in diabetic retinopathy.

    PubMed

    Kim, Seong-Jae; Yoo, Woong-Sun; Kim, Hwajin; Kwon, Jeong Eun; Hong, Eun-Kyung; Choi, Meeyoung; Han, Yongseop; Chung, Inyoung; Seo, Seongwook; Park, Jongmoon; Yoo, Ji-Myong; Choi, Wan-Sung

    2015-01-01

    The present study addresses the role of tonicity response element binding protein (TonEBP) in retinal ganglion cell (RGC) death in diabetic retinopathy and the impact of Aralia elata extract on the TonEBP/RGC interaction. Diabetes was induced in C57BL/6 mice by intraperitoneal injection of streptozotocin (STZ). Control mice received phosphate-buffered saline. After five injections of STZ or saline buffer, A. elata extract was administered by daily oral tube feeding for 7 weeks. All mice were killed at 2 months after the last injection of STZ or saline and the extent of cell death together with the protein expression levels of TonEBP, aldose reductase (AR) and nuclear factor-kappa B (NF-κB) were examined. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive signals were colocalized with TonEBP-immunoreactive RGCs. The apoptotic cell death of RGCs and the expression levels of TonEBP, AR and NF-κB were significantly increased in the retinas of diabetic mice compared with controls at 2 months after the induction of diabetes. However, these changes were effectively blocked by the administration of A. elata extract. These results indicate that A. elata prevents diabetes-induced RGC apoptosis and downregulates TonEBP expression. Therefore, A. elata extract may have therapeutic potential to prevent diabetes-induced retinal neurodegeneration in diabetic retinopathy. © 2015 S. Karger AG, Basel.

  1. Advanced glycation end products increase carbohydrate responsive element binding protein expression and promote cancer cell proliferation.

    PubMed

    Chen, Hanbei; Wu, Lifang; Li, Yakui; Meng, Jian; Lin, Ning; Yang, Dianqiang; Zhu, Yemin; Li, Xiaoyong; Li, Minle; Xu, Ye; Wu, Yuchen; Tong, Xuemei; Su, Qing

    2014-09-01

    Diabetic patients have increased levels of advanced glycation end products (AGEs) and the role of AGEs in regulating cancer cell proliferation is unclear. Here, we found that treating colorectal and liver cancer cells with AGEs promoted cell proliferation. AGEs stimulated both the expression and activation of a key transcription factor called carbohydrate responsive element binding protein (ChREBP) which had been shown to promote glycolytic and anabolic activity as well as proliferation of colorectal and liver cancer cells. Using siRNAs or the antagonistic antibody for the receptor for advanced glycation end-products (RAGE) blocked AGEs-induced ChREBP expression or cell proliferation in cancer cells. Suppressing ChREBP expression severely impaired AGEs-induced cancer cell proliferation. Taken together, these results demonstrate that AGEs-RAGE signaling enhances cancer cell proliferation in which AGEs-mediated ChREBP induction plays an important role. These findings may provide new explanation for increased cancer progression in diabetic patients. Copyright © 2014. Published by Elsevier Ireland Ltd.

  2. CTCF Binding Elements Mediate Control of V(D)J Recombination

    PubMed Central

    Guo, Chunguang; Yoon, Hye Suk; Franklin, Andrew; Jain, Suvi; Ebert, Anja; Cheng, Hwei-Ling; Hansen, Erica; Despo, Orion; Bossen, Claudia; Vettermann, Christian; Bates, Jamie G.; Richards, Nicholas; Myers, Darienne; Patel, Harin; Gallagher, Michael; Schlissel, Mark S.; Murre, Cornelis; Busslinger, Meinrad; Giallourakis, Cosmas C.; Alt, Frederick W.

    2012-01-01

    Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7 megabase (Mb) mouse IgH locus (IgH), V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Herein, we report a key IgH V(D)J recombination regulatory region, termed InterGenic Control Region-1 (IGCR1), that lies between the VH and D clusters. Functionally, IGCR1 employs CTCF looping/insulator factor binding elements and, correspondingly, mediates IgH loops containing distant enhancers. IGCR1 promotes normal B cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VHs and promoting rearrangement of distal VHs. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination, respectively, by suppressing VH joining to Ds not joined to JHs and VH to DJH joins in thymocytes. IGCR1 also is required to allow feedback regulation and allelic exclusion of proximal VH to DJH recombination. Our studies elucidate a long-sought IgH V(D)J recombination control region and implicate a new role for the generally expressed CTCF protein. PMID:21909113

  3. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

    PubMed Central

    Bhattacharya, Bonhi S.; Sweby, Peter K.; Minihane, Anne-Marie; Jackson, Kim G.; Tindall, Marcus J.

    2014-01-01

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature. PMID:24444765

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

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

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

  5. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    DOE Data Explorer

    Loots, Gabriela G. [LLNL; Ovcharenko, I. [LLNL

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. This database of evolutionary conserved regions (ECRs) in vertebrate genomes features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a comprehensive collection of promoters in all vertebrate genomes generated using multiple sources of gene annotation. The database also contains a collection of annotated transcription factor binding sites (TFBSs) in evolutionary conserved and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and fugu genomes. (taken from paper in Journal: Bioinformatics, November 7, 2006, pp. 122-124

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

    PubMed Central

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

    2016-01-01

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

  7. Specific binding of atrial natriuretic factor in brain microvessels

    SciTech Connect

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

    1987-04-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. Efficient translation of Dnmt1 requires cytoplasmic polyadenylation and Musashi binding elements.

    PubMed

    Rutledge, Charlotte E; Lau, Ho-Tak; Mangan, Hazel; Hardy, Linda L; Sunnotel, Olaf; Guo, Fan; MacNicol, Angus M; Walsh, Colum P; Lees-Murdock, Diane J

    2014-01-01

    Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3'UTR of Dnmt1 mRNA identical between humans and Xenopus and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of Dnmt1 during periods of oocyte growth in mouse and during oocyte activation in Xenopus. Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.

  12. The Next Generation of Transcription Factor Binding Site Prediction

    PubMed Central

    Mathelier, Anthony; Wasserman, Wyeth W.

    2013-01-01

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

  13. Discovery of new binding elements in DPP-4 inhibition and their applications in novel DPP-4 inhibitor design.

    PubMed

    Liang, Gui-Bai; Qian, Xiaoxia; Biftu, Tesfaye; Singh, Suresh; Gao, Ying-Duo; Scapin, Giovanna; Patel, Sangita; Leiting, Barbara; Patel, Reshma; Wu, Joseph; Zhang, Xiaoping; Thornberry, Nancy A; Weber, Ann E

    2008-07-01

    Probing with tool molecules, and by modeling and X-ray crystallography the binding modes of two structurally distinct series of DPP-4 inhibitors led to the discovery of a rare aromatic fluorine H-bond and the spatial requirement for better biaryl binding in the DPP-4 enzyme active site. These newly found binding elements were successfully incorporated into novel DPP-4 inhibitors.

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

    PubMed Central

    2013-01-01

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

  15. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus

    ERIC Educational Resources Information Center

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.

    2007-01-01

    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

  16. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus

    ERIC Educational Resources Information Center

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.

    2007-01-01

    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-03-29

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

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

    PubMed

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

    2017-06-30

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

  20. An in silico strategy identified the target gene candidates regulated by dehydration responsive element binding proteins (DREBs) in Arabidopsis genome.

    PubMed

    Wang, Shichen; Yang, Shuo; Yin, Yuejia; Guo, Xiaosen; Wang, Shan; Hao, Dongyun

    2009-01-01

    Identification of downstream target genes of stress-relating transcription factors (TFs) is desirable in understanding cellular responses to various environmental stimuli. However, this has long been a difficult work for both experimental and computational practices. In this research, we presented a novel computational strategy which combined the analysis of the transcription factor binding site (TFBS) contexts and machine learning approach. Using this strategy, we conducted a genome-wide investigation into novel direct target genes of dehydration responsive element binding proteins (DREBs), the members of AP2-EREBPs transcription factor super family which is reported to be responsive to various abiotic stresses in Arabidopsis. The genome-wide searching yielded in total 474 target gene candidates. With reference to the microarray data for abiotic stresses-inducible gene expression profile, 268 target gene candidates out of the total 474 genes predicted, were induced during the 24-h exposure to abiotic stresses. This takes about 57% of total predicted targets. Furthermore, GO annotations revealed that these target genes are likely involved in protein amino acid phosphorylation, protein binding and Endomembrane sorting system. The results suggested that the predicted target gene candidates were adequate to meet the essential biological principle of stress-resistance in plants.

  1. Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHβ.

    PubMed

    Roybal, Lacey L; Hambarchyan, Arpi; Meadows, Jason D; Barakat, Nermeen H; Pepa, Patricia A; Breen, Kellie M; Mellon, Pamela L; Coss, Djurdjica

    2014-10-01

    We previously identified FOXL2 as a critical component in FSHβ gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHβ transcription. There is a stronger induction of FSHβ reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHβ, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHβ expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHβ by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHβ promoter.

  2. Roles of Binding Elements, FOXL2 Domains, and Interactions With cJUN and SMADs in Regulation of FSHβ

    PubMed Central

    Roybal, Lacey L.; Hambarchyan, Arpi; Meadows, Jason D.; Barakat, Nermeen H.; Pepa, Patricia A.; Breen, Kellie M.; Mellon, Pamela L.

    2014-01-01

    We previously identified FOXL2 as a critical component in FSHβ gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHβ transcription. There is a stronger induction of FSHβ reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHβ, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHβ expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHβ by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHβ promoter. PMID:25105693

  3. Soluble spiroperidol binding factors from bovine caudate nucleus.

    PubMed

    Winkler, M H; Berl, S

    1982-09-01

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

  4. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  5. Maturation and activity of sterol regulatory element binding protein 1 is inhibited by acyl-CoA binding domain containing 3.

    PubMed

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

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

    PubMed Central

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

    2012-01-01

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

  7. An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

    PubMed Central

    Molkentin, J D; Markham, B E

    1994-01-01

    Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy. Images PMID:8035789

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

    PubMed Central

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

    2012-01-01

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

  9. The trehalose/maltose-binding protein as the sensitive element of a glucose biosensor

    NASA Astrophysics Data System (ADS)

    Fonin, A. V.; Povarova, O. I.; Staiano, M.; D'Auria, S.; Turoverov, K. K.; Kuznetsova, I. M.

    2014-08-01

    The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D-trehalose/D-maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis. We investigated the physical-chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo, the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP-glucose complex is in the range of 3-8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms ТМВР/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/C182S/A14C-BADAN-Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes.

  10. Thyroglobulin repression of thyroid transcription factor 1 (TTF-1) gene expression is mediated by decreased DNA binding of nuclear factor I proteins which control constitutive TTF-1 expression.

    PubMed

    Nakazato, M; Chung, H K; Ulianich, L; Grassadonia, A; Suzuki, K; Kohn, L D

    2000-11-01

    Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp -264 and -153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp -264 to -153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.

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

    PubMed

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

    1991-10-15

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

  12. Predicting DNA-Binding Specificities of Eukaryotic Transcription Factors

    PubMed Central

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

    2010-01-01

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

  13. Variable structure motifs for transcription factor binding sites.

    PubMed

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

    2010-01-14

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

  14. The mouse albumin enhancer contains a negative regulatory element that interacts with a novel DNA-binding protein.

    PubMed Central

    Herbst, R S; Boczko, E M; Darnell, J E; Babiss, L E

    1990-01-01

    The far-upstream mouse albumin enhancer (-10.5 to -8.43 kilobases) has both positive and negative regulatory domains which contribute to the rate and tissue specificity of albumin gene transcription. (R. S. Herbst, N. Friedman, J. E. Darnell, Jr., and L. E. Babiss, Proc. Natl. Acad. Sci. USA 86:1553-1557). In this work, the negative regulatory region has been functionally localized to sequences -8.7 to -8.43 kilobases upstream of the albumin gene cap site. In the absence of the albumin-modulating region (in which there are binding sites for the transcription factor C/EBP), the negative region can suppress a neighboring positive-acting element, thereby interfering with albumin enhancer function. The negative region is also capable of negating the positive action of the heterologous transthyretin enhancer in an orientation-independent fashion. Within this negative-acting region we can detect two DNA-binding sites, both of which are recognized by a protein present in all cell types tested. This DNA-binding activity is not competed for by any of a series of known DNA-binding sites, and hence this new protein is a candidate for a role in suppressing the albumin gene in nonhepatic cells. Images PMID:2370857

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

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

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

  16. Factors influencing trace element composition in human teeth

    SciTech Connect

    Tandon, L.; Iyengar, G.V.

    1997-12-01

    The authors recently compiled and reviewed the literature published in or after 1978 for 45 major, minor, and trace elements in human teeth as a part of an International Atomic Energy Agency (IAEA) study. The purpose of this paper is to discuss the various factors that influence the concentration levels of certain trace elements in human teeth. The sampling practices and analytical techniques that are applicable for trace element analysis are also discussed. It is also our intention to identify reference range of values, where data permit such conclusions. The scrutiny was designed to identify only the healthy permanent teeth, and values from teeth with fillings, caries, or periodontal diseases were eliminated.

  17. Prolactin regulatory element-binding protein is involved in suppression of the adiponectin gene in vivo.

    PubMed

    Zhang, X Z; Imachi, H; Lyu, J Y; Fukunaga, K; Sato, S; Ibata, T; Kobayashi, T; Yoshimoto, T; Kikuchi, F; Dong, T; Murao, K

    2017-04-01

    Prolactin regulatory element-binding protein (PREB), a member of the WD-repeat protein family, has been recognized as a transcriptional factor that regulates prolactin promoter activity in the anterior pituitary of rats. PREB is expressed not only in the pituitary but also in various other tissues, including the adipose tissue. Previous studies have shown that PREB acts as a transcriptional regulator and suppresses the expression of the adiponectin gene in cultured 3T3L1 preadipocytes. The aim of this study was to further examine the potential role of PREB in adipose tissue in vivo. Transgenic mice that overexpressing PREB (PREB transgenic mice) were generated. Insulin resistance was evaluated in PREB transgenic mice using glucose and insulin tolerance tests. Adiponectin expression in the adipose tissue was examined by western blot analysis and quantitative polymerase chain reaction (qPCR). The expression levels of stearoyl-CoA desaturase (Scd) and adiponectin receptor 2(ADIPOR2) were quantified by qPCR. Glucose and insulin tolerance tests revealed insulin resistance in PREB transgenic mice. Serum adiponectin and leptin concentrations were decreased. Adiponectin gene expression was decreased in the adipose tissue, which was confirmed by the downregulation of the adiponectin-dependent hepatic Scd gene and upregulation of the ADIPOR2 gene in the liver of PREB transgenic mice. We also found that pioglitazone, an agonist for the peroxisome proliferator-activated receptor-r, improved the insulin resistance in the PREB transgenic mice after a 10-day feeding period. These results demonstrated that PREB might contribute to the regulation of adiponectin gene expression in vivo.

  18. OCTAMER-BINDING TRANSCRIPTION FACTORS: GENOMICS AND FUNCTIONS

    PubMed Central

    Zhao, Feng-Qi

    2015-01-01

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

  19. Evaluation of the effectiveness of DNA-binding drugs to inhibit transcription using the c-fos serum response element as a target.

    PubMed

    White, C M; Heidenreich, O; Nordheim, A; Beerman, T A

    2000-10-10

    Previous work has demonstrated that sequence-selective DNA-binding drugs can inhibit transcription factors from binding to their target sites on gene promoters. In this study, the potency and effectiveness of DNA-binding drugs to inhibit transcription were assessed using the c-fos promoter's serum response element (SRE) as a target. The drugs chosen for analysis included the minor groove binding agents chromomycin A(3) and Hoechst 33342, which bind to G/C-rich and A/T-rich regions, respectively, and the intercalating agent nogalamycin, which binds G/C-rich sequences in the major groove. The transcription factors targeted, Elk-1 and serum response factor (SRF), form a ternary complex (TC) on the SRE that is necessary and sufficient for induction of c-fos by serum. The drugs' abilities to prevent TC formation on the SRE in vitro were nogalamycin > Hoechst 33342 > chromomycin. Their potencies in inhibiting cell-free transcription and endogenous c-fos expression in NIH3T3 cells, however, were chromomycin > nogalamycin > Hoechst 33342. The latter order of potency was also obtained for the drugs' cytotoxicity and inhibition of general transcription as measured by [(3)H]uridine incorporation. These systematic analyses provide insight into how drug and transcription factor binding characteristics are related to drugs' effectiveness in inhibiting gene expression.

  20. Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.

    PubMed Central

    Zeng, C; Pinsonneault, J; Gellon, G; McGinnis, N; McGinnis, W

    1994-01-01

    How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E. Images PMID:7910795

  1. Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

    PubMed Central

    Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

    2016-01-01

    Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

  2. The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.

    PubMed

    Yahata, T; de Caestecker, M P; Lechleider, R J; Andriole, S; Roberts, A B; Isselbacher, K J; Shioda, T

    2000-03-24

    The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.

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

    PubMed Central

    Lee, L S

    1981-01-01

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

  4. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    NASA Astrophysics Data System (ADS)

    Lee, L. S.

    1981-02-01

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

  5. Characterization of sequence-specific DNA binding by the transcription factor Oct-1.

    PubMed

    Lundbäck, T; Chang, J F; Phillips, K; Luisi, B; Ladbury, J E

    2000-06-27

    The DNA-binding domain of the Oct-1 transcription factor, POU, recognizes a defined DNA sequence known as the octamer element to regulate the expression of both general and cell-type-specific genes. The two-part DNA-binding domain partially encircles the DNA to recognize the eight base pairs of the octamer element. We have characterized the binding of Oct-1/POU to an octamer element using isothermal titration calorimetry. As found for other cognate protein/DNA complexes, the formation of the Oct-1 POU/DNA complex is associated with a large negative heat capacity change, DeltaC(p)()(, obs). However, the observed change is much greater than expected by empirical relationships with buried surface area. Supported by data from proteolysis studies on the free and DNA-bound protein, we propose that the discrepancy in heat capacity arises principally from the partial folding of the Oct-1 POU protein upon complex formation. Formation of the Oct-1 POU/DNA complex is strongly dependent on ionic strength, and the detailed quantification of this relationship suggests that six charged contacts are made between the protein and the phosphate groups of the DNA. This agrees with observations from the crystal structure of an Oct-1 POU/DNA complex.

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

    PubMed Central

    Qin, Qian

    2017-01-01

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

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

    PubMed

    Qin, Qian; Feng, Jianxing

    2017-02-01

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

  8. Binding.

    ERIC Educational Resources Information Center

    Rebsamen, Werner

    1981-01-01

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

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

    PubMed

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

    2013-01-01

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

  10. Juvenile hormone regulation of an insect gene: a specific transcription factor and a DNA response element.

    PubMed

    Zhang, J; Saleh, D S; Wyatt, G R

    1996-08-30

    We have used locust fat body nuclear protein extracts and upstream DNA of the juvenile hormone (JH)-inducible locust gene, jhp21, to examine the regulation of specific transcription by JH. Promoter activity was assayed with G-free cassette reporter constructs. Nuclear extracts from adult female fat body, previously exposed to JH or an analog, actively transcribe from the jhp21 promoter and a control adenovirus major late (AdML) promoter, whereas extracts from JH-deprived female fat body, or other tissues, transcribe strongly from the AdML promoter but weakly or not at all from the jhp21 promoter. Transcription is enhanced by sequences between -140 and -211 nt from the jhp21 transcription start point (tsp), which include a CAAT box, and also by sequences between -1056 and -1200. A 15-nt partially palindromic sequence element found at -1152, resembling known hormone response elements, was shown to stimulate transcription when restored to truncated jhp21 DNA. Two very similar sequences occur further upstream. In electrophoretic mobility shift assays (EMSA), the same sequence element was shown to specifically bind a protein that was present in nuclear extracts from JH-exposed, but not from JH-deprived, fat body. Several lines of evidence suggest that the DNA element may be a JH response element (JHRE). The JH-induced protein that binds to it appears to be a transcription factor that activates the initiation of JH target gene (jhp21) transcription, and could be a JH receptor.

  11. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

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

    PubMed

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

    2015-06-29

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    1994-05-13

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

  15. The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example

    PubMed Central

    Pan, Yongping; Nussinov, Ruth

    2011-01-01

    What is the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA? The IFN-β enhanceosome provides a good model system: it is small; its components' crystal structures are available; and there are biochemical and cellular data. In the IFN-β enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization. PMID:21698143

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

    PubMed

    Bach, Leon A

    2015-12-01

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

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

    PubMed

    Bach, Leon A

    2015-10-01

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

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

    PubMed Central

    Spivakov, Mikhail

    2014-01-01

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

  19. Characterization of Purine-Rich Element Binding Protein B as a Novel Biomarker in Acute Myelogenous Leukemia Prognostication.

    PubMed

    Kelm, Robert J; Lamba, Gurpreet S; Levis, Jamie E; Holmes, Chris E

    2017-08-23

    Acute myelogenous leukemia (AML)(1) is an aggressive hematologic cancer characterized by infiltration of proliferative, clonal, abnormally differentiated cells of myeloid lineage in the bone marrow and blood. Malignant cells in AML often exhibit chromosomal and other genetic or epigenetic abnormalities that are useful in prognostic risk assessment. In this study, the relative expression and novel single-stranded DNA (ssDNA) binding function of purine-rich element binding proteins A and B (Purα and Purβ) were systematically evaluated in established leukemia cell lines and in lineage committed myeloid cells isolated from patients diagnosed with a hematologic malignancy. Western blotting revealed that the Purα and Purβ are markedly elevated in CD33(+) /CD66b(+) cells from AML patients compared to healthy subjects and to patients with other types of myeloid cell disorders. Results of in silico database analysis of PURA and PURB mRNA expression during hematopoiesis in conjunction with the quantitative immunoassay of the ssDNA-binding activities of Purα and Purβ in transformed leukocyte cell lines pointed to Purβ as the more distinguishing biomarker of myeloid cell differentiation status. Purβ ssDNA-binding activity was significantly increased in myeloid cells from AML patients but not from individuals with other myeloid-related diseases. The highest levels of Purβ activity were detected in myeloid cells from primary AML patients and from AML patients displaying other risk factors forecasting a poor prognosis. Collectively, these findings suggest that the enhanced ssDNA-binding activity of Purβ in transformed myeloid cells may serve as a unique and measurable phenotypic trait for improving prognostic risk stratification in AML. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding.

    PubMed Central

    Simmons, D T; Loeber, G; Tegtmeyer, P

    1990-01-01

    By mutational analysis, we have identified a motif critical to the proper recognition and binding of simian virus 40 large tumor antigen (T antigen) to virus DNA sequences at the origin of DNA replication. This motif is tripartite and consists of two elements (termed A1 and B2) that are necessary for sequence-specific binding of the origin and a central element (B1) which is required for nonspecific DNA-binding activity. Certain amino acids in elements A1 (residues 152 to 155) and B2 (203 to 207) may make direct contact with the GAGGC pentanucleotide sequences in binding sites I and II on the DNA. Alternatively, these two elements could determine the proper structure of the DNA-binding domain, although for a number of reasons we favor the first possibility. In contrast, element B1 (183 to 187) is most likely important for recognizing a general structural feature of DNA. Elements A1 and B2 are nearly identical in all known papovavirus T antigens, whereas B1 is identical only in the closely related papovaviruses simian virus 40, BK virus, and JC virus. In addition to these three elements, a fourth (B3; residues 215 to 219) is necessary for the binding of T antigen to site II but not to site I. We propose that additional contact sites on T antigen are involved in the interaction with site II to initiate the replication of the viral DNA. PMID:2157865

  1. Binding of stereognostically designed ligands to trivalent, pentavalent, and hexavalent f-block elements

    SciTech Connect

    Sinkov, Sergey I.; Lumetta, Gregg J.; Warner, Marvin G.; Pittman, Jonathan W.

    2012-03-26

    Stability constants were determined for the complexes formed from two stereognostically designed ligands and the f-block elements Nd(III), Np(V), and Pu(VI). The ligands investigated were tris[3-(2-carboxyphenoxy)propyl]amine (NPB) and tris-N,N',N''-[2-(2-carboxy-4-ethyl-phenoxy)ethyl]-1,4,7-triazacyclononane (EETAC). A stereognostically blind ligand, nitrilotriacetic acid (NTA), was also investigated for comparison. The results suggest that there is no significant stereognostic effect for complexation of NPB or EETAC to Np(V). On the other hand, a modest stereognostic effect is seen for the NPB ligand when complexed to Pu(VI), leading to an approximately 8-fold increase in the binding strength. A more significant effect is observed for the EETAC system in which a 250-fold increase in binding is observed for Pu(VI) versus Nd(III).

  2. Dual function of a nuclear factor I binding site in MMTV transcription regulation.

    PubMed Central

    Buetti, E; Kühnel, B; Diggelmann, H

    1989-01-01

    Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. Images PMID:2542892

  3. ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

    PubMed

    Chen, Jiang; Yi, Qiang; Cao, Yao; Wei, Bin; Zheng, Lanjie; Xiao, Qianling; Xie, Ying; Gu, Yong; Li, Yangping; Huang, Huanhuan; Wang, Yongbin; Hou, Xianbin; Long, Tiandan; Zhang, Junjie; Liu, Hanmei; Liu, Yinghong; Yu, Guowu; Huang, Yubi

    2016-03-01

    Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.

  4. Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

    PubMed Central

    Zhou, Weixin; Chung, Yang Jo; Parrilla Castellar, Edgardo R.; Zheng, Ying; Chung, Hye-Jung; Bandle, Russell; Liu, Juhong; Tessarollo, Lino; Batchelor, Eric; Aplan, Peter D.; Levens, David

    2017-01-01

    The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1−/−) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1−/− hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1−/− hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1−/− embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression. PMID:26774856

  5. Conservation of transcription factor binding specificities across 600 million years of bilateria evolution

    PubMed Central

    Nitta, Kazuhiro R; Jolma, Arttu; Yin, Yimeng; Morgunova, Ekaterina; Kivioja, Teemu; Akhtar, Junaid; Hens, Korneel; Toivonen, Jarkko; Deplancke, Bart; Furlong, Eileen E M; Taipale, Jussi

    2015-01-01

    Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells. DOI: http://dx.doi.org/10.7554/eLife.04837.001 PMID:25779349

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

    SciTech Connect

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

    1987-12-01

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

  7. Metal loading effect on rare earth element binding to humic acid: Experimental and modelling evidence

    NASA Astrophysics Data System (ADS)

    Marsac, Rémi; Davranche, Mélanie; Gruau, Gérard; Dia, Aline

    2010-03-01

    The effect of metal loading on the binding of rare earth elements (REE) to humic acid (HA) was studied by combining ultrafiltration and Inductively Coupled Plasma Mass Spectrometry techniques. REE-HA complexation experiments were performed at pH 3 for REE/C molar ratios ranging from ca 4 × 10 -4 to 2.7 × 10 -2. Results show that the relative amount of REE bound to HA strongly increases with decreasing REE/C. A middle-REE (MREE) downward concavity is shown by patterns at high metal loading, whereas patterns at low metal loading display a regular increase from La to Lu. Humic Ion Model VI modelling are close to the experimental data variations, provided that (i) the ΔLK 2 parameter (i.e. the Model VI parameter taken into account the presence of strong but low density binding sites) is allowed to increase regularly from La to Lu (from 1.1 to 2.1) and (ii) the published log KMA values (i.e. the REE-HA binding constants specific to Model VI) are slightly modified, in particular with respect to heavy REE. Modelling approach provided evidence that logKdREE patterns with varying REE/C likely arises because REE binding to HA occurs through two types of binding sites in different density: (i) a few strong sites that preferentially complex the heavy REE and thus control the logKdREE atterns at low REE/C; (ii) a larger amount of weaker binding sites that preferentially complex the middle-REE and thus control the logKdREE pattern at high REE/C. Hence, metal loading exerts a major effect on HA-mediated REE binding, which could explain the diversity of published conditional constants for REE binding with HA. A literature survey suggests that the few strong sites activated at low REE/C could be multidentate carboxylic sites, or perhaps N-, or P-functional groups. Finally, an examination of the literature field data proposed that the described loading effect could account for much of the variation in REE patterns observed in natural organic-rich waters (DOC > 5 mg L -1 and 4

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

    PubMed

    Zabet, Nicolae Radu; Adryan, Boris

    2015-01-01

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

  9. Role of non-coding RNA transcription around gene regulatory elements in transcription factor recruitment

    PubMed Central

    Ohta, Kunihiro

    2017-01-01

    ABSTRACT Eukaryotic cells produce a variety of non-coding RNAs (ncRNAs), many of which have been shown to play pivotal roles in biological processes such as differentiation, maintenance of pluripotency of stem cells, and cellular response to various stresses. Genome-wide analyses have revealed that many ncRNAs are transcribed around regulatory DNA elements located proximal or distal to gene promoters, but their biological functions are largely unknown. Recently, it has been demonstrated in yeast and mouse that ncRNA transcription around gene promoters and enhancers facilitates DNA binding of transcription factors to their target sites. These results suggest universal roles of promoter/enhancer-associated ncRNAs in the recruitment of transcription factors to their binding sites. PMID:27763805

  10. Production of element correction factors for thermoluminescent dosimeters

    SciTech Connect

    Plato, P.; Miklos, J.

    1985-11-01

    Approximately 80 processors of personal dosimetry in the United States use thermoluminescent dosimeters (TLDs). Recent demands that dosimetry processors be able to measure radiation doses to within +/- 50% of the correct value have focused attention on the reproducibility of the TL elements within each TLD. The phosphors for these TLDs are manufactured by three companies. A dosimetry processor faces three options concerning the quality of the TL elements purchased; trust the supplier's quality control program, screen new TL elements and discard those that are extremely bad, or use element correction factors (ECFs). The first option results in dosimetry processors failing the +/- 50% accuracy requirement due to excessive variability among the TL elements. The second option still permits large precision errors that come close to the +/- 50% accuracy requirement. This paper advocates the third option and presents a 10-step procedure to produce ECFs. The procedure ensures that the ECFs represent only variations among the TL elements and not variations caused by stability problems with the TLD reader. Following is an example of ECF production for 3000 TLDs.

  11. Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology.

    PubMed Central

    Katsani, K R; Hajibagheri, M A; Verrijzer, C P

    1999-01-01

    The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodelling and human cancers. Here, we study the role of the POZ domain of the GAGA transcription factor in promoter recognition. Natural target promoters for GAGA typically contain multiple GAGA-binding elements. Our results show that the POZ domain mediates strong co-operative binding to multiple sites but inhibits binding to single sites. Protein cross-linking and gel filtration chromatography experiments established that the POZ domain is required for GAGA oligomerization into higher order complexes. Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy revealed that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. Our results indicate a novel mode of DNA binding by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. We suggest a model in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation. PMID:9927429

  12. Sterol Regulatory Element Binding Protein Is a Principal Regulator of Anaerobic Gene Expression in Fission Yeast†

    PubMed Central

    Todd, Bridget L.; Stewart, Emerson V.; Burg, John S.; Hughes, Adam L.; Espenshade, Peter J.

    2006-01-01

    Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced ≥2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption. PMID:16537923

  13. Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

    PubMed

    Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

    2017-08-01

    Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

  14. All-atom structural models of insulin binding to the insulin receptor in the presence of a tandem hormone-binding element.

    PubMed

    Vashisth, Harish; Abrams, Cameron F

    2013-06-01

    Insulin regulates blood glucose levels in higher organisms by binding to and activating insulin receptor (IR), a constitutively homodimeric glycoprotein of the receptor tyrosine kinase (RTK) superfamily. Therapeutic efforts in treating diabetes have been significantly impeded by the absence of structural information on the activated form of the insulin/IR complex. Mutagenesis and photo-crosslinking experiments and structural information on insulin and apo-IR strongly suggest that the dual-chain insulin molecule, unlike the related single-chain insulin-like growth factors, binds to IR in a very different conformation than what is displayed in storage forms of the hormone. In particular, hydrophobic residues buried in the core of the folded insulin molecule engage the receptor. There is also the possibility of plasticity in the receptor structure based on these data, which may in part be due to rearrangement of the so-called CT-peptide, a tandem hormone-binding element of IR. These possibilities provide opportunity for large-scale molecular modeling to contribute to our understanding of this system. Using various atomistic simulation approaches, we have constructed all-atom structural models of hormone/receptor complexes in the presence of CT in its crystallographic position and a thermodynamically favorable displaced position. In the "displaced-CT" complex, many more insulin-receptor contacts suggested by experiments are satisfied, and our simulations also suggest that R-insulin potentially represents the receptor-bound form of hormone. The results presented in this work have further implications for the design of receptor-specific agonists/antagonists.

  15. Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression.

    PubMed

    Vernimmen, Douglas; Begon, Dominique; Salvador, Christophe; Gofflot, Stéphanie; Grooteclaes, Madeleine; Winkler, Rosita

    2003-02-15

    The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element in the ERBB2 promoter which is involved in the gene's overexpression. This cis element, located 501 bp upstream from the main ERBB2 transcription initiation site, binds a transcription factor called HTF (HER2 transcription factor). We report here the identification of HTF as an AP-2 (activator protein-2) transcription factor. The new cis element is bound by AP-2 with high affinity, compared with a previously described AP-2 binding site located 284 bp downstream. Co-transfection of an AP-2alpha expression vector with a reporter vector containing the newly identified AP-2 binding site in front of a minimal ERBB2 promoter induced a dose-dependent increase in transcriptional activity. We examined the contribution of the new AP-2 binding site to ERBB2 overexpression. For this purpose we abolished the new and/or the previously described AP-2 binding sequence by site-directed mutagenesis. The results show that the two functional AP-2 sites in the first 700 bp of the ERBB2 promoter co-operate to achieve maximal transcriptional activity.

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

    PubMed

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

    2017-02-09

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

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

    PubMed Central

    Rooj, AK.; Bronisz, A.

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization.

    PubMed

    Lee, Chic; Huang, Chun-Hsi

    2013-03-01

    The release of ChIP-seq data from the ENCyclopedia Of DNA Elements (ENCODE) and Model Organism ENCyclopedia Of DNA Elements (modENCODE) projects has significantly increased the amount of transcription factor (TF) binding affinity information available to researchers. However, scientists still routinely use TF binding site (TFBS) search tools to scan unannotated sequences for TFBSs, particularly when searching for lesser-known TFs or TFs in organisms for which ChIP-seq data are unavailable. The sequence analysis often involves multiple steps such as TF model collection, promoter sequence retrieval, and visualization; thus, several different tools are required. We have developed a novel integrated web tool named LASAGNA-Search that allows users to perform TFBS searches without leaving the web site. LASAGNA-Search uses the LASAGNA (Length-Aware Site Alignment Guided by Nucleotide Association) algorithm for TFBS alignment. Important features of LASAGNA-Search include (i) acceptance of unaligned variable-length TFBSs, (ii) a collection of 1726 TF models, (iii) automatic promoter sequence retrieval, (iv) visualization in the UCSC Genome Browser, and (v) gene regulatory network inference and visualization based on binding specificities. LASAGNA-Search is freely available at http://biogrid.engr.uconn.edu/lasagna_search/.

  20. The cis-acting elements involved in endonucleolytic cleavage of the 3' UTR of human IGF-II mRNAs bind a 50 kDa protein.

    PubMed Central

    Scheper, W; Holthuizen, P E; Sussenbach, J S

    1996-01-01

    Site-specific cleavage of human insulin-like growth factor II mRNAs requires two cis-acting elements, I and II, that are both located in the 3' untranslated region and separated by almost 2 kb. These elements can interact and form a stable RNA-RNA stem structure. In this study we have initiated the investigation of transacting factors involved in the cleavage of IGF-II mRNAs. The products of the cleavage reaction accumulate in the cytoplasm, suggesting that cleavage occurs in this cellular compartment. By electrophoretic mobility shift assays, we have identified a cytoplasmic protein with an apparent molecular weight of 48-50 kDa, IGF-II cleavage unit binding protein (ICU-BP), that binds to the stem structure formed by interaction of parts of the cis-acting elements I and II. The binding is resistant to high K+ concentrations and is dependent on Mg2+. In addition, ICU-BP binding is dependent on the cell density and correlates inversely with the IGM-II mRNA levels. In vivo cross-linking data show that this protein is associated with IGF-II mRNAs in vivo. PMID:8604329

  1. General expressions for the matrix elements of the tight-binding operator within the Racah-Wigner algebra*

    NASA Astrophysics Data System (ADS)

    Möller, Thomas

    2016-12-01

    General expressions for the matrix elements of the tight-binding operator are presented using the Racah-Wigner algebra, where the wave functions are expressed as coupled multiplet wave functions within a given angular momentum coupling scheme. The knowledge of all possible Slater determinants is not necessary and the matrix elements can be written as compact expressions computable with arbitrary accuracy.

  2. Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters.

    PubMed

    Srivastava, Surabhi; Puri, Deepika; Garapati, Hita Sony; Dhawan, Jyotsna; Mishra, Rakesh K

    2013-04-22

    Hox genes impart segment identity to body structures along the anterior-posterior axis and are crucial for the proper development of all organisms. Multiple regulatory elements, best defined in Drosophila melanogaster, ensure that Hox expression patterns follow the spatial and temporal colinearity reflected in their tight genomic organization. However, the precise mechanisms that regulate colinear patterns of Hox gene expression remain unclear, especially in higher vertebrates where it is not fully determined how the distinct activation domains of the tightly clustered Hox genes are defined independently of each other. Here, we report the identification of a large number of novel cis-elements at mammalian Hox clusters that can help in regulating their precise expression pattern. We have identified DNA elements at all four murine Hox clusters that show poor association with histone H3 in chromatin immunoprecipitation (ChIP)-chip tiling arrays. The majority of these elements lie in the intergenic regions segregating adjacent Hox genes; we demonstrate that they possess efficient enhancer-blocking activity in mammalian cells. Further, we find that these histone-free intergenic regions bear GA repeat motifs and associate with the vertebrate homolog of the GAGA binding boundary factor. This suggests that they can act as GAGA factor-dependent chromatin boundaries that create independent domains, insulating each Hox gene from the influence of neighboring regulatory elements. Our results reveal a large number of potential regulatory elements throughout the murine Hox clusters. We further demarcate the precise location of several novel cis-elements bearing chromatin boundary activity that appear to segregate successive Hox genes. This reflects a pattern reminiscent of the organization of homeotic genes in Drosophila, where such regulatory elements have been characterized. Our findings thus provide new insights into the regulatory processes and evolutionarily conserved

  3. Jaccard index based similarity measure to compare transcription factor binding site models

    PubMed Central

    2013-01-01

    Background Positional weight matrix (PWM) remains the most popular for quantification of transcription factor (TF) binding. PWM supplied with a score threshold defines a set of putative transcription factor binding sites (TFBS), thus providing a TFBS model. TF binding DNA fragments obtained by different experimental methods usually give similar but not identical PWMs. This is also common for different TFs from the same structural family. Thus it is often necessary to measure the similarity between PWMs. The popular tools compare PWMs directly using matrix elements. Yet, for log-odds PWMs, negative elements do not contribute to the scores of highly scoring TFBS and thus may be different without affecting the sets of the best recognized binding sites. Moreover, the two TFBS sets recognized by a given pair of PWMs can be more or less different depending on the score thresholds. Results We propose a practical approach for comparing two TFBS models, each consisting of a PWM and the respective scoring threshold. The proposed measure is a variant of the Jaccard index between two TFBS sets. The measure defines a metric space for TFBS models of all finite lengths. The algorithm can compare TFBS models constructed using substantially different approaches, like PWMs with raw positional counts and log-odds. We present the efficient software implementation: MACRO-APE (MAtrix CompaRisOn by Approximate P-value Estimation). Conclusions MACRO-APE can be effectively used to compute the Jaccard index based similarity for two TFBS models. A two-pass scanning algorithm is presented to scan a given collection of PWMs for PWMs similar to a given query. Availability and implementation MACRO-APE is implemented in ruby 1.9; software including source code and a manual is freely available at http://autosome.ru/macroape/ and in supplementary materials. PMID:24074225

  4. Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking*

    PubMed Central

    Xu, SongXiao; Wong, Catherine C. L.; Tong, Edith H. Y.; Chung, Stephen S. M.; Yates, John R.; Yin, YiBing; Ko, Ben C. B.

    2008-01-01

    The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1α1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process. PMID:18411282

  5. Phosphorylation by casein kinase 1 regulates tonicity-induced osmotic response element-binding protein/tonicity enhancer-binding protein nucleocytoplasmic trafficking.

    PubMed

    Xu, SongXiao; Wong, Catherine C L; Tong, Edith H Y; Chung, Stephen S M; Yates, John R; Yin, YiBing; Ko, Ben C B

    2008-06-20

    The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

  6. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

    PubMed Central

    Müller, Gerd A.; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Padi, Megha; Fischer, Martin; Litovchick, Larisa; DeCaprio, James A.; Engeland, Kurt

    2012-01-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G0/G1. It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G0. Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G0/G1, but also for activation in S, G2 and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle. PMID:22064854

  7. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes.

    PubMed

    Müller, Gerd A; Quaas, Marianne; Schümann, Michael; Krause, Eberhard; Padi, Megha; Fischer, Martin; Litovchick, Larisa; DeCaprio, James A; Engeland, Kurt

    2012-02-01

    Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G(0)/G(1). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G(0). Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G(0)/G(1), but also for activation in S, G(2) and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle.

  8. The housekeeping promoter from the mouse CpG island HTF9 contains multiple protein-binding elements that are functionally redundant.

    PubMed Central

    Somma, M P; Pisano, C; Lavia, P

    1991-01-01

    The mouse CpG-rich island HTF9 harbours the divergent RNA initiation sites shared by two genes that are both expressed in a housekeeping fashion. In this work we have analyzed the architecture of the HTF9 promoter. Gel shift assays were first employed to locate nuclear factor-binding sites within HTF9. Multiple protein-binding sites were identified across a 500 bp-long region, two of which appear to interact with novel factors. Deletion analysis was used to determine the requirements for the different sites in transient expression of a CAT reporter gene. Although multiple elements contributed to the overall promoter strength in each orientation, extensive deletions failed to affect the basal level of transcription from HTF9 in either direction. Thus, only a subset of elements is necessary to activate transcription from HTF9. Functional redundancy may be a general feature of housekeeping CpG-rich promoters. Images PMID:1711672

  9. NICER elements: a family of nerve growth factor-inducible cAMP-extinguishable retrovirus-like elements.

    PubMed Central

    Cho, K O; Minsk, B; Wagner, J A

    1990-01-01

    We have shown previously that the transcription of the gene designated d5 is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original d5 cDNA hybridized to numerous restriction fragments in genomic DNA; (ii) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members of this family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements. Images PMID:2160077

  10. Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

    PubMed

    Hirose, F; Yamaguchi, M; Matsukage, A

    1999-09-01

    The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

  11. Strategy for molecular beacon binding readout: separating molecular recognition element and signal reporter.

    PubMed

    Wang, Yongxiang; Li, Jishan; Jin, Jianyu; Wang, Hao; Tang, Hongxing; Yang, Ronghua; Wang, Kemin

    2009-12-01

    A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. Hg2+ was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of Hg2+ between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing Hg2+ ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 microM, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter.

  12. Protein Phosphatase 2A (PP2A) Regulates Low Density Lipoprotein Uptake through Regulating Sterol Response Element-binding Protein-2 (SREBP-2) DNA Binding*

    PubMed Central

    Rice, Lyndi M.; Donigan, Melissa; Yang, Muhua; Liu, Weidong; Pandya, Devanshi; Joseph, Biny K.; Sodi, Valerie; Gearhart, Tricia L.; Yip, Jenny; Bouchard, Michael; Nickels, Joseph T.

    2014-01-01

    LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake. PMID:24770487

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

    PubMed

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

    2017-02-07

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

  14. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    SciTech Connect

    Loots, G; Ovcharenko, I

    2006-08-08

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes entitled ECRbase that is constructed from a collection of pairwise vertebrate genome alignments produced by the ECR Browser database. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a collection of promoters in all vertebrate genomes presented in the database. The database also contains a collection of annotated transcription factor binding sites (TFBS) in all ECRs and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and two pufferfish genomes. It is freely accessible at http://ECRbase.dcode.org.

  15. Identifying combinatorial regulation of transcription factors and binding motifs

    PubMed Central

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

    2004-01-01

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

  16. A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    PubMed Central

    Vallström, Anna; Olofsson, Annelie; Öhman, Carina; Rakhimova, Lena; Borén, Thomas; Engstrand, Lars; Brännström, Kristoffer; Arnqvist, Anna

    2014-01-01

    During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors. PMID:24991812

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

    SciTech Connect

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

    2007-06-01

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

  18. Detection of active transcription factor binding sites with the combination of DNase hypersensitivity and histone modifications.

    PubMed

    Gusmao, Eduardo G; Dieterich, Christoph; Zenke, Martin; Costa, Ivan G

    2014-11-15

    The identification of active transcriptional regulatory elements is crucial to understand regulatory networks driving cellular processes such as cell development and the onset of diseases. It has recently been shown that chromatin structure information, such as DNase I hypersensitivity (DHS) or histone modifications, significantly improves cell-specific predictions of transcription factor binding sites. However, no method has so far successfully combined both DHS and histone modification data to perform active binding site prediction. We propose here a method based on hidden Markov models to integrate DHS and histone modifications occupancy for the detection of open chromatin regions and active binding sites. We have created a framework that includes treatment of genomic signals, model training and genome-wide application. In a comparative analysis, our method obtained a good trade-off between sensitivity versus specificity and superior area under the curve statistics than competing methods. Moreover, our technique does not require further training or sequence information to generate binding location predictions. Therefore, the method can be easily applied on new cell types and allow flexible downstream analysis such as de novo motif finding. Our framework is available as part of the Regulatory Genomics Toolbox. The software information and all benchmarking data are available at http://costalab.org/wp/dh-hmm. ivan.costa@rwth-aachen.de or eduardo.gusmao@rwth-aachen.de Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

    PubMed Central

    Hamada, K; Gleason, S L; Levi, B Z; Hirschfeld, S; Appella, E; Ozato, K

    1989-01-01

    Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor. Images PMID:2554307

  20. Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone:Receptor Binding Specificity.

    PubMed

    Křížková, Květoslava; Chrudinová, Martina; Povalová, Anna; Selicharová, Irena; Collinsová, Michaela; Vaněk, Václav; Brzozowski, Andrzej M; Jiráček, Jiří; Žáková, Lenka

    2016-05-31

    Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain.

  1. Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage

    PubMed Central

    Ross, Heather L.; Nonnemacher, Michael R.; Hogan, Tricia H.; Quiterio, Shane J.; Henderson, Andrew; McAllister, John J.; Krebs, Fred C.; Wigdahl, Brian

    2001-01-01

    Recent observations have shown two CCAAT/enhancer binding protein (C/EBP) binding sites to be critically important for efficient human immunodeficiency virus type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a cell type likely involved in transport of the virus to the brain. Additionally, sequence variation at C/EBP site I, which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB), has been shown to affect HIV-1 long terminal repeat (LTR) activity. Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family, we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I. Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent, naturally occurring, low-affinity C/EBP site. This biophysical interaction appears to occur via at least two mechanisms. First, low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites. In addition, CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites. This interaction is reciprocal, since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB. Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms. Most importantly, sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation, a treatment that leads to increases in C/EBP activation. Thus, HIV-1 LTR ATF/CREB binding site sequence

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

    PubMed

    Séralini, G E

    1996-01-01

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

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

    SciTech Connect

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

    1989-10-01

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

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

    PubMed Central

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

    2005-01-01

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

  5. Effects of cytosine methylation on transcription factor binding sites

    PubMed Central

    2014-01-01

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

  6. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

    PubMed

    Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

    1999-03-01

    The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

  7. Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

    PubMed

    Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

    2017-07-18

    Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca(2+)-binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca(2+) but detaches from DRE under Ca(2+) stimulation, allowing gene expression. The Ca(2+) binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca(2+) binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca(2+). Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca(2+)-binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca(2+) signal to CREB-mediated gene transcription.

  8. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells

    PubMed Central

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation. PMID:26941778

  9. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function.

    PubMed

    Ciolkowski, Ingo; Wanke, Dierk; Birkenbihl, Rainer P; Somssich, Imre E

    2008-09-01

    WRKY transcription factors have been shown to play a major role in regulating, both positively and negatively, the plant defense transcriptome. Nearly all studied WRKY factors appear to have a stereotypic binding preference to one DNA element termed the W-box. How specificity for certain promoters is accomplished therefore remains completely unknown. In this study, we tested five distinct Arabidopsis WRKY transcription factor subfamily members for their DNA binding selectivity towards variants of the W-box embedded in neighboring DNA sequences. These studies revealed for the first time differences in their binding site preferences, which are partly dependent on additional adjacent DNA sequences outside of the TTGACY-core motif. A consensus WRKY binding site derived from these studies was used for in silico analysis to identify potential target genes within the Arabidopsis genome. Furthermore, we show that even subtle amino acid substitutions within the DNA binding region of AtWRKY11 strongly impinge on its binding activity. Additionally, all five factors were found localized exclusively to the plant cell nucleus and to be capable of trans-activating expression of a reporter gene construct in vivo.

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

    PubMed

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

    2016-06-01

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

  11. Epstein–Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

    PubMed Central

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B.; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G.; Sinclair, Alison J.

    2015-01-01

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

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

    SciTech Connect

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

    2004-10-28

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

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

    PubMed

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

    1999-02-05

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

  14. Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

    PubMed

    Mitton, B; Chae, H-D; Hsu, K; Dutta, R; Aldana-Masangkay, G; Ferrari, R; Davis, K; Tiu, B C; Kaul, A; Lacayo, N; Dahl, G; Xie, F; Li, B X; Breese, M R; Landaw, E M; Nolan, G; Pellegrini, M; Romanov, S; Xiao, X; Sakamoto, K M

    2016-12-01

    The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

  15. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    PubMed Central

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  16. Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

    PubMed Central

    Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

  17. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation.

    PubMed

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-08-14

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

  18. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    PubMed Central

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  19. Core-level binding-energy shifts for the metallic elements

    NASA Astrophysics Data System (ADS)

    Johansson, Börje; Mårtensson, Nils

    1980-05-01

    A general treatment of core-level binding-energy shifts in metals relative to the free atom is introduced and applied to all elemental metals in the Periodic Table. The crucial ingredients of the theoretical description are (a) the assumption of a fully screened final state in the metallic case and (b) the (Z+1) approximation for the screening valence charge distribution around the core-ionized site. This core-ionized site is, furthermore, treated as an impurity in an otherwise perfect metal. The combination of the complete screening picture and the (Z+1) approximation makes it possible to introduce a Born-Haber cycle which connects the initial state with the final state of the core-ionization process. From this cycle it becomes evident that the main contributions to the core-level shift are the cohesive energy difference between the (Z+1) and Z metal and an appropriate ionization energy of the (Z+1) atom (usually the first ionization potential). The appearance of the ionization potential in the shift originates from the assumption of a charge-neutral final state, while the contribution from the cohesive energies essentially describes the change of bonding properties between the initial and final state of the site. The calculated shifts show very good agreement with available experimental values (at present, for 19 elements). For the other elements we have made an effort to combine experimental ionization potentials with theoretical calculations in order to obtain accurate estimates of some of the atomic-core-level binding energies. Such energies together with measured metallic binding energies give "pseudoexperimental" shifts for many elements. Our calculated core-level shifts agree exceedingly well also with these data. For some of the transition elements the core-level shift shows a deviating behavior in comparison with that of neighboring elements. This is shown to be due to a difference in the atomic ground-state configuration, such as, for example, d5s in

  20. MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

    PubMed Central

    Horie, Takahiro; Nishino, Tomohiro; Baba, Osamu; Kuwabara, Yasuhide; Nakao, Tetsushi; Nishiga, Masataka; Usami, Shunsuke; Izuhara, Masayasu; Sowa, Naoya; Yahagi, Naoya; Shimano, Hitoshi; Matsumura, Shigenobu; Inoue, Kazuo; Marusawa, Hiroyuki; Nakamura, Tomoyuki; Hasegawa, Koji; Kume, Noriaki; Yokode, Masayuki; Kita, Toru; Kimura, Takeshi; Ono, Koh

    2013-01-01

    MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo. PMID:24300912

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

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

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

  2. GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes

    PubMed Central

    Bannert, Norbert; Avots, Andris; Baier, Michael; Serfling, Edgar; Kurth, Reinhard

    1999-01-01

    Interleukin 16 (IL-16) is a chemotactic cytokine that binds to the CD4 receptor and affects the activation of T cells and replication of HIV. It is expressed as a large 67-kDa precursor protein (pro-IL-16) in lymphocytes, macrophages, and mast cells, as well as in airway epithelial cells from asthmatics after challenge with allergen. This pro-IL-16 is subsequently processed to the mature cytokine of 13 kDa. To study the expression of IL-16 at the transcriptional level, we cloned the human chromosomal IL-16 gene and analyzed its promoter. The human IL-16 gene consists of seven exons and six introns. The 5′ sequences up to nucleotide −120 of the human and murine IL-16 genes share >84% sequence homology and harbor promoter elements for constitutive and inducible transcription in T cells. Although both promoters lack any TATA box, they contain two CAAT box-like motifs and three binding sites of GA-binding protein (GABP) transcription factors. Two of these motifs are part of a highly conserved and inducible dyad symmetry element shown previously to control a remote IL-2 enhancer and the CD18 promoter. In concert with the coactivator CREB binding protein/p300, which interacts with GABPα, the binding of GABPα and -β to the dyad symmetry element controls the induction of IL-16 promoter in T cells. Supplementing the data on the processing of pro-IL-16, our results indicate the complexity of IL-16 expression, which is tightly controlled at the transcriptional and posttranslational levels in T lymphocytes. PMID:9990060

  3. Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein.

    PubMed

    Li, Wencheng; Liu, Jiao; Hammond, Sean L; Tjalkens, Ronald B; Saifudeen, Zubaida; Feng, Yumei

    2015-07-15

    We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

  4. Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein

    PubMed Central

    Li, Wencheng; Liu, Jiao; Hammond, Sean L.; Tjalkens, Ronald B.; Saifudeen, Zubaida

    2015-01-01

    We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter. PMID:25994957

  5. Cyclic adenosine monophosphate-response element-binding protein mediates the proangiogenic or proinflammatory activity of gremlin.

    PubMed

    Corsini, Michela; Moroni, Emanuela; Ravelli, Cosetta; Andrés, Germán; Grillo, Elisabetta; Ali, Imran H; Brazil, Derek P; Presta, Marco; Mitola, Stefania

    2014-01-01

    Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs). Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin. Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Similar effects were induced by the canonical VEGFR2 ligand VEGF-A165. Together, the results underline the tight cross-talk between angiogenesis and inflammation and demonstrate a crucial role of CREB activation in the modulation of the VEGFR2-mediated proinflammatory/proangiogenic response of ECs to gremlin.

  6. Mutations and Binding Sites of Human Transcription Factors

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2000-01-01

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

  8. Phosphate binding protein as the biorecognition element in a biosensor for phosphate

    NASA Technical Reports Server (NTRS)

    Salins, Lyndon L E.; Deo, Sapna K.; Daunert, Sylvia

    2004-01-01

    This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the

  9. Phosphate binding protein as the biorecognition element in a biosensor for phosphate

    NASA Technical Reports Server (NTRS)

    Salins, Lyndon L E.; Deo, Sapna K.; Daunert, Sylvia

    2004-01-01

    This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the

  10. Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements

    PubMed Central

    Fiorentino, Gabriella; Cannio, Raffaele; Rossi, Mosè; Bartolucci, Simonetta

    2003-01-01

    A transcriptionally active region has been identified in the 5′ flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of ∼16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes. PMID:12813087

  11. Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to its Heparin-Binding Site

    PubMed Central

    Karuturi, Rajesh; Al-Horani, Rami A.; Mehta, Shrenik C.; Gailani, David; Desai, Umesh R.

    2013-01-01

    To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs. PMID:23451707

  12. Human Research Program: Space Human Factors and Habitability Element

    NASA Technical Reports Server (NTRS)

    Russo, Dane M.

    2007-01-01

    The three project areas of the Space Human Factors and Habitability Element work together to achieve a working and living environment that will keep crews healthy, safe, and productive throughout all missions -- from Earth orbit to Mars expeditions. The Advanced Environmental Health (AEH) Project develops and evaluates advanced habitability systems and establishes requirements and health standards for exploration missions. The Space Human Factors Engineering (SHFE) Project s goal is to ensure a safe and productive environment for humans in space. With missions using new technologies at an ever-increasing rate, it is imperative that these advances enhance crew performance without increasing stress or risk. The ultimate goal of Advanced Food Technology (AFT) Project is to develop and deliver technologies for human centered spacecraft that will support crews on missions to the moon, Mars, and beyond.

  13. Identification of novel factors that bind to the PRD I region of the human beta-interferon promoter.

    PubMed Central

    Whiteside, S T; Visvanathan, K V; Goodbourn, S

    1992-01-01

    Treatment of cells with virus or synthetic double-stranded RNA (dsRNA) leads to the transient transcriptional activation of the beta-interferon gene. Genetic analysis has revealed that the 5' regulatory sequence responsible for this induction contains multiple positive and negative elements. One of these, Positive Regulatory Domain I (PRD I), has been shown to bind the positively-acting transcription factor IRF-1. In this study we show that this element is inducible under conditions where IRF-1 cannot be detected, suggesting that additional cellular factors are involved in the induction process. To investigate the existence of such factors we have analysed the range and properties of PRD I-binding activities present in HeLa cells. In addition to the repressor protein IRF-2, several novel factors can bind to PRD I in uninduced cells: two of these have properties consistent with a role in negative regulation; levels of two others increase upon priming, and may be alternative candidates for activators. Upon induction we also observe a novel factor whose appearance does not depend upon de novo protein synthesis, and which appears to be a truncated form of IRF-2. The potential involvement of these factors in regulating the beta-interferon gene is discussed. Images PMID:1579446

  14. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  15. The Binding of Syndapin SH3 Domain to Dynamin Proline-rich Domain Involves Short and Long Distance Elements.

    PubMed

    Luo, Lin; Xue, Jing; Kwan, Ann; Gamsjaeger, Roland; Wielens, Jerome; von Kleist, Lisa; Cubeddu, Liza; Guo, Zhong; Stow, Jennifer L; Parker, Michael W; Mackay, Joel P; Robinson, Phillip J

    2016-04-29

    Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction.

  16. A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

    PubMed Central

    Baßler, Jochen; Paternoga, Helge; Holdermann, Iris; Thoms, Matthias; Granneman, Sander; Barrio-Garcia, Clara; Nyarko, Afua; Stier, Gunter; Clark, Sarah A.; Schraivogel, Daniel; Kallas, Martina; Beckmann, Roland; Tollervey, David

    2014-01-01

    Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation. PMID:25404745

  17. DNA-binding mechanism of the Hippo pathway transcription factor TEAD4.

    PubMed

    Shi, Z; He, F; Chen, M; Hua, L; Wang, W; Jiao, S; Zhou, Z

    2017-07-27

    TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarily conserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain-DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

  18. Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat.

    PubMed Central

    Winandy, S; Renjifo, B; Li, Y; Hopkins, N

    1992-01-01

    Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation. Images PMID:1501272

  19. An intact DNA-binding domain is not required for peroxisome proliferator-activated receptor gamma (PPARgamma) binding and activation on some PPAR response elements.

    PubMed

    Temple, Karla A; Cohen, Ronald N; Wondisford, Sarah R; Yu, Christine; Deplewski, Dianne; Wondisford, Fredric E

    2005-02-04

    Peroxisome proliferator-activated receptor gamma (PPARgamma) interacts with retinoid X receptor (RXR) on PPAR response elements (PPREs) to regulate transcription of PPAR-responsive genes. To investigate the binding of PPARgamma and RXR to PPREs, three mutations were constructed in the DNA-binding domains of PPARgamma; two of the mutants maintained the structure of zinc finger I (PPARgamma-GS and PPARgamma-AA), and a third mutation disrupted the protein structure of zinc finger I (PPARgamma-CS). Results indicated that the mutations of PPARgamma that maintained intact zinc fingers were capable of binding to a variety of PPREs in the presence of RXR and could activate transcription on several PPREs. In parallel, a mutation was created in the DNA-binding domain of RXRalpha that maintained the structure of the zinc fingers (RXR-GS) but did not bind DNA and was transcriptionally inactive. Examination of the 3' half-site of several PPREs revealed that variations from the consensus sequence reduced or abolished transcriptional activity, but conversion to consensus improved transcriptional activity with PPARgamma-GS and PPARgamma-AA. Examination of the 5' half-site indicated that the upstream three nucleotides were more important for transcriptional activity than the downstream three nucleotides. Our data demonstrated that stringent binding of RXR to the 3' half-site of a PPRE is more influential on the binding of the PPARgamma/RXR heterodimer than the ability of PPARgamma to bind DNA. Thus, unlike RXR, PPARgamma exhibits promiscuity in binding on a PPRE, suggesting that the definition of a PPRE for PPARgamma may need to be expanded.

  20. Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.

    PubMed

    Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu; Vishnivetskiy, Sergey A; Iverson, Tina M; Gurevich, Vsevolod V; Sanders, Charles R

    2013-01-15

    Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ~80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.

  1. Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

    PubMed Central

    Hales, L M; Gumport, R I; Gardner, J F

    1996-01-01

    DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF. PMID:8650000

  2. Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

    PubMed

    Hales, L M; Gumport, R I; Gardner, J F

    1996-05-01

    DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.

  3. Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5

    PubMed Central

    Currie, Simon L.; Lau, Desmond K. W.; Doane, Jedediah J.; Whitby, Frank G.; Okon, Mark; McIntosh, Lawrence P.

    2017-01-01

    Abstract Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein–protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins. PMID:28161714

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

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

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

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

    PubMed

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

    2008-03-01

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

  6. Genetic variation in T-box binding element functionally affects SCN5A/SCN10A enhancer.

    PubMed

    van den Boogaard, Malou; Wong, L Y Elaine; Tessadori, Federico; Bakker, Martijn L; Dreizehnter, Lisa K; Wakker, Vincent; Bezzina, Connie R; 't Hoen, Peter A C; Bakkers, Jeroen; Barnett, Phil; Christoffels, Vincent M

    2012-07-01

    The contraction pattern of the heart relies on the activation and conduction of the electrical impulse. Perturbations of cardiac conduction have been associated with congenital and acquired arrhythmias as well as cardiac arrest. The pattern of conduction depends on the regulation of heterogeneous gene expression by key transcription factors and transcriptional enhancers. Here, we assessed the genome-wide occupation of conduction system-regulating transcription factors TBX3, NKX2-5, and GATA4 and of enhancer-associated coactivator p300 in the mouse heart, uncovering cardiac enhancers throughout the genome. Many of the enhancers colocalized with ion channel genes repressed by TBX3, including the clustered sodium channel genes Scn5a, essential for cardiac function, and Scn10a. We identified 2 enhancers in the Scn5a/Scn10a locus, which were regulated by TBX3 and its family member and activator, TBX5, and are functionally conserved in humans. We also provided evidence that a SNP in the SCN10A enhancer associated with alterations in cardiac conduction patterns in humans disrupts TBX3/TBX5 binding and reduces the cardiac activity of the enhancer in vivo. Thus, the identification of key regulatory elements for cardiac conduction helps to explain how genetic variants in noncoding regulatory DNA sequences influence the regulation of cardiac conduction and the predisposition for cardiac arrhythmias.

  7. Ectopic expression of dehydration responsive element binding proteins (StDREB2) confers higher tolerance to salt stress in potato.

    PubMed

    Bouaziz, Donia; Pirrello, Julien; Ben Amor, Hela; Hammami, Asma; Charfeddine, Mariam; Dhieb, Amina; Bouzayen, Mondher; Gargouri-Bouzid, Radhia

    2012-11-01

    Dehydration responsive element binding proteins (DREB) are members of a larger family of transcription factors, many of which have been reported to contribute to plant responses to abiotic stresses in several species. While, little is known about their role in potato (Solanum tuberosum). This report describes the cloning and characterization of a DREB transcription factor cDNA, StDREB2, isolated from potato (cv Nicola) plants submitted to salt treatment. Based on a multiple sequence alignment, this protein was classified into the A-5 group of DREB subfamily. Expression studies revealed that StDREB2 was induced in leaves, roots and stems upon various abiotic stresses and in response to exogenous treatment with abscisic acid (ABA). In agreement with this expression pattern, over-expression of StDREB2 in transgenic potato plants resulted in enhanced tolerance to salt stress. These data suggest that the isolated StDREB2 encodes a functional protein involved in plant response to different abiotic stresses. An electrophoretic mobility shift assay (EMSA) indicated that the StDREB2 protein bound specifically to the DRE core element (ACCGAGA) in vitro. Moreover, Semi quantitative RT-PCR analysis revealed that the transcript level of a putative target gene i.e. δ(1)-pyrroline-5-carboxylate synthase (P5CS) was up-regulated in transgenic plants submitted to salt stress conditions. A concomitant increase in proline accumulation was also observed under these conditions. Taking together, all these data suggest that StDREB2 takes part in the processes underlying plant responses to abiotic stresses probably via the regulation of ABA hormone signaling and through a mechanism allowing proline synthesis. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

    PubMed

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

    1999-06-15

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

  9. Repressor element-1 silencing transcription factor (REST) is present in human control and Huntington's disease neurones.

    PubMed

    Schiffer, Davide; Caldera, Valentina; Mellai, Marta; Conforti, Paola; Cattaneo, Elena; Zuccato, Chiara

    2014-12-01

    The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients. Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis. REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed. These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD. © 2014 British Neuropathological Society.

  10. Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

    PubMed Central

    Garrity, P A; Chen, D; Rothenberg, E V; Wold, B J

    1994-01-01

    Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using

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

    PubMed Central

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

    2013-01-01

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

  12. Bioadsorption of rare earth elements through cell surface display of lanthanide binding tags

    SciTech Connect

    Park, Dan M.; Reed, David W.; Yung, Mimi C.; Eslamimanesh, Ali; Lencka, Malgorzata M.; Anderko, Andrzej; Fujita, Yoshiko; Riman, Richard E.; Navrotsky, Alexandra; Jiao, Yongqin

    2016-02-02

    In this study, with the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb3+ could be effectively recovered using citrate, consistent with thermodynamic speciation calculations that predicted strong complexation of Tb3+ by citrate. No reduction in Tb3+ adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.

  13. Bioadsorption of rare earth elements through cell surface display of lanthanide binding tags

    DOE PAGES

    Park, Dan M.; Reed, David W.; Yung, Mimi C.; ...

    2016-02-02

    In this study, with the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb3+ could be effectively recovered using citrate,more » consistent with thermodynamic speciation calculations that predicted strong complexation of Tb3+ by citrate. No reduction in Tb3+ adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.« less

  14. Tonicity response element binding protein associated with neuronal cell death in the experimental diabetic retinopathy

    PubMed Central

    Kim, Seong-Jae; Kim, Hwajin; Park, Jeongsook; Chung, Inyoung; Kwon, Hyug-Moo; Choi, Wan-Sung; Yoo, Ji-Myong

    2014-01-01

    AIM To study the contribution of tonicity response element binding protein (TonEBP) in retinal ganglion cell (RGC) death of diabetic retinopathy (DR). METHODS Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received vehicle (phosphate-buffered saline). All mice were killed 2mo after injections, and the extent of cell death and the protein expression levels of TonEBP and aldose reductase (AR) were examined. RESULTS The TonEBP and AR protein levels and the death of RGC were significantly increased in the retinas of diabetic mice compared with controls 2mo after the induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive signals co-localized with TonEBP immunoreactive RGC. These changes were increased in the diabetic retinas compared with controls. CONCLUSION The present data show that AR and TonEBP are upregulated in the DR and TonEBP may contribute to apoptosis of RGC in the DR. PMID:25540742

  15. Tonicity response element binding protein associated with neuronal cell death in the experimental diabetic retinopathy.

    PubMed

    Kim, Seong-Jae; Kim, Hwajin; Park, Jeongsook; Chung, Inyoung; Kwon, Hyug-Moo; Choi, Wan-Sung; Yoo, Ji-Myong

    2014-01-01

    To study the contribution of tonicity response element binding protein (TonEBP) in retinal ganglion cell (RGC) death of diabetic retinopathy (DR). Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received vehicle (phosphate-buffered saline). All mice were killed 2mo after injections, and the extent of cell death and the protein expression levels of TonEBP and aldose reductase (AR) were examined. The TonEBP and AR protein levels and the death of RGC were significantly increased in the retinas of diabetic mice compared with controls 2mo after the induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive signals co-localized with TonEBP immunoreactive RGC. These changes were increased in the diabetic retinas compared with controls. The present data show that AR and TonEBP are upregulated in the DR and TonEBP may contribute to apoptosis of RGC in the DR.

  16. Bioadsorption of Rare Earth Elements through Cell Surface Display of Lanthanide Binding Tags.

    PubMed

    Park, Dan M; Reed, David W; Yung, Mimi C; Eslamimanesh, Ali; Lencka, Malgorzata M; Anderko, Andrzej; Fujita, Yoshiko; Riman, Richard E; Navrotsky, Alexandra; Jiao, Yongqin

    2016-03-01

    With the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb(3+) could be effectively recovered using citrate, consistent with thermodynamic speciation calculations that predicted strong complexation of Tb(3+) by citrate. No reduction in Tb(3+) adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.

  17. Modeling RNA-ligand interactions: the Rev-binding element RNA-aminoglycoside complex.

    PubMed

    Leclerc, F; Cedergren, R

    1998-01-15

    An approach to the modeling of ligand-RNA complexes has been developed by combining three-dimensional structure-activity relationship (3D-SAR) computations with a docking protocol. The ability of 3D-SAR to predict bound conformations of flexible ligands was first assessed by attempting to reconstruct the known, bound conformations of phenyloxazolines complexed with human rhinovirus 14 (HRV14) RNA. Subsequently, the same 3D-SAR analysis was applied to the identification of bound conformations of aminoglycosides which associate with the Rev-binding element (RBE) RNA. Bound conformations were identified by parsing ligand conformational data sets with pharmacophores determined by the 3D-SAR analysis. These "bioactive" structures were docked to the receptor RNA, and optimization of the complex was undertaken by extensive searching of ligand conformational space coupled with molecular dynamics computations. The similarity between the bound conformations of the ligand from the 3D-SAR analysis and those found in the docking protocol suggests that this methodology is valid for the prediction of bound ligand conformations and the modeling of the structure of the ligand-RNA complexes.

  18. Interference on cytoplasmic polyadenylation element-binding proteins affects the invasion ability of glioma stem cells.

    PubMed

    Liu, H L; Huo, J F; Liu, Z J; Chen, X B

    2015-10-28

    Glioma stem cells derived from primary cultures were divided into an experiment group, a control group, and a blank group and subjected to cytoplasmic polyadenilation element-binding protein (CPEBs) interference, transfection with empty vector, and normal culture, respectively, to compare their invasion abilities. Western blotting showed that siRNA-3 had the strongest interfering effect on CPEBs. CPEBs were expressed in the experiment group with green fluorescence at an expression rate of over 70%. Significantly lower CPEB expression was observed in the experiment group compared to in the control and blank groups (P < 0.05). After 48-h treatment, the apoptotic rate in the experiment group was 21.43%, which was significantly higher than that in the blank (0.51%) and control (1.43%) groups (P < 0.05). After 3 days of treatment, the experiment group grew significantly more slowly than did the control and blank groups (P < 0.05). The transwell invasion assay showed that significantly fewer cells in the experiment group penetrated the membrane than did cells in the control and blank groups (P < 0.05). After CPEB interference, the growth, proliferation, and invasion of glioma stem cells were substantially inhibited, providing support for targeted therapy of glioma and for improving prognosis.

  19. The Ewing sarcoma protein (EWS) binds directly to the proximal elements of the macrophage-specific promoter of the CSF-1 receptor (csf1r) gene.

    PubMed

    Hume, David A; Sasmono, Tedjo; Himes, S Roy; Sharma, Sudarshana M; Bronisz, Agnieszka; Constantin, Myrna; Ostrowski, Michael C; Ross, Ian L

    2008-05-15

    Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.

  20. Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.

    PubMed

    Saha, Abhijit; Kizaki, Seiichiro; De, Debojyoti; Endo, Masayuki; Kim, Kyeong Kyu; Sugiyama, Hiroshi

    2016-08-19

    Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

    PubMed Central

    Zechel, C; Shen, X Q; Chambon, P; Gronemeyer, H

    1994-01-01

    We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs. Images PMID:8137825

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

    PubMed Central

    Cabrer, Bartolomé; Vázquez, David; Modolell, Juan

    1972-01-01

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

  3. 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

  4. Finite element methods of studying mechanical factors in blood flow.

    PubMed

    Davids, N

    1981-01-01

    This paper reviews some biomechanical analyses of blood flow in large arteries based on a general computer modeling using the finite element method. We study the following question: What is the role played by the interrelated factors of mechanical stress, flow irregularities, and diffusion through the endothelium on the etiology of atherosclerosis or the aggravation of vascular injury. It presents the computational features of the method and stresses the physiological significance of the results, such as the effect of geometric complexities, material nonlinearities, and non-Newtonian rheology of the blood. The specific mechanical and fluid dynamic factors analyzed are wall shear stress, flow profiles, and pressure variations. After simulating tubes of circular cross section, we apply the analysis to a number of physiological situations of significance, including blood flow in the entrance region, at bifurcations, in the annular region between an inserted catheter of varying diameter and the vessel. A model study of pulsatile flow in a 60 degree bifurcated channel of velocity profiles provided corroborative measurements of these processes with special emphasis on reversed or distributed flow conditions. The corresponding analysis was extended to the situation in which flow separates and reverses in the neighborhood of stagnation points. This required developing the nonlinear expression for the convective velocity change in the medium. A computer algorithm was developed to handle simultaneous effects of pressure and viscous forces on velocity change across the element and applied to the canine prebranch arterial segment. For mean physiological flow conditions, low shear stresses (0-10 dynes/cm2) are predicted near the wall in the diverging plane, higher values (50 dynes/cm2) along the converging sides of the wall. Backflow is predicted along the outer wall, pressure recovery prior to and into the branches, and a peak shear at the divider lip.

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

    PubMed

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

    2015-08-20

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

  6. Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

    PubMed

    Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

    2017-09-08

    Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

  7. Factorizing the factorization - a spectral-element solver for elliptic equations with linear operation count

    NASA Astrophysics Data System (ADS)

    Huismann, Immo; Stiller, Jörg; Fröhlich, Jochen

    2017-10-01

    The paper proposes a novel factorization technique for static condensation of a spectral-element discretization matrix that yields a linear operation count of just 13N multiplications for the residual evaluation, where N is the total number of unknowns. In comparison to previous work it saves a factor larger than 3 and outpaces unfactored variants for all polynomial degrees. Using the new technique as a building block for a preconditioned conjugate gradient method yields linear scaling of the runtime with N which is demonstrated for polynomial degrees from 2 to 32. This makes the spectral-element method cost effective even for low polynomial degrees. Moreover, the dependence of the iterative solution on the element aspect ratio is addressed, showing only a slight increase in the number of iterations for aspect ratios up to 128. Hence, the solver is very robust for practical applications.

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

    PubMed Central

    Andrilenas, Kellen K.; Penvose, Ashley

    2015-01-01

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

  9. Two distinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcription.

    PubMed Central

    Klug, J; Knapp, S; Castro, I; Beato, M

    1994-01-01

    The rabbit uteroglobin gene is expressed in a variety of epithelial cell types like the lung Clara cells and the glandular and luminal epithelial cells of the endometrium. Expression in Clara cells is on a high constitutive level, whereas expression in the rabbit endometrium is under tight hormonal control. One important element of the rabbit uteroglobin gene mediating its efficient transcription in two epithelial cell lines from human endometrium (Ishikawa) and lung (NCI-H441) is its noncanonical TATA box (TACA). Here, we show that two factors (TATA core factor [TCF] and TATA palindrome factor [TPF]) different from the TATA-box binding protein bind to the DNA major groove at two adjacent sites within the uteroglobin TATA-box region and that one of them (TCF) is specifically expressed in cell lines derived from uteroglobin-expressing tissues. The binding sites for TCF and TPF, respectively, are both required for efficient transcription in Ishikawa and NCI-H441 cells. Mutation of the TACA box, which we show is a poor TATA box in functional terms, to a canonical TATA motif does not affect TCF and TPF binding. Therefore, we suggest that the function of the unusual cytosine could be to reduce rabbit uteroglobin expression in cells lacking TCF and that the interaction of TATA-box binding protein with the weak TACA site is facilitated in TCF- and TPF-positive cells. Images PMID:8065353

  10. DNA-binding domains of plant-specific transcription factors: structure, function, and evolution.

    PubMed

    Yamasaki, Kazuhiko; Kigawa, Takanori; Seki, Motoaki; Shinozaki, Kazuo; Yokoyama, Shigeyuki

    2013-05-01

    The families of the plant-specific transcription factors (TFs) are defined by their characteristic DNA-binding domains (DBDs), such as AP2/ERF, B3, NAC, SBP, and WRKY. Recently, three-dimensional structures of the DBDs, including those in complexes with DNA, were determined by NMR spectroscopy and X-ray crystallography. In this review we summarize the functional and evolutionary implications arising from structure analyses. The unexpected structural similarity between B3 and the noncatalytic DBD of the restriction endonuclease EcoRII allowed us to build structural models of the B3/DNA complex. Most of the DBDs of plant-specific TFs are likely to have originated from endonucleases associated with transposable elements. After the DBDs have been established in unicellular eukaryotes, they experienced extensive plant-specific expansion, by acquiring new functions.

  11. Differential recognition of heat shock elements by members of the heat shock transcription factor family.

    PubMed

    Yamamoto, Noritaka; Takemori, Yukiko; Sakurai, Mayumi; Sugiyama, Kazuhisa; Sakurai, Hiroshi

    2009-04-01

    Heat shock transcription factor (HSF), an evolutionarily conserved stress response regulator, forms trimers and binds to heat shock element (HSE), comprising at least three continuous inverted repeats of the sequence 5'-nGAAn-3'. The single HSF of yeast is also able to bind discontinuously arranged nGAAn units. We investigated interactions between three human HSFs and various HSE types in vitro, in yeast cells, and in HeLa cells. Human HSF1, a stress-activated regulator, preferentially bound to continuous HSEs rather than discontinuous HSEs, and heat shock of HeLa cells caused expression of reporter genes containing continuous HSEs. HSF2, whose function is implicated in neuronal specification and spermatogenesis, exhibited a slightly higher binding affinity to discontinuous HSEs than did HSF1. HSF4, a protein required for ocular lens development, efficiently recognized discontinuous HSEs in a trimerization-dependent manner. Among four human gamma-crystallin genes encoding structural proteins of the lens, heat-induced HSF1 preferred HSEs on the gammaA-crystallin and gammaB-crystallin promoters, whereas HSF4 preferred HSE on the gammaC-crystallin promoter. These results suggest that the HSE architecture is an important determinant of which HSF members regulate genes in diverse cellular processes.

  12. Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus.

    PubMed

    Nibuya, M; Nestler, E J; Duman, R S

    1996-04-01

    The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.

  13. CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle*

    PubMed Central

    Liu, Yongfeng; Sun, Li-Yan; Singer, Diane V.; Ginnan, Roman; Singer, Harold A.

    2013-01-01

    One transcription factor mediator of Ca2+-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca2+/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser142 and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser142 without affecting Ser133 phosphorylation. CREB-Ser142 phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser142 phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells. PMID:24106266

  14. An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between Vδ and Dδ Gene Segments.

    PubMed

    Chen, Liang; Zhao, Lijuan; Alt, Frederick W; Krangel, Michael S

    2016-10-15

    Chromatin looping mediated by the CCCTC binding factor (CTCF) regulates V(D)J recombination at Ag receptor loci. CTCF-mediated looping can influence recombination signal sequence (RSS) accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate long-distance interactions between RSSs, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG tracking, and RAG-independent long-distance interactions. In this study, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. The ectopic CBE also limited directional RAG tracking from the Tcrd recombination center, demonstrating that a single CBE can impact the distribution of RAG proteins along chromatin. However, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent contacts between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility, and RAG tracking. Copyright © 2016 by The American Association of Immunologists, Inc.

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

    PubMed

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

    2015-08-01

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

  16. Estrogen-mediated regulation of Igf1 transcription and uterine growth involves direct binding of estrogen receptor alpha to estrogen-responsive elements.

    PubMed

    Hewitt, Sylvia C; Li, Yin; Li, Leping; Korach, Kenneth S

    2010-01-22

    Estrogen enables uterine proliferation, which depends on synthesis of the IGF1 growth factor. This proliferation and IGF1 synthesis requires the estrogen receptor (ER), which binds directly to target DNA sequences (estrogen-responsive elements or EREs), or interacts with other transcription factors, such as AP1, to impact transcription. We observe neither uterine growth nor an increase in Igf1 transcript in a mouse with a DNA-binding mutated ER alpha (KIKO), indicating that both Igf1 regulation and uterine proliferation require the DNA binding function of the ER. We identified several potential EREs in the Igf1 gene, and chromatin immunoprecipitation analysis revealed ER alpha binding to these EREs in wild type but not KIKO chromatin. STAT5 is also reported to regulate Igf1; uterine Stat5a transcript is increased by estradiol (E(2)), but not in KIKO or alpha ERKO uteri, indicating ER alpha- and ERE-dependent regulation. ER alpha binds to a potential Stat5a ERE. We hypothesize that E(2) increases Stat5a transcript through ERE binding; that ER alpha, either alone or together with STAT5, then acts to increase Igf1 transcription; and that the resulting lack of IGF1 impairs KIKO uterine growth. Treatment with exogenous IGF1, alone or in combination with E(2), induces proliferation in wild type but not KIKO uteri, indicating that IGF1 replacement does not rescue the KIKO proliferative response. Together, these observations suggest in contrast to previous in vitro studies of IGF-1 regulation involving AP1 motifs that direct ER alpha-DNA interaction is required to increase Igf1 transcription. Additionally, full ER alpha function is needed to mediate other cellular signals of the growth factor for uterine growth.

  17. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-10-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

  18. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed Central

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-01-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase. PMID:12095415

  19. D-phenylglycinol-derived non-covalent factor Xa inhibitors: effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity.

    PubMed

    Klimkowski, Valentine J; Watson, Brian M; Wiley, Michael R; Liebeschuetz, John; Franciskovich, Jeffry B; Marimuthu, Jothirajah; Bastian, Jolie A; Sall, Daniel J; Smallwood, Jeffrey K; Chirgadze, Nikolay Y; Smith, Gerald F; Foster, Ronald S; Craft, Trelia; Sipes, Philip; Chastain, Marcia; Sheehan, Scott M

    2007-11-01

    Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.

  20. Stb3 binds to ribosomal RNA processing element motifs that control transcriptional responses to growth in Saccharomyces cerevisiae.

    PubMed

    Liko, Dritan; Slattery, Matthew G; Heideman, Warren

    2007-09-07

    Transfer of quiescent Saccharomyces cerevisiae cells to fresh medium rapidly induces hundreds of genes needed for growth. A large subset of these genes is regulated via a DNA sequence motif known as the ribosomal RNA processing element (RRPE). However, no RRPE-binding proteins have been identified. We screened a panel of 6144 glutathione S-transferase-open reading frame fusions for RRPE-binding proteins and identified Stb3 as a specific RRPE-binding protein, both in vitro and in vivo. Chromatin immunoprecipitation experiments showed that glucose increases Stb3 binding to RRPE-containing promoters. Microarray experiments demonstrated that the loss of Stb3 inhibits the transcriptional response to fresh glucose, especially for genes with RRPE motifs. However, these experiments also showed that not all genes containing RRPEs were dependent on Stb3 for expression. Overall our data support a model in which Stb3 plays an important but not exclusive role in the transcriptional response to growth conditions.

  1. Genome-wide analysis reveals positional-nucleosome-oriented binding pattern of pioneer factor FOXA1

    PubMed Central

    Ye, Zhenqing; Chen, Zhong; Sunkel, Benjamin; Frietze, Seth; Huang, Tim H.-M.; Wang, Qianben; Jin, Victor X.

    2016-01-01

    The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, ‘well-positioned’ configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis. PMID:27458208

  2. A General Pairwise Interaction Model Provides an Accurate Description of In Vivo Transcription Factor Binding Sites

    PubMed Central

    Santolini, Marc; Mora, Thierry; Hakim, Vincent

    2014-01-01

    The identification of transcription factor binding sites (TFBSs) on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs), in which each DNA base pair contributes independently to the transcription factor (TF) binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM), a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting TFBSs beyond

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

    PubMed Central

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

    2008-01-01

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

  4. Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats.

    PubMed

    Li, Hong-Ye; Chye, Mee-Len

    2004-01-01

    Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.

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

    PubMed

    Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

    2013-04-01

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

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

    PubMed Central

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

    2012-01-01

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

  7. The enhancer factor R of Epstein-Barr virus (EBV) is a sequence-specific DNA binding protein.

    PubMed Central

    Gruffat, H; Manet, E; Rigolet, A; Sergeant, A

    1990-01-01

    In cells latently infected with EBV, the switch from latency to productive infection is linked to the expression of two EBV transcription factors called EB1 (or Z) and R. EB1 is an upstream element factor which has partial homology to the AP1/ATF family, whereas R is an enhancer factor. In the R-responsive enhancer of the replication origin only active during the EBV lytic cycle (ORIIyt), R-responsive elements are located in a region of about 70 bp (RRE-DR). Here we show that R, produced either by in vitro translation, or present in nuclear extracts from HeLa cells constitutively producing R, binds directly to and protects against DNAase I digestion, two regions in RRE-DR. Using mobility shift assay and DMS interference, we have characterized the contact-points between R and the DNA. Two binding sites, RRE-DR1 and RRE-DR2, were characterized and are contiguous in RRE-DR. R binds to these two sites probably by simultaneously contacting two sequences within the sites, which are separated by 7 bp in RRE-DR1, cctGTGCCttgtcccGTGGACaatgtccc, and by 6bp in RRE-DR2, caatGTCCCtccagcGTGGTGgctg. Direct interaction of R with its cognate sequences is conferred by its N-terminal 355 amino-acids. Directed mutagenesis in RRE-DR, of either R-binding site, impaired binding of R in vitro and, as assayed by transient expression in HeLa cells, impaired R-activation by a factor of two. This suggests that RRE-DR1 and RRE-DR2 do not respond cooperatively to R. Images PMID:2175879

  8. Protein kinase C phosphorylates the cAMP response element binding protein in the hypothalamic paraventricular nucleus during morphine withdrawal

    PubMed Central

    Martín, F; Mora, L; Laorden, ML; Milanés, MV

    2011-01-01

    BACKGROUND AND PURPOSE Exposure to drugs of abuse or stress results in adaptation in the brain involving changes in gene expression and transcription factors. Morphine withdrawal modulates gene expression through various second-messenger signal transduction systems. Here, we investigated changes in activation of the transcription factor, cAMP-response element binding protein (CREB), in the hypothalamic paraventricular nucleus (PVN) and the kinases that may mediate the morphine withdrawal-triggered activation of CREB and the response of the hypothalamic-pituitary-adrenocortical (HPA) axis after naloxone-induced morphine withdrawal. EXPERIMENTAL APPROACH The effects of morphine dependence and withdrawal, phosphorylated CREB (pCREB), corticotrophin-releasing factor (CRF) expression in the PVN and HPA axis activity were measured using immunoblotting, immunohistochemistry and radioimmunoassay in controls and in morphine-dependent rats, withdrawn with naloxone and pretreated with vehicle, calphostin C, chelerythrine (inhibitors of protein kinase C (PKC) or SL-327 [inhibitor of extracellular signal regulated kinase (ERK) kinase]. In addition, changes in PKCα and PKCγ immunoreactivity were measured after 60 min of withdrawal. KEY RESULTS In morphine-withdrawn rats, pCREB immunoreactivity was increased within CRF immunoreactive neurons in the PVN and plasma corticosterone levels were raised. SL-327, at doses that reduced the augmented pERK levels in the PVN, did not attenuate the rise in pCREB immunoreactivity or plasma corticosterone secretion. In contrast, PKC inhibition reduced the withdrawal-triggered rise in pCREB, pERK1/2 and corticosterone secretion. CONCLUSIONS AND IMPLICATIONS PKC mediated, in part, both CREB activation and the HPA response to morphine withdrawal. The ERK kinase/ERK pathway might not be necessary for either activation of CREB or HPA axis hyperactivity. PMID:21615389

  9. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway*

    PubMed Central

    Zhou, Jie; Zhai, Yonggong; Mu, Ying; Gong, Haibiao; Uppal, Hirdesh; Toma, David; Ren, Songrong; Evans, Ronald M.; Xie, Wen

    2014-01-01

    The pregnane X receptor (PXR) was isolated as a xenosensor regulating xenobiotic responses. In this study, we show that PXR plays an endobiotic role by impacting lipid homeostasis. Expression of an activated PXR in the livers of transgenic mice resulted in an increased hepatic deposit of triglycerides. This PXR-mediated lipid accumulation was independent of the activation of the lipogenic transcriptional factor SREBP-1c (sterol regulatory element-binding protein 1c) and its primary lipogenic target enzymes, including fatty-acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC-1). Instead, the lipid accumulation in transgenic mice was associated with an increased expression of the free fatty acid transporter CD36 and several accessory lipogenic enzymes, such as stearoyl-CoA desaturase-1 (SCD-1) and long chain free fatty acid elongase. Studies using transgenic and knock-out mice showed that PXR is both necessary and sufficient for Cd36 activation. Promoter analyses revealed a DR-3-type of PXR-response element in the mouse Cd36 promoter, establishing Cd36 as a direct transcriptional target of PXR. The hepatic lipid accumulation and Cd36 induction were also seen in the hPXR “humanized” mice treated with the hPXR agonist rifampicin. The activation of PXR was also associated with an inhibition of pro-β-oxidative genes, such as peroxisome proliferator-activated receptor α (PPARα) and thiolase, and an up-regulation of PPARγ, a positive regulator of CD36. The cross-regulation of CD36 by PXR and PPARγ suggests that this fatty acid transporter may function as a common target of orphan nuclear receptors in their regulation of lipid homeostasis. PMID:16556603

  10. Identification of the mismatch repair genes PMS2 and MLH1 as p53 target genes by using serial analysis of binding elements

    PubMed Central

    Chen, Jiguo; Sadowski, Ivan

    2005-01-01

    The ability to determine the global location of transcription factor binding sites in vivo is important for a comprehensive understanding of gene regulation in human cells. We have developed a technology, called serial analysis of binding elements (SABE), involving subtractive hybridization of chromatin immunoprecipitation-enriched DNA fragments followed by the generation and analysis of concatamerized sequence tags. We applied the SABE technology to search for p53 target genes in the human genome, and have identified several previously described p53 targets in addition to numerous potentially novel targets, including the DNA mismatch repair genes MLH1 and PMS2. Both of these genes were determined to be responsive to DNA damage and p53 activation in normal human fibroblasts, and have p53-response elements within their first intron. These two genes may serve as a sensor in DNA repair mechanisms and a critical determinant for the decision between cell-cycle arrest and apoptosis. These results also demonstrate the potential for use of SABE as a broadly applicable means to globally identify regulatory elements for human transcription factors in vivo. PMID:15781865

  11. Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

    PubMed Central

    Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2016-01-01

    Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661

  12. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

    PubMed

    Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin; Choi, Ah-Reum; Lee, Sook-Jeong; Hoe, Kwang-Lae; Kim, Dong-Uk

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    SciTech Connect

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li . E-mail: lfang@utmb.edu; Li Junfa . E-mail: junfali@cpums.edu.cn

    2006-02-10

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning.

  14. Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation.

    PubMed Central

    Dorsman, J C; van Heeswijk, W C; Grivell, L A

    1990-01-01

    GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts. Images PMID:2187179

  15. A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX.

    PubMed

    Li, X; Chen, S; Wang, Q; Zack, D J; Snyder, S H; Borjigin, J

    1998-02-17

    The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene (Atp7b) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5' regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE.

  16. A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX

    PubMed Central

    Li, Xiaodong; Chen, Shiming; Wang, Qingliang; Zack, Donald J.; Snyder, Solomon H.; Borjigin, Jimo

    1998-01-01

    The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene (Atp7b) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5′ regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE. PMID:9465110

  17. Growth Suppression of Lung Cancer Cells by Targeting Cyclic AMP Response Element-Binding Protein

    PubMed Central

    Aggarwal, Sita; Kim, Seung-Wook; Ryu, Seung-Hee; Chung, Wen-Cheng; Koo, Ja Seok

    2010-01-01

    Genes regulated by cyclic AMP response element-binding protein (CREB) have been reported to suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis. However, it is not clear whether CREB is critically involved in the carcinogenesis of lung cancer. We found that non-small cell lung cancer (NSCLC) cell lines exhibited elevated constitutive activity in CREB; in its immediate upstream kinases, ribosomal s6 kinase and extracellular signal kinase; and in the CREB-regulated cell survival proteins, Bcl-2 and Bcl-xL. We hypothesized that constitutively active CREB is important to lung cancer cell growth and survival and therefore could be a potential therapeutic target for NSCLC. Ectopic expression of dominant-repressor CREB and transfection with small interfering RNA against CREB suppressed the growth and survival of NSCLC cells and induced apoptotic cell death. Furthermore, treating H1734 NSCLC cells with an inhibitor of the CREB signaling pathway, Ro-31-8220, inhibited CREB activation by blocking the activity of extracellular signal kinase and ribosomal s6 kinase, arrested the cell cycle at the G2/M phase, and subsequently induced apoptosis with the suppression of Bcl-2 and Bcl-xL expression. Ro-31-8220 suppressed both the anchorage-dependent and the independent growth of NSCLC cells, but its cytotoxic effect was much less prominent in normal bronchial epithelial cells. Our results indicate that active CREB plays an important role in NSCLC cell growth and survival. Thus, agents that suppress CREB activation could have potential therapeutic value for NSCLC treatment. PMID:18281471

  18. The patterns of histone modifications in the vicinity of transcription factor binding sites in human lymphoblastoid cell lines.

    PubMed

    Nie, Yumin; Liu, Hongde; Sun, Xiao

    2013-01-01

    Transcription factor (TF) binding at specific DNA sequences is the fundamental step in transcriptional regulation and is highly dependent on the chromatin structure context, which may be affected by specific histone modifications and variants, known as histone marks. The lack of a global binding map for hundreds of TFs means that previous studies have focused mainly on histone marks at binding sites for several specific TFs. We therefore studied 11 histone marks around computationally-inferred and experimentally-determined TF binding sites (TFBSs), based on 164 and 34 TFs, respectively, in human lymphoblastoid cell lines. For H2A.Z, methylation of H3K4, and acetylation of H3K27 and H3K9, the mark patterns exhibited bimodal distributions and strong pairwise correlations in the 600-bp region around enriched TFBSs, suggesting that these marks mainly coexist within the two nucleosomes proximal to the TF sites. TFs competing with nucleosomes to access DNA at most binding sites, contributes to the bimodal distribution, which is a common feature of histone marks for TF binding. Mark H3K79me2 showed a unimodal distribution on one side of TFBSs and the signals extended up to 4000 bp, indicating a longer-distance pattern. Interestingly, H4K20me1, H3K27me3, H3K36me3 and H3K9me3, which were more diffuse and less enriched surrounding TFBSs, showed unimodal distributions around the enriched TFBSs, suggesting that some TFs may bind to nucleosomal DNA. Besides, asymmetrical distributions of H3K36me3 and H3K9me3 indicated that repressors might establish a repressive chromatin structure in one direction to repress gene expression. In conclusion, this study demonstrated the ranges of histone marks associated with TF binding, and the common features of these marks around the binding sites. These findings have epigenetic implications for future analysis of regulatory elements.

  19. Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.

    PubMed Central

    Kamachi, Y; Ogawa, E; Asano, M; Ishida, S; Murakami, Y; Satake, M; Ito, Y; Shigesada, K

    1990-01-01

    We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2. Images PMID:2168969

  20. In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.

    PubMed Central

    Harel-Bellan, A; Brini, A T; Ferris, D K; Robin, P; Farrar, W L

    1989-01-01

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer. Images PMID:2740211

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

    PubMed

    Babbitt, G A

    2010-10-15

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

  2. The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein.

    PubMed

    Marrugo, J; Marsh, D G; Ghosh, B

    1996-10-01

    The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

  3. Identification of a minimal promoter element of the mouse epidermal growth factor gene.

    PubMed Central

    Pascall, J C; Brown, K D

    1997-01-01

    We have previously generated a transgenic mouse line (EGF/Tag) in which simian virus 40 (SV40) T-antigen expression is directed by the mouse epidermal growth factor (EGF) gene promoter. In these mice, cellular hyperproliferation is observed in the submaxillary gland associated with SV40 T-antigen expression. In addition, SV40 T-antigen-expressing tumours of prostatic origin are seen. We have now derived immortalized cell lines from these tissues and have used the cells to perform a functional analysis of the EGF gene promoter. Cells were transfected with EGF promoter/reporter constructs, and an element located between 51 and 35 bases upstream of the EGF mRNA start site required for basal activity of the promoter was identified. Electrophoretic mobility-shift analysis suggests that three proteins bind to this region, one of which is either Sp1 or a closely related protein. PMID:9210411

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

    PubMed

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

    2016-05-05

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

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

    PubMed Central

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

    2016-01-01

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

  6. The human RNA-binding protein and E3 ligase MEX-3C binds the MEX-3-recognition element (MRE) motif with high affinity.

    PubMed

    Yang, Lingna; Wang, Chongyuan; Li, Fudong; Zhang, Jiahai; Nayab, Anam; Wu, Jihui; Shi, Yunyu; Gong, Qingguo

    2017-09-29

    MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans, and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype (HLA-A2) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells.

    PubMed

    Laurette, Patrick; Strub, Thomas; Koludrovic, Dana; Keime, Céline; Le Gras, Stéphanie; Seberg, Hannah; Van Otterloo, Eric; Imrichova, Hana; Siddaway, Robert; Aerts, Stein; Cornell, Robert A; Mengus, Gabrielle; Davidson, Irwin

    2015-03-24

    Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

  8. The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation.

    PubMed

    Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H; Ghosal, Suman; Sun, Hong-Wei; Kazemier, Hinke G; Paeschke, Katrin; Hafner, Markus; Juranek, Stefan A

    2017-03-21

    The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

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

    PubMed Central

    Walker, P; Reeder, R H

    1988-01-01

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

  10. Sterols regulate 3β-hydroxysterol Δ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by Sterol Regulatory Element Binding Proteins.

    PubMed

    Zerenturk, Eser J; Sharpe, Laura J; Brown, Andrew J

    2012-10-01

    3β-Hydroxysterol Δ24-reductase (DHCR24) catalyzes a final step in cholesterol synthesis, and has been ascribed diverse functions, such as being anti-apoptotic and anti-inflammatory. How this enzyme is regulated transcriptionally by sterols is currently unclear. Some studies have suggested that its expression is regulated by Sterol Regulatory Element Binding Proteins (SREBPs) while another suggests it is through the Liver X Receptor (LXR). However, these transcription factors have opposing effects on cellular sterol levels, so it is likely that one predominates. Here we establish that sterol regulation of DHCR24 occurs predominantly through SREBP-2, and identify the particular region of the DHCR24 promoter to which SREBP-2 binds. We demonstrate that sterol regulation is mediated by two sterol regulatory elements (SREs) in the promoter of the gene, assisted by two nearby NF-Y binding sites. Moreover, we present evidence that the dual SREs work cooperatively to regulate DHCR24 expression by comparison to two known SREBP target genes, the LDL receptor with one SRE, and farnesyl-diphosphate farnesyltransferase 1, with two SREs. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Fkh1 and Fkh2 Bind Multiple Chromosomal Elements in the S. cerevisiae Genome with Distinct Specificities and Cell Cycle Dynamics

    PubMed Central

    Knott, Simon R. V.; Fox, Catherine A.; Tavaré, Simon; Aparicio, Oscar M.

    2014-01-01

    Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating mating-type silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycle-regulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome. PMID:24504085

  12. The DNA unwinding element binding protein DUE-B interacts with Cdc45 in preinitiation complex formation.

    PubMed

    Chowdhury, A; Liu, G; Kemp, M; Chen, X; Katrangi, N; Myers, S; Ghosh, M; Yao, J; Gao, Y; Bubulya, P; Leffak, M

    2010-03-01

    Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

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

    PubMed

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

    1998-06-09

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

  14. Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns.

    PubMed

    Kirby, Marie K; Ramaker, Ryne C; Roberts, Brian S; Lasseigne, Brittany N; Gunther, David S; Burwell, Todd C; Davis, Nicholas S; Gulzar, Zulfiqar G; Absher, Devin M; Cooper, Sara J; Brooks, James D; Myers, Richard M

    2017-04-17

    Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer. We measured genome-wide DNA methylation patterns in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array. We overlaid the most significantly differentially methylated sites in the genome with transcription factor binding sites measured by the Encyclopedia of DNA Elements consortium. We used logistic regression and receiver operating characteristic curves to assess the performance of candidate diagnostic models. We identified methylation patterns that have a high predictive power for distinguishing malignant prostate tissue from benign-adjacent prostate tissue, and these methylation signatures were validated using data from The Cancer Genome Atlas Project. Furthermore, by overlaying ENCODE transcription factor binding data, we observed an enrichment of enhancer of zeste homolog 2 binding in gene regulatory regions with higher DNA methylation in malignant prostate tissues. DNA methylation patterns are greatly altered in prostate cancer tissue in comparison to benign-adjacent tissue. We have discovered patterns of DNA methylation marks that can distinguish prostate cancers with high specificity and sensitivity in multiple patient tissue cohorts, and we have identified transcription factors binding in these differentially methylated regions that may play important roles in prostate cancer development.

  15. Molecular biology of beta-estradiol-estrogen receptor complex binding to estrogen response element and the effect on cell proliferation.

    PubMed

    Heger, Zbynek; Zitka, Ondrej; Krizkova, Sona; Beklova, Miroslava; Kizek, Rene; Adam, Vojtech

    2013-01-01

    Group of estrogen pollutants, where the highest estrogen activity is reported at estradiol, is characterized by the fact that even at very low concentrations have potential to cause xenoestrogenic effects. During exposure of excessive amounts of estradiols may be produced undesirable effects resulting in the feminization of males of water organisms. The presence of estradiols in drinking water implies also a risk for the human population in the form of cancers of endocrine systems, abnormalities in reproduction or dysfunctions of neuronal and immune system. Currently, the research is focused mainly to uncover the relationship between the estrogen receptors binding affinity with an estrogen response element and estradiol. In this review we summarized facts about molecular biological principles of β estradiol-estrogen receptor complex binding with estrogen response element and its successive effect on cancer genes expression.

  16. Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo

    PubMed Central

    Schulz, Katharine N.; Bondra, Eliana R.; Moshe, Arbel; Villalta, Jacqueline E.; Lieb, Jason D.; Kaplan, Tommy; McKay, Daniel J.; Harrison, Melissa M.

    2015-01-01

    The transition from a specified germ cell to a population of pluripotent cells occurs rapidly following fertilization. During this developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Open chromatin is associated with Zelda-bound loci, as well as more generally with regions of active transcription. Nonetheless, the extent to which Zelda influences chromatin accessibility across the genome is largely unknown. Here we used formaldehyde-assisted isolation of regulatory elements to determine the role of Zelda in regulating regions of open chromatin in the early embryo. We demonstrate that Zelda is essential for hundreds of regions of open chromatin. This Zelda-mediated chromatin accessibility facilitates transcription-factor recruitment and early gene expression. Thus, Zelda possesses some key characteristics of a pioneer factor. Unexpectedly, chromatin at a large subset of Zelda-bound regions remains open even in the absence of Zelda. The GAGA factor-binding motif and embryonic GAGA factor binding are specifically enriched in these regions. We propose that both Zelda and GAGA factor function to specify sites of open chromatin and together facilitate the remodeling of the early embryonic genome. PMID:26335634

  17. Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

    PubMed

    Yoffe, Yael; Zuberek, Joanna; Lerer, Asaf; Lewdorowicz, Magdalena; Stepinski, Janusz; Altmann, Michael; Darzynkiewicz, Edward; Shapira, Michal

    2006-12-01

    The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

  18. Highly accessible AU-rich regions in 3’ untranslated regions are hotspots for binding of regulatory factors

    PubMed Central

    2017-01-01

    Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3’UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing “free” target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer

  19. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    SciTech Connect

    Istrate, Monica A.; Nussler, Andreas K.; Eichelbaum, Michel; Burk, Oliver

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  20. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

    PubMed Central

    Schauwaers, Kris; De Gendt, Karel; Saunders, Philippa T. K.; Atanassova, Nina; Haelens, Annemie; Callewaert, Leen; Moehren, Udo; Swinnen, Johannes V.; Verhoeven, Guido; Verrijdt, Guy; Claessens, Frank

    2007-01-01

    Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction. PMID:17360365

  1. Structure and function of the c-myc DNA-unwinding element-binding protein DUE-B.

    PubMed

    Kemp, Michael; Bae, Brian; Yu, John Paul; Ghosh, Maloy; Leffak, Michael; Nair, Satish K

    2007-04-06

    Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

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

    PubMed Central

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

    1985-01-01

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

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

    PubMed

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

    2015-07-31

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

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

    PubMed Central

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

    2015-01-01

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

  5. Transcription initiation at the TATA-less spliced leader RNA gene promoter requires at least two DNA-binding proteins and a tripartite architecture that includes an initiator element.

    PubMed

    Luo, H; Gilinger, G; Mukherjee, D; Bellofatto, V

    1999-11-05

    Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m(7)G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

  6. Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes.

    PubMed

    Yalley, Akua; Schill, Daniel; Hatta, Mitsutoki; Johnson, Nicole; Cirillo, Lisa Ann

    2016-04-15

    FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding.

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

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. The Soft Touch: Low-