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Sample records for gli3 repressor controls

  1. Kif3a controls murine nephron number via GLI3 repressor, cell survival, and gene expression in a lineage-specific manner.

    PubMed

    Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-Chung; Rosenblum, Norman D

    2013-01-01

    The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3(Δ699/+) ) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms.

  2. Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

    PubMed Central

    Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

    2013-01-01

    The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

  3. Urogenital development in Pallister–Hall syndrome is disrupted in a cell-lineage-specific manner by constitutive expression of GLI3 repressor

    PubMed Central

    Blake, Joshua; Hu, Di; Cain, Jason E.; Rosenblum, Norman D.

    2016-01-01

    Pallister–Hall syndrome (PHS) is a rare disorder caused by mutations in GLI3 that produce a transcriptional repressor (GLI3R). Individuals with PHS present with a variably penetrant variety of urogenital system malformations, including renal aplasia or hypoplasia, hydroureter, hydronephrosis or a common urogenital sinus. The embryologic mechanisms controlled by GLI3R that result in these pathologic phenotypes are undefined. We demonstrate that germline expression of GLI3R causes renal hypoplasia, associated with decreased nephron number, and hydroureter and hydronephrosis, caused by blind-ending ureters. Mice with obligate GLI3R expression also displayed duplication of the ureters that was caused by aberrant common nephric duct patterning and ureteric stalk outgrowth. These developmental abnormalities are associated with suppressed Hedgehog signaling activity in the cloaca and adjacent vesicular mesenchyme. Mice with conditional expression of GLI3R were utilized to identify lineage-specific effects of GLI3R. In the ureteric bud, GLI3R expression decreased branching morphogenesis. In Six2-positive nephrogenic progenitors, GLI3R decreased progenitor cell proliferation reducing the number of nephrogenic precursor structures. Using mutant mice with Gli3R and Gli3 null alleles, we demonstrate that urogenital system patterning and development is controlled by the levels of GLI3R and not by an absence of full-length GLI3. We conclude that the urogenital system phenotypes observed in PHS are caused by GLI3R-dependent perturbations in nephric duct patterning, renal branching morphogenesis and nephrogenic progenitor self-renewal. PMID:26604140

  4. Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3

    PubMed Central

    Dai, Ping; Shinagawa, Toshie; Nomura, Teruaki; Harada, Jun; Kaul, Sunil C.; Wadhwa, Renu; Khan, Md Matiullah; Akimaru, Hiroshi; Sasaki, Hiroshi; Colmenares, Clemencia; Ishii, Shunsuke

    2002-01-01

    Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. Here we report that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation. PMID:12435627

  5. Gata6-Dependent GLI3 Repressor Function is Essential in Anterior Limb Progenitor Cells for Proper Limb Development

    PubMed Central

    Hayashi, Shinichi; Akiyama, Ryutaro; Wong, Julia; Tahara, Naoyuki; Kawakami, Hiroko; Kawakami, Yasuhiko

    2016-01-01

    Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod. PMID:27352137

  6. The role of primary cilia in corpus callosum formation is mediated by production of the Gli3 repressor.

    PubMed

    Laclef, Christine; Anselme, Isabelle; Besse, Laurianne; Catala, Martin; Palmyre, Aurélien; Baas, Dominique; Paschaki, Marie; Pedraza, Maria; Métin, Christine; Durand, Bénédicte; Schneider-Maunoury, Sylvie

    2015-09-01

    Agenesis of the corpus callosum (AgCC) is a frequent brain disorder found in over 80 human congenital syndromes including ciliopathies. Here, we report a severe AgCC in Ftm/Rpgrip1l knockout mouse, which provides a valuable model for Meckel-Grüber syndrome. Rpgrip1l encodes a protein of the ciliary transition zone, which is essential for ciliogenesis in several cell types in mouse including neuroepithelial cells in the developing forebrain. We show that AgCC in Rpgrip1l(-/-) mouse is associated with a disturbed location of guidepost cells in the dorsomedial telencephalon. This mislocalization results from early patterning defects and abnormal cortico-septal boundary (CSB) formation in the medial telencephalon. We demonstrate that all these defects primarily result from altered GLI3 processing. Indeed, AgCC, together with patterning defects and mispositioning of guidepost cells, is rescued by overexpressing in Rpgrip1l(-/-) embryos, the short repressor form of the GLI3 transcription factor (GLI3R), provided by the Gli3(Δ699) allele. Furthermore, Gli3(Δ699) also rescues AgCC in Rfx3(-/-) embryos deficient for the ciliogenic RFX3 transcription factor that regulates the expression of several ciliary genes. These data demonstrate that GLI3 processing is a major outcome of primary cilia function in dorsal telencephalon morphogenesis. Rescuing CC formation in two independent ciliary mutants by GLI3(Δ699) highlights the crucial role of primary cilia in maintaining the proper level of GLI3R required for morphogenesis of the CC.

  7. Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning.

    PubMed

    Magnani, Dario; Hasenpusch-Theil, Kerstin; Benadiba, Carine; Yu, Tian; Basson, M Albert; Price, David J; Lebrand, Cécile; Theil, Thomas

    2014-01-01

    The corpus callosum (CC) represents the major forebrain commissure connecting the 2 cerebral hemispheres. Midline crossing of callosal axons is controlled by several glial and neuronal guideposts specifically located along the callosal path, but it remains unknown how these cells acquire their position. Here, we show that the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn) displays agenesis of the CC and mislocation of the glial and neuronal guidepost cells. Using transplantation experiments, we demonstrate that agenesis of the CC is primarily caused by midline defects. These defects originate during telencephalic patterning and involve an up-regulation of Slit2 expression and altered Fgf and Wnt/β-catenin signaling. Mutations in sprouty1/2 which mimic the changes in these signaling pathways cause a disorganization of midline guideposts and CC agenesis. Moreover, a partial recovery of midline abnormalities in Pdn/Pdn;Slit2(-/-) embryos mutants confirms the functional importance of correct Slit2 expression levels for callosal development. Hence, Gli3 controlled restriction of Fgf and Wnt/β-catenin signaling and of Slit2 expression is crucial for positioning midline guideposts and callosal development.

  8. T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number

    PubMed Central

    Emechebe, Uchenna; Kumar P, Pavan; Rozenberg, Julian M; Moore, Bryn; Firment, Ashley; Mirshahi, Tooraj; Moon, Anne M

    2016-01-01

    Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. Mechanisms whereby T-box3 regulates limb development are poorly understood. We discovered requirements for T-box at multiple stages of mouse limb development and distinct molecular functions in different tissue compartments. Early loss of T-box3 disrupts limb initiation, causing limb defects that phenocopy Sonic Hedgehog (Shh) mutants. Later ablation of T-box3 in posterior limb mesenchyme causes digit loss. In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor. Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function. DOI: http://dx.doi.org/10.7554/eLife.07897.001 PMID:27046536

  9. Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

    PubMed Central

    Krauß, Sybille; So, Joyce; Hambrock, Melanie; Köhler, Andrea; Kunath, Melanie; Scharff, Constance; Wessling, Martina; Grzeschik, Karl-Heinz; Schneider, Rainer; Schweiger, Susann

    2009-01-01

    Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3

  10. The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia.

    PubMed

    Caparrós-Martín, Jose A; Valencia, María; Reytor, Edel; Pacheco, María; Fernandez, Margarita; Perez-Aytes, Antonio; Gean, Esther; Lapunzina, Pablo; Peters, Heiko; Goodship, Judith A; Ruiz-Perez, Victor L

    2013-01-01

    Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

  11. GLI3 Links Environmental Arsenic Exposure and Human Fetal Growth.

    PubMed

    Winterbottom, Emily F; Fei, Dennis L; Koestler, Devin C; Giambelli, Camilla; Wika, Eric; Capobianco, Anthony J; Lee, Ethan; Marsit, Carmen J; Karagas, Margaret R; Robbins, David J

    2015-06-01

    Although considerable evidence suggests that in utero arsenic exposure affects children's health, these data are mainly from areas of the world where groundwater arsenic levels far exceed the World Health Organization limit of 10 μg/L. We, and others, have found that more common levels of in utero arsenic exposure may also impact children's health. However, the underlying molecular mechanisms are poorly understood. To address this issue, we analyzed the expression of key developmental genes in fetal placenta in a birth cohort of women using unregulated water supplies in a US region with elevated groundwater arsenic. We identified several genes whose expression associated with maternal arsenic exposure in a fetal sex-specific manner. In particular, expression of the HEDGEHOG pathway component, GLI3, in female placentae was both negatively associated with arsenic exposure and positively associated with infant birth weight. This suggests that modulation of GLI3 in the fetal placenta, and perhaps in other fetal tissues, contributes to arsenic's detrimental effects on fetal growth. We showed previously that arsenic-exposed NIH3T3 cells have reduced GLI3 repressor protein. Together, these studies identify GLI3 as a key signaling node that is affected by arsenic, mediating a subset of its effects on developmental signaling and fetal health.

  12. A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy

    PubMed Central

    Matera, Ivana; Watkins-Chow, Dawn E.; Loftus, Stacie K.; Hou, Ling; Incao, Arturo; Silver, Debra L.; Rivas, Cecelia; Elliott, Eugene C.; Baxter, Laura L.; Pavan, William J.

    2008-01-01

    Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10LacZ/+). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10LacZ/+ mice. Analysis of 230 pedigrees identified three modifiers, named modifier of Sox10 neurocristopathies (Mos1, Mos2 and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4 and 3, respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog(HH)-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3Xt-J) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10LacZ/+;Gli3Mos1/+ double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3Mos1/Mos1 embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other HH signaling components as modifiers of human neurocristopathies. PMID:18397875

  13. Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10.

    PubMed Central

    Wray, L V; Reznikoff, W S

    1983-01-01

    The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Deletions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry. PMID:6315680

  14. Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

    PubMed

    Abbasi, Amir A; Minhas, Rashid; Schmidt, Ansgar; Koch, Sabine; Grzeschik, Karl-Heinz

    2013-10-01

    The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs.

  15. Abrogation of Gli3 expression suppresses the growth of colon cancer cells via activation of p53

    SciTech Connect

    Kang, Han Na; Oh, Sang Cheul; Kim, Jun Suk

    2012-03-10

    p53, the major human tumor suppressor, appears to be related to sonic hedgehog (Shh)-Gli-mediated tumorigenesis. However, the role of p53 in tumor progression by the Shh-Gli signaling pathway is poorly understood. Herein we investigated the critical regulation of Gli3-p53 in tumorigenesis of colon cancer cells and the molecular mechanisms underlying these effects. RT-PCR analysis indicated that the mRNA level of Shh and Gli3 in colon tumor tissues was significantly higher than corresponding normal tissues (P < 0.001). The inhibition of Gli3 by treatment with Gli3 siRNA resulted in a clear decrease in cell proliferation and enhanced the level of expression of p53 proteins compared to treatment with control siRNA. The half-life of p53 was dramatically increased by treatment with Gli3 siRNA. In addition, treatment with MG132 blocked MDM2-mediated p53 ubiquitination and degradation, and led to accumulation of p53 in Gli3 siRNA-overexpressing cells. Importantly, ectopic expression of p53 siRNA reduced the ability of Gli3 siRNA to suppress proliferation of those cells compared with the cells treated with Gli3 siRNA alone. Moreover, Gli3 siRNA sensitized colon cancer cells to treatment with anti-cancer agents (5-FU and bevacizumab). Taken together, our studies demonstrate that loss of Gli3 signaling leads to disruption of the MDM2-p53 interaction and strongly potentiate p53-dependent cell growth inhibition in colon cancer cells, indicating a basis for the rational use of Gli3 antagonists as a novel treatment option for colon cancer.

  16. The interaction of RNA polymerase and lac repressor with the lac control region.

    PubMed Central

    Schmitz, A; Galas, D J

    1979-01-01

    We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex. Images PMID:370784

  17. Transcriptional Analysis of Gli3 Mutants Identifies Wnt Target Genes in the Developing Hippocampus

    PubMed Central

    Hasenpusch-Theil, Kerstin; Magnani, Dario; Amaniti, Eleni-Maria; Han, Lin; Armstrong, Douglas

    2012-01-01

    Early development of the hippocampus, which is essential for spatial memory and learning, is controlled by secreted signaling molecules of the Wnt gene family and by Wnt/β-catenin signaling. Despite its importance, little is known, however, about Wnt-regulated genes during hippocampal development. Here, we used the Gli3 mutant mouse extra-toes (XtJ), in which Wnt gene expression in the forebrain is severely affected, as a tool in a microarray analyses to identify potential Wnt target genes. This approach revealed 53 candidate genes with restricted or graded expression patterns in the dorsomedial telencephalon. We identified conserved Tcf/Lef-binding sites in telencephalon-specific enhancers of several of these genes, including Dmrt3, Gli3, Nfia, and Wnt8b. Binding of Lef1 to these sites was confirmed using electrophoretic mobility shift assays. Mutations in these Tcf/Lef-binding sites disrupted or reduced enhancer activity in vivo. Moreover, ectopic activation of Wnt/β-catenin signaling in an ex vivo explant system led to increased telencephalic expression of these genes. Finally, conditional inactivation of Gli3 results in defective hippocampal growth. Collectively, these data strongly suggest that we have identified a set of direct Wnt target genes in the developing hippocampus and provide inside into the genetic hierarchy underlying Wnt-regulated hippocampal development. PMID:22235033

  18. Suppressor of Fused Is Required for Determining Digit Number and Identity via Gli3/Fgfs/Gremlin

    PubMed Central

    Cui, Ying; Yang, Xueqin; Li, Yan; Zhang, Xiaoyun; Qiu, Mengsheng; Zhang, Ze; Zhang, Zunyi

    2015-01-01

    The anterior-posterior patterning of the vertebrate limb bud requires closely coordinated signaling interactions, including Sonic Hedgehog (Shh)-mediated counteraction of the Gli3 transcription factor in the distal and posterior mesenchyme of the limb bud. Suppressor of Fused (Sufu), an intracellular negative regulator of Shh signaling via Gli2 and Gli3, is implicated in early development of the mouse limb bud. However, how Sufu is involved in the genetic regulation of limb bud patterning still remains elusive. In this study, we show that the conditional deletion of Sufu in the mesenchyme of the early limb bud results in polydactyly with loss of digit identity and supernumerary bones in the wrist and the ankle. These pattern alterations are associated with anterior expansion of HoxD genes located at the 5’ end of the cluster. By focusing on gene expression analysis of Shh/Gremlin1/Fgf signaling critical for the establishment and maintenance of anterior-posterior patterning, we show that early response to loss of Sufu involves anterior prolongation of Fgf4 and Fgf8 expression in the apical ectodermal ridge at E10.5. We also reveal the anterior activation of Shh-dependent posterior markers Ptc1, Gli1 and Gremlin in limb buds lacking Sufu. Furthermore, we find that loss of Sufu leads to attenuated levels of repressor Gli2 and repressor Gli3 in the early limb bud. Moreover, expression of Hand2 is activated in the entire limb bud at the early outgrowth stage in the mutant lacking Sufu. Thus, we provide evidence that Sufu is involved in the genetic network that restricts the posterior expression of Gli2/3/Hand2 and Gremlin/Fgf in limb bud patterning. PMID:26001200

  19. 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.

  20. Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.

    PubMed Central

    Wu, J; Anderton-Loviny, T; Smith, C A; Hartley, B S

    1985-01-01

    Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation. PMID:3891331

  1. Molecular and Clinical Analyses of Greig Cephalopolysyndactyly and Pallister-Hall Syndromes: Robust Phenotype Prediction from the Type and Position of GLI3 Mutations

    PubMed Central

    Johnston, Jennifer J.; Olivos-Glander, Isabelle; Killoran, Christina; Elson, Emma; Turner, Joyce T.; Peters, Kathryn F.; Abbott, Margaret H.; Aughton, David J.; Aylsworth, Arthur S.; Bamshad, Michael J.; Booth, Carol; Curry, Cynthia J.; David, Albert; Dinulos, Mary Beth; Flannery, David B.; Fox, Michelle A.; Graham, John M.; Grange, Dorothy K.; Guttmacher, Alan E.; Hannibal, Mark C.; Henn, Wolfram; Hennekam, Raoul C. M.; Holmes, Lewis B.; Hoyme, H. Eugene; Leppig, Kathleen A.; Lin, Angela E.; MacLeod, Patrick; Manchester, David K.; Marcelis, Carlo; Mazzanti, Laura; McCann, Emma; McDonald, Marie T.; Mendelsohn, Nancy J.; Moeschler, John B.; Moghaddam, Billur; Neri, Giovanni; Newbury-Ecob, Ruth; Pagon, Roberta A.; Phillips, John A.; Sadler, Laurie S.; Stoler, Joan M.; Tilstra, David; Walsh Vockley, Catherine M.; Zackai, Elaine H.; Zadeh, Touran M.; Brueton, Louise; Black, Graeme Charles M.; Biesecker, Leslie G.

    2005-01-01

    Mutations in the GLI3 zinc-finger transcription factor gene cause Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS), which are variable but distinct clinical entities. We hypothesized that GLI3 mutations that predict a truncated functional repressor protein cause PHS and that functional haploinsufficiency of GLI3 causes GCPS. To test these hypotheses, we screened patients with PHS and GCPS for GLI3 mutations. The patient group consisted of 135 individuals: 89 patients with GCPS and 46 patients with PHS. We detected 47 pathological mutations (among 60 probands); when these were combined with previously published mutations, two genotype-phenotype correlations were evident. First, GCPS was caused by many types of alterations, including translocations, large deletions, exonic deletions and duplications, small in-frame deletions, and missense, frameshift/nonsense, and splicing mutations. In contrast, PHS was caused only by frameshift/nonsense and splicing mutations. Second, among the frameshift/nonsense mutations, there was a clear genotype-phenotype correlation. Mutations in the first third of the gene (from open reading frame [ORF] nucleotides [nt] 1–1997) caused GCPS, and mutations in the second third of the gene (from ORF nt 1998–3481) caused primarily PHS. Surprisingly, there were 12 mutations in patients with GCPS in the 3′ third of the gene (after ORF nt 3481), and no patients with PHS had mutations in this region. These results demonstrate a robust correlation of genotype and phenotype for GLI3 mutations and strongly support the hypothesis that these two allelic disorders have distinct modes of pathogenesis. PMID:15739154

  2. Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites.

    PubMed

    Merulla, Davide; van der Meer, Jan Roelof

    2016-01-15

    Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

  3. Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

    DOE PAGES

    Liang, Yan; Richardson, Sarah; Yan, Jingwei; ...

    2017-01-17

    © 2017 American Chemical Society. Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. Meeting these challenges will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5′UTR of mRNA, we developed a two-component expression-repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can bemore » used to repress transgene expression by > 400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Using a bioinformatics approach, we identified 54 orthologous systems from various bacteria, and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

  4. The quaternary structure of Tet repressors bound to the Tn10-encoded tet gene control region determined by neutron solution scattering.

    PubMed Central

    Lederer, H; Tovar, K; Baer, G; May, R P; Hillen, W; Heumann, H

    1989-01-01

    The spatial arrangement of Tet repressor dimer, both free and in complex with an 80 bp DNA fragment spanning the wild-type Tn10-encoded tet transcriptional control sequence containing a tandem repeat of two operators, has been determined by neutron small-angle scattering. The active, free Tet repressor dimer is an elongated and flat molecule with a maximum dimension of 11 +/- 1.5 mm which can be approximated by an ellipsoid with the half-axes 6 nm, 2.5 nm and 1 nm. The overall conformation undergoes no detectable change when the repressor dimer is bound to a DNA fragment containing a single tet operator. The normal distance between the centre of gravity of the protein and the DNA axis is 3.0 +/- 0.1 nm, indicating that the repressor dimer is mainly located on one side of the DNA. When bound to the wild type tet control DNA, the two repressor dimers have a centre-to-centre distance of 11.0 +/- 0.5 nm. Their minimal distance is 5 +/- 2 nm. Protein-protein contacts via loop formation of the DNA by repressor binding is excluded. The repressors are well separated and have no direct contact. A model is proposed where the two repressor dimers are located on opposite sides of the DNA and the DNA is not strongly bent in the complex. Images PMID:2545442

  5. A repressor-antirepressor pair links two loci controlling light-induced carotenogenesis in Myxococcus xanthus.

    PubMed

    López-Rubio, José Juan; Elías-Arnanz, Montserrat; Padmanabhan, S; Murillo, Francisco José

    2002-03-01

    The light-inducible carB operon encodes all but one of the structural genes for carotenogenesis in Myxococcus xanthus. It is transcriptionally controlled by two proteins expressed from two unlinked genetic loci: CarS from the light-inducible carQRS operon, and CarA from the light-independent carA operon. CarA represses transcription from the carB promoter (P(B)) in the dark, and CarS counteracts this on illumination. The CarA sequence revealed a helix-turn-helix DNA-binding motif of the type found in bacterial MerR transcriptional factors, whereas CarS contains no known DNA-binding motif. Here, we examine the molecular interplay between CarA and CarS. We demonstrate the following. (i) Whereas CarS exhibits no DNA binding in vitro, CarA binds specifically to a region encompassing P(B) to form at least two distinct complexes. (ii) A palindrome located between positions -46 and -63 relative to the transcription start point is essential but not sufficient for the formation of the two CarA-DNA complexes observed. (iii) CarS abrogates the specific DNA binding of CarA. CarA is therefore a repressor and CarS an antirepressor. (iv) CarS physically interacts with CarA; thus, the functional interaction between them is mediated by protein-protein interactions.

  6. Control of gene expression in Helicobacter pylori using the Tet repressor

    PubMed Central

    McClain, Mark S.; Duncan, Stacy S.; Gaddy, Jennifer A.; Cover, Timothy L.

    2013-01-01

    The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis. PMID:24113399

  7. A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230.

    PubMed

    Yang, K; Han, L; He, J; Wang, L; Vining, L C

    2001-11-28

    A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.

  8. p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

    PubMed Central

    Ferrándiz, Nuria; Caraballo, Juan M.; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M. Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M. Jose; Blanco, Rosa; Reyes, Jose C.; Agell, Neus; Delgado, M. Dolores; Dotto, G. Paolo; León, Javier

    2012-01-01

    It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

  9. Molecular analysis expands the spectrum of phenotypes associated with GLI3 mutations

    PubMed Central

    Johnston, Jennifer J.; Sapp, Julie C.; Turner, Joyce T.; Amor, David; Aftimos, Salim; Aleck, Kyrieckos A.; Bocian, Maureen; Bodurtha, Joann N.; Cox, Gerald F.; Curry, Cynthia J.; Day, Ruth; Donnai, Dian; Field, Michael; Fujiwara, Ikuma; Gabbett, Michael; Gal, Moran; Graham, John M.; Hedera, Peter; Hennekam, Raoul C.M.; Hersh, Joseph H.; Hopkin, Robert J.; Kayserili, Hülya; Kidd, Alexa M.J.; Kimonis, Virginia; Lin, Angela E.; Lynch, Sally Ann; Maisenbacher, Melissa; Mansour, Sahar; McGaughran, Julie; Mehta, Lakshmi; Murphy, Helen; Raygada, Margarita; Robin, Nathaniel H.; Rope, Alan F.; Rosenbaum, Kenneth N.; Schaefer, G. Bradley; Shealy, Amy; Smith, Wendy; Soller, Maria; Sommer, Annmarie; Stalker, Heather J.; Steiner, Bernhard; Stephan, Mark J.; Tilstra, David; Tomkins, Susan; Trapane, Pamela; Tsai, Anne Chun-Hui; Van Allen, Margot I.; Vasudevan, Pradeep C.; Zabel, Bernhard; Zunich, Janice; Black, Graeme C.M.; Biesecker, Leslie G.

    2010-01-01

    A range of phenotypes including Greig cephalopolysyndactyly and Pallister-Hall syndromes (GCPS, PHS) are caused by pathogenic mutation of the GLI3 gene. To characterize the clinical variability of GLI3 mutations, we present a subset of a cohort of 174 probands referred for GLI3 analysis. Eighty-one probands with typical GCPS or PHS were previously reported, and we report the remaining ninety-three probands here. This includes nineteen probands (twelve mutations) who fulfilled clinical criteria for GCPS or PHS, forty-eight probands (sixteen mutations) with features of GCPS or PHS but who did not meet the clinical criteria (sub-GCPS and sub-PHS), twenty-one probands (six mutations) with features of PHS or GCPS and oral-facial-digital syndrome and five probands (one mutation) with non-syndromic polydactyly. These data support previously identified genotype-phenotype correlations and demonstrate a more variable degree of severity than previously recognized. The finding of GLI3 mutations in patients with features of oral-facial-digital syndrome supports the observation that GLI3 interacts with cilia. We conclude that the phenotypic spectrum of GLI3 mutations is broader than that encompassed by the clinical diagnostic criteria, but the phenotype-genotype correlation persists. Individuals with features of either GCPS or PHS should be screened for mutations in GLI3 even if they do not fulfill clinical criteria. PMID:20672375

  10. New insights into genotype-phenotype correlation for GLI3 mutations.

    PubMed

    Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent-Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

    2015-01-01

    The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

  11. New insights into genotype–phenotype correlation for GLI3 mutations

    PubMed Central

    Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent- Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

    2015-01-01

    The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister–Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype–phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype–phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues. PMID:24736735

  12. Control of developmentally primed erythroid genes by combinatorial co-repressor actions

    PubMed Central

    Stadhouders, Ralph; Cico, Alba; Stephen, Tharshana; Thongjuea, Supat; Kolovos, Petros; Baymaz, H. Irem; Yu, Xiao; Demmers, Jeroen; Bezstarosti, Karel; Maas, Alex; Barroca, Vilma; Kockx, Christel; Ozgur, Zeliha; van Ijcken, Wilfred; Arcangeli, Marie-Laure; Andrieu-Soler, Charlotte; Lenhard, Boris; Grosveld, Frank; Soler, Eric

    2015-01-01

    How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2–IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. PMID:26593974

  13. Novel frame-shift mutations of GLI3 gene in non-syndromic postaxial polydactyly patients.

    PubMed

    Wang, Zhigang; Wang, Jian; Li, Yuchan; Geng, Juan; Fu, Qihua; Xu, Yunlan; Shen, Yiping

    2014-06-10

    Polydactyly is a common congenital limb deformity. This anomaly may occur in isolation (non-syndromic) or as part of a syndrome. The glioma-associated oncogene family zinc finger 3 (GLI3) is known to be associated with both syndromic and non-syndromic polydactyly. GLI3 plays a predominant role in the pathogenesis of syndromic polydactyly: mutations have been identified in 68% of patients with Greig cephalopolysyndactyly syndrome and 91% of patients with Pallister-Hall syndrome. The knowledge regarding the contribution of GLI3 in non-syndromic polydactyly is currently very limited. In this study, we assembled a cohort of individuals of Chinese ethnicity with non-syndromic postaxial polydactyly. We presented the clinical features and molecular evaluations of 19 probands. GLI3 mutations were identified in 15.8% of probands (3/19) including two novel frame-shift mutations c.3855dupC (p.Met1286HisfsTer18) and c.4141delA (p.Arg1381GlyfsTer38) detected in sporadic cases and one previously reported nonsense mutation (c.1927C>T/p.Arg643Ter) in a familial case. Of note, GLI3 mutations were exclusively detected in patients with bilateral polydactyly affecting both hands and feet. Three out of five (60%) probands with bilateral polydactyly on both hands and feet carried pathogenic mutations in GLI3. Our study demonstrated the role of GLI3 in a significant fraction of patients with non-syndromic bilateral polydactyly affecting both hands and feet.

  14. A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate▿

    PubMed Central

    del Castillo, Teresa; Duque, Estrella; Ramos, Juan L.

    2008-01-01

    Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism. PMID:18245293

  15. GLI3 mutations in syndromic and non-syndromic polydactyly in two Indian families.

    PubMed

    Patel, Rashmi; Singh, Chandra Bhan; Bhattacharya, Visweswar; Singh, Subodh Kumar; Ali, Akhtar

    2016-03-01

    The GLI3 protein is a zinc finger transcription factor, expressed early in development. The GLI3 gene exhibits allelic heterogeneity as mutations in this gene are associated with several developmental syndromic and non-syndromic polydactyly. The present study reports two cases: first, a familial case of Greig Cephalopolysyndactyly Syndrome (GCPS); the second is a sporadic case with both postaxial polydactyly (PAP) type A and B. Resequencing of GLI3 gene reveals a previously reported nonsense truncation mutation g.42007251G > A (p.R792X; rs121917714) in the GCPS family and a novel single nucleotide insertion g.42004239_42004240insA (p.E1478X) in the sporadic case of postaxial polydactyly (PAP). Both nonsense truncation mutations; p.R792X (GCPS) and p.E1478X (PAP) introduce a premature stop codon leading to loss of C-terminal domains.

  16. Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus

    PubMed Central

    Haddad-Tóvolli, Roberta; Paul, Fabian A.; Zhang, Yuanfeng; Zhou, Xunlei; Theil, Thomas; Puelles, Luis; Blaess, Sandra; Alvarez-Bolado, Gonzalo

    2015-01-01

    Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance—the Shh-Gli code—is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it. PMID:25859185

  17. Inhibition of HIV-1 enhancer-controlled transcription by artificial enhancer-binding peptides derived from bacteriophage 434 repressor.

    PubMed

    Caderas, G; Klauser, S; Liu, N; Bienz, A; Gutte, B

    1999-12-01

    An artificial HIV-1 enhancer-binding 42-residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Städler, K. & Gutte, B. (1993) FEBS Lett. 315, 51-55; Caderas, G. (1997) PhD Thesis, University of Zürich]. Here we show that, after N-terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60-70% reduction of the rate of transcription of an HIV-1 enhancer-carrying plasmid in COS-1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF-kappaBp50 indicated that the binding sites of nucR42 and NF-kappaB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF-kappaBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor-derived nine-residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper- or retro-leucine zipper-mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer-binding peptides as intracellular inhibitors of HIV replication.

  18. The Influence of Repressor DNA Binding Site Architecture on Transcriptional Control

    PubMed Central

    Park, Dan M.

    2014-01-01

    ABSTRACT How the architecture of DNA binding sites dictates the extent of repression of promoters is not well understood. Here, we addressed the importance of the number and information content of the three direct repeats (DRs) in the binding and repression of the icdA promoter by the phosphorylated form of the global Escherichia coli repressor ArcA (ArcA-P). We show that decreasing the information content of the two sites with the highest information (DR1 and DR2) eliminated ArcA binding to all three DRs and ArcA repression of icdA. Unexpectedly, we also found that DR3 occupancy functions principally in repression, since mutation of this low-information-content site both eliminated DNA binding to DR3 and significantly weakened icdA repression, despite the fact that binding to DR1 and DR2 was intact. In addition, increasing the information content of any one of the three DRs or addition of a fourth DR increased ArcA-dependent repression but perturbed signal-dependent regulation of repression. Thus, our data show that the information content and number of DR elements are critical architectural features for maintaining a balance between high-affinity binding and signal-dependent regulation of icdA promoter function in response to changes in ArcA-P levels. Optimization of such architectural features may be a common strategy to either dampen or enhance the sensitivity of DNA binding among the members of the large OmpR/PhoB family of regulators as well as other transcription factors. PMID:25161193

  19. Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: Measurement of DNA-bound Escherichia coli lac repressor in vivo*

    PubMed Central

    Kao-Huang, Ying; Revzin, Arnold; Butler, Andrew P.; O'Conner, Pamela; Noble, Daniel W.; Von Hippel, Peter H.

    1977-01-01

    Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of Escherichia coli lac repressor molecules bound in vivo to the operator region and to nonoperator sites on the E. coli chromosome is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent E. coli cells, we find that less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum in vivo value of the association constant for repressor to bulk nonoperator DNA (KRD) calculated from these results is about 103 M-1, and analysis of the sources of error in the minicell experiment suggests that the actual in vivo value of KRD could be substantially greater. The value of KRD, coupled with in vitro data on the ionic strength dependence of this parameter, can be used to estimate that the effective intracellular cation activity of E. coli is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of sodium ion equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (KRID) is [unk] KRDin vivo. These results are used to calculate minimum in vivo values of KRO and KRIO (association constants for repressor and for inducer-repressor complex binding to operator) of about 1012 M-1 and about 109 M-1, respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression [von Hippel, P. H., Revzin, A., Gross, C. A. & Wang, A. C

  20. Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.

    PubMed

    Bockamp, Ernesto; Christel, Cerstin; Hameyer, Dorothe; Khobta, Andriy; Maringer, Marko; Reis, Marco; Heck, Rosario; Cabezas-Wallscheid, Nina; Epe, Bernd; Oesch-Bartlomowicz, Barbara; Kaina, Bernd; Schmitt, Steffen; Eshkind, Leonid

    2007-04-01

    Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined. Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background. The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression. Copyright (c) 2007 John Wiley & Sons, Ltd.

  1. Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals

    PubMed Central

    Fu, Lin; Wu, Hailong; Cheng, Steven Y; Gao, Daming; Zhang, Lei; Zhao, Yun

    2016-01-01

    Hedgehog signaling plays very important roles in development and cancers. Vertebrates have three transcriptional factors, Gli1, Gli2 and Gli3. Among them, Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus (Ci, in Drosophila) structurally and functionally as a ‘double agent’ for Shh target gene expression. Here we show that Gli3 full-length, but not the truncated form, can be methylated at K436 and K595. This methylation is specifically catalyzed by Set7, a lysine methyltransferase (KMT). Methylation at K436 and K595 respectively increases the stability and DNA binding ability of Gli3, resulting in an enhancement of Shh signaling activation. Furthermore, functional experiments indicate that the Gli3 methylation contributes to the tumor growth and metastasis in non-small cell lung cancer in vitro and in vivo. Therefore, we propose that Set7 mediated methylation is a novel PTM of Gli3, which positively regulates the transactivity of Gli3 and the activation of Shh signaling. DOI: http://dx.doi.org/10.7554/eLife.15690.001 PMID:27146893

  2. The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

    PubMed

    Su, Liyan; Bassa, Carole; Audran, Corinne; Mila, Isabelle; Cheniclet, Catherine; Chevalier, Christian; Bouzayen, Mondher; Roustan, Jean-Paul; Chervin, Christian

    2014-11-01

    Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

  3. MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3

    PubMed Central

    Yao, Chencheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Chen, Zheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility PMID:26755652

  4. CaAP2 transcription factor is a candidate gene for a flowering repressor and a candidate for controlling natural variation of flowering time in Capsicum annuum.

    PubMed

    Borovsky, Yelena; Sharma, Vinod K; Verbakel, Henk; Paran, Ilan

    2015-06-01

    The APETALA2 transcription factor homolog CaAP2 is a candidate gene for a flowering repressor in pepper, as revealed by induced-mutation phenotype, and a candidate underlying a major QTL controlling natural variation in flowering time. To decipher the genetic control of transition to flowering in pepper (Capsicum spp.) and determine the extent of gene function conservation compared to model species, we isolated and characterized several ethyl methanesulfonate (EMS)-induced mutants that vary in their flowering time compared to the wild type. In the present study, we report on the isolation of an early-flowering mutant that flowers after four leaves on the primary stem compared to nine leaves in the wild-type 'Maor'. By genetic mapping and sequencing of putative candidate genes linked to the mutant phenotype, we identified a member of the APETALA2 (AP2) transcription factor family, CaAP2, which was disrupted in the early-flowering mutant. CaAP2 is a likely ortholog of AP2 that functions as a repressor of flowering in Arabidopsis. To test whether CaAP2 has an effect on controlling natural variation in the transition to flowering in pepper, we performed QTL mapping for flowering time in a cross between early and late-flowering C. annuum accessions. We identified a major QTL in a region of chromosome 2 in which CaAP2 was the most significant marker, explaining 52 % of the phenotypic variation of the trait. Sequence comparison of the CaAP2 open reading frames in the two parents used for QTL mapping did not reveal significant variation. In contrast, significant differences in expression level of CaAP2 were detected between near-isogenic lines that differ for the flowering time QTL, supporting the putative function of CaAP2 as a major repressor of flowering in pepper.

  5. Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements.

    PubMed

    Akiyama, Ryutaro; Kawakami, Hiroko; Wong, Julia; Oishi, Isao; Nishinakamura, Ryuichi; Kawakami, Yasuhiko

    2015-04-21

    Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

  6. The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine1

    PubMed Central

    Cavallini, Erika; Matus, José Tomás; Finezzo, Laura; Zenoni, Sara; Loyola, Rodrigo; Guzzo, Flavia; Schlechter, Rudolf; Ageorges, Agnès; Arce-Johnson, Patricio

    2015-01-01

    Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of

  7. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    SciTech Connect

    Wang, Shucai; Chang, Ying; Guo, Jianjun; Zeng, Qingning; Ellis, Brian; Chen, Jay

    2011-01-01

    BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  8. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    PubMed Central

    Guo, Jianjun; Zeng, Qingning; Ellis, Brian E.; Chen, Jin-Gui

    2011-01-01

    Background The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. Methodology/Principal Findings We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. Conclusions/Significance Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  9. Arabidopsis ovate family proteins, a novel transcriptional repressor family, control multiple aspects of plant growth and development.

    PubMed

    Wang, Shucai; Chang, Ying; Guo, Jianjun; Zeng, Qingning; Ellis, Brian E; Chen, Jin-Gui

    2011-01-01

    The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed

  10. Involvement of PpDof1 transcriptional repressor in the nutrient condition-dependent growth control of protonemal filaments in Physcomitrella patens

    PubMed Central

    Sugiyama, Takumi; Ishida, Tetsuya; Tabei, Nobumitsu; Shigyo, Mikao; Konishi, Mineko; Yoneyama, Tadakatsu; Yanagisawa, Shuichi

    2012-01-01

    In higher plants, the Dof transcription factors that harbour a conserved plant-specific DNA-binding domain function in the regulation of diverse biological processes that are unique to plants. Although these factors are present in both higher and lower plants, they have not yet been characterized in lower plants. Here six genes encoding Dof transcription factors in the moss Physcomitrella patens are characterized and two of these genes, PpDof1 and PpDof2, are functionally analysed. The targeted disruption of PpDof1 caused delayed or reduced gametophore formation, accompanied by an effect on development of the caulonema from the chloronema. Furthermore, the ppdof1 disruptants were found to form smaller colonies with a reduced frequency of branching of protonemal filaments, depending on the nutrients in the media. Most of these phenotypes were not apparent in the ppdof2 disruptant, although the ppdof2 disruptants also formed smaller colonies on a particular medium. Transcriptional repressor activity of PpDof1 and PpDof2 and modified expression of a number of genes in the ppdof disruptant lines were also shown. These results thus suggest that the PpDof1 transcriptional repressor has a role in controlling nutrient-dependent filament growth. PMID:22345635

  11. Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner☆

    PubMed Central

    Collette, Nicole M.; Yee, Cristal S.; Murugesh, Deepa; Sebastian, Aimy; Taher, Leila; Gale, Nicholas W.; Economides, Aris N.; Harland, Richard M.; Loots, Gabriela G.

    2013-01-01

    WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1–/– mice lack any obvious limb or skeletal defects, Sost–/– mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost–/–; Sostdc1–/– mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost–/– and Sost–/–; Sostdc1–/– mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling. PMID:23994639

  12. Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function

    SciTech Connect

    Michaud III, Edward J; Haycraft, Courtney J; Aydin Son, Yesim; Zhang, Qihong; Yoder, Bradley

    2005-01-01

    Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh)signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  13. A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome.

    PubMed

    Schrader, Erin K; Harstad, Kristine G; Holmgren, Robert A; Matouschek, Andreas

    2011-11-11

    The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome.

  14. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

    PubMed Central

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Rao, Kummara Madhusudana; Park, Soo Yong; Chung, Ildoo; Ha, Chang-Sik; Kim, Sang-Woo; Yun, Yang H.; Jung, Youngmi

    2016-01-01

    Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis. PMID:27001906

  15. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine.

    PubMed

    Cavallini, Erika; Matus, José Tomás; Finezzo, Laura; Zenoni, Sara; Loyola, Rodrigo; Guzzo, Flavia; Schlechter, Rudolf; Ageorges, Agnès; Arce-Johnson, Patricio; Tornielli, Giovanni Battista

    2015-04-01

    Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of

  16. AraC-Type Regulator Rbf Controls the Staphylococcus epidermidis Biofilm Phenotype by Negatively Regulating the icaADBC Repressor SarR.

    PubMed

    Rowe, Sarah E; Campbell, Christopher; Lowry, Colm; O'Donnell, Sinead T; Olson, Michael E; Lindgren, Jill K; Waters, Elaine M; Fey, Paul D; O'Gara, James P

    2016-11-01

    Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating

  17. Recent insights into Groucho co-repressor recruitment and function.

    PubMed

    Kaul, Aamna K; Schuster, Eugene F; Jennings, Barbara H

    2015-01-01

    Gene expression is often controlled by transcriptional repressors during development. Many transcription factors lack intrinsic repressive activity but recruit co-factors that inhibit productive transcription. Here we discuss new insights and models for repression mediated by the Groucho/Transducin-Like Enhancer of split (Gro/TLE) family of co-repressor proteins.

  18. Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho.

    PubMed

    Peden, Erin; Kimberly, Elizabeth; Gengyo-Ando, Keiko; Mitani, Shohei; Xue, Ding

    2007-12-01

    Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. Here we report the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. Our study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37.

  19. The NAC-like gene ANTHER INDEHISCENCE FACTOR acts as a repressor that controls anther dehiscence by regulating genes in the jasmonate biosynthesis pathway in Arabidopsis

    PubMed Central

    Yang, Chang-Hsien

    2014-01-01

    ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, β-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7–9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis. PMID:24323506

  20. On the ion selectivity in Ca-binding proteins: the cyclo(-L-Pro-Gly-)3 peptide as a model.

    PubMed Central

    Sussman, F; Weinstein, H

    1989-01-01

    Calcium plays a crucial role in many cellular processes. Its functions are directly dependent on the high specificity for Ca2+ exhibited by the proteins and ion carriers that bind divalent ions. To elucidate the basis for this specificity we have calculated the relative energies of solvation of calcium and magnesium ions in complexes with cyclo(-L-Pro-Gly-)3, a small synthetic peptide that binds Ca2+ with an affinity comparable to those of the naturally occurring proteins. The results show that the ion selectivity of the peptide resides in the difference in the solvation energies of the competing ions in water. Although the peptide is able to complex Mg2+ better than Ca2+ in the stoichiometries in which cyclo(-L-Pro-Gly-)3 binds divalent ions, it is not always able to provide as much stabilization for Mg2+ as water does. These results also explain why cyclo(-L-Pro-Gly-)3 binds Ca2+ and Mg2+ with different stoichiometries and indicate the source for expected differences in the structures of complexes of the two ions. Images PMID:2813364

  1. The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae

    PubMed Central

    Higuchi-Sanabria, Ryo; Vevea, Jason D.; Charalel, Joseph K.; Sapar, Maria L.; Pon, Liza A.

    2016-01-01

    Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae. Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2∆ yeast, and extends the replicative lifespan and cellular health span of sir2∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast. PMID:28357337

  2. Dueling regulatory properties of a transcriptional activator (MtrA) and repressor (MtrR) that control efflux pump gene expression in Neisseria gonorrhoeae.

    PubMed

    Zalucki, Yaramah M; Dhulipala, Vijaya; Shafer, William M

    2012-12-04

    MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20-30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci. Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a "superbug." One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump

  3. The Conserved MAPK Site in E(spl)-M8, an Effector of Drosophila Notch Signaling, Controls Repressor Activity during Eye Development

    PubMed Central

    Bandyopadhyay, Mohna; Bishop, Clifton P.

    2016-01-01

    The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader

  4. Polymorphism of collagen triple helix revealed by 19F NMR of model peptide [Pro-4(R)-hydroxyprolyl-Gly]3-[Pro-4(R)-fluoroprolyl-Gly]-[Pro-4(R)-hydroxyprolyl-Gly]3.

    PubMed

    Kawahara, Kazuki; Nemoto, Nobuaki; Motooka, Daisuke; Nishi, Yoshinori; Doi, Masamitsu; Uchiyama, Susumu; Nakazawa, Takashi; Nishiuchi, Yuji; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

    2012-06-14

    We have characterized various structures of (Pro-Hyp(R)-Gly)(3)-Pro-fPro(R)-Gly-(Pro-Hyp(R)-Gly)(3) in the process of cis-trans isomerization and helix-coil transition by exploiting the sole (19)F NMR probe in 4(R)-fluoroproline (fPro(R)). Around the transition temperature (T(m)), we detected a species with a triple helical structure distinct from the ordinary one concerning the alignment of three strands. The (19)F-(19)F exchange spectroscopy showed that this misaligned and that the ordinary triple helices were interchangeable only indirectly via an extended monomer strand with all-trans peptide bonds at Pro-fPro(R), Pro-Hyp(R), and Gly-Pro in the central segment. This finding demonstrates that the helix-coil transition of collagen peptides is not described with a simple two-state model. We thus elaborated a scheme for the transition mechanism of (Pro-Hyp(R)-Gly)(n) that the most extended monomer strand can be the sole source both to the misaligned and correctly folded triple-helices. The staggered ends could help misaligned triple helices to self-assemble to higher-order structures. We have also discussed the possible relationship between the misaligned triple helix accumulating maximally at T(m) and the kinetic hysteresis associated with the helix-coil transition of collagen.

  5. Response to Copper Stress in Streptomyces lividans Extends beyond Genes under Direct Control of a Copper-sensitive Operon Repressor Protein (CsoR)*

    PubMed Central

    Dwarakanath, Srivatsa; Chaplin, Amanda K.; Hough, Michael A.; Rigali, Sébastien; Vijgenboom, Erik; Worrall, Jonathan A. R.

    2012-01-01

    A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoRSl) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and CuI-CsoRSl to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoRSl reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P1-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon. PMID:22451651

  6. Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

    PubMed

    Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

    2017-09-15

    Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

  7. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  8. An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5′Hoxd–Gli3 antagonism

    PubMed Central

    Huang, Bau-Lin; Trofka, Anna; Furusawa, Aki; Norrie, Jacqueline L.; Rabinowitz, Adam H.; Vokes, Steven A.; Mark Taketo, M.; Zakany, Jozsef; Mackem, Susan

    2016-01-01

    The number of phalanges and joints are key features of digit ‘identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5′Hoxd–Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5′Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation. PMID:27713395

  9. The transcriptional repressor Bcl6 controls the stability of regulatory T cells by intrinsic and extrinsic pathways.

    PubMed

    Sawant, Deepali V; Wu, Hao; Yao, Weiguo; Sehra, Sarita; Kaplan, Mark H; Dent, Alexander L

    2015-05-01

    Foxp3(+) regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6(-/-) ) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6(-/-) mice, increased numbers of Foxp3-negative 'exTreg' cells were found, particularly in the CD25(+) population. ExTreg cells from Bcl6(-/-) mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6(-/-) mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6(Foxp3-/-) ) mice were analysed. Bcl6(Foxp3-/-) mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6(-/-) mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6(Foxp3-/-) mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6(+) Treg cells. Bcl6(Foxp3-/-) mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses. © 2014 John Wiley & Sons Ltd.

  10. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

    PubMed

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-07-05

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

  11. Adult Gli2+/–;Gli3Δ699/+ Male and Female Mice Display a Spectrum of Genital Malformation

    PubMed Central

    He, Fei; Akbari, Pedram; Mo, Rong; Zhang, Jennifer J.; Hui, Chi-Chung; Kim, Peter C.; Farhat, Walid A.

    2016-01-01

    Disorders of sexual development (DSD) encompass a broad spectrum of urogenital malformations and are amongst the most common congenital birth defects. Although key genetic factors such as the hedgehog (Hh) family have been identified, a unifying postnatally viable model displaying the spectrum of male and female urogenital malformations has not yet been reported. Since human cases are diagnosed and treated at various stages postnatally, equivalent mouse models enabling analysis at similar stages are of significant interest. Additionally, all non-Hh based genetic models investigating DSD display normal females, leaving female urogenital development largely unknown. Here, we generated compound mutant mice, Gli2+/–;Gli3Δ699/+, which exhibit a spectrum of urogenital malformations in both males and females upon birth, and also carried them well into adulthood. Analysis of embryonic day (E)18.5 and adult mice revealed shortened anogenital distance (AGD), open ventral urethral groove, incomplete fusion of scrotal sac, abnormal penile size and structure, and incomplete testicular descent with hypoplasia in male mice, whereas female mutant mice displayed reduced AGD, urinary incontinence, and a number of uterine anomalies such as vaginal duplication. Male and female fertility was also investigated via breeding cages, and it was identified that male mice were infertile while females were unable to deliver despite becoming impregnated. We propose that Gli2+/–;Gli3Δ699/+ mice can serve as a genetic mouse model for common DSD such as cryptorchidism, hypospadias, and incomplete fusion of the scrotal sac in males, and a spectrum of uterine and vaginal abnormalities along with urinary incontinence in females, which could prove essential in revealing new insights into their equivalent diseases in humans. PMID:27814383

  12. Complex postaxial polydactyly types A and B with camptodactyly, hypoplastic third toe, zygodactyly and other digit anomalies caused by a novel GLI3 mutation.

    PubMed

    Mumtaz, Sara; Yıldız, Esra; Lal, Karmoon; Tolun, Aslıhan; Malik, Sajid

    2017-03-14

    Polydactyly is a phenotypically and genetically highly heterogeneous limb malformation with preaxial and postaxial subtypes and subtypes A and B. Most polydactyly entities are associated with GLI3 mutation. We report on 10 affected individuals from a large Pakistani kindred initially evaluated as a possible new condition. The phenotype is postaxial polydactyly types A and B associated with zygodactyly, postaxial webbing of toes and additional features not previously reported for isolated polydactyly such as camptodactyly, hypoplasia of third toe, and wide space between hallux and second toe. Hypothesizing that the disorder could have resulted from a mutation in a novel gene responsible for polydactyly, we launched a genetic investigation. By linkage mapping and exome sequencing in the most severe case, we identified novel heterozygous frameshift mutation NM_000168.5 (GLI3): c.3635delG (p.(Gly1212Alafs*18)) but did not detect any other possibly deleterious mutation that could explain the unusual features of camptodactyly, hypoplasia of third toe and wide space between first and second toes. Our findings further expand the phenotypic variability of GLI3 polydactyly. We also present a review of GLI3-associated isolated limb anomalies, which indicates that GLI3 mutation leads primarily to two well-established polydactyly types: postaxial types A and B and crossed polydactyly type I. In addition, a variety of other minor digit anomalies generally accompany polydactyly, and there is no straightforward genotype-polydactyly phenotype correlation.

  13. Mechanism of promoter repression by Lac repressor-DNA loops.

    PubMed

    Becker, Nicole A; Peters, Justin P; Maher, L James; Lionberger, Troy A

    2013-01-07

    The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.

  14. MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

    SciTech Connect

    Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

    2013-05-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC

  15. Central role of the flowering repressor ZCCT2 in the redox control of freezing tolerance and the initial development of flower primordia in wheat

    PubMed Central

    2014-01-01

    Background As both abiotic stress response and development are under redox control, it was hypothesised that the pharmacological modification of the redox environment would affect the initial development of flower primordia and freezing tolerance in wheat (Triticum aestivum L.). Results Pharmacologically induced redox changes were monitored in winter (T. ae. ssp. aestivum cv. Cheyenne, Ch) and spring (T. ae. ssp. spelta; Tsp) wheat genotypes grown after germination at 20/17°C for 9 d (chemical treatment: last 3 d), then at 5°C for 21 d (chemical treatment: first 4 d) and subsequently at 20/17°C for 21 d (recovery period). Thiols and their disulphide forms were measured and based on these data reduction potentials were calculated. In the freezing-tolerant Ch the chemical treatments generally increased both the amount of thiol disulphides and the reduction potential after 3 days at 20/17°C. In the freezing-sensitive Tsp a similar effect of the chemicals on these parameters was only observed after the continuation of the treatments for 4 days at 5°C. The applied chemicals slightly decreased root fresh weight and increased freezing tolerance in Ch, whereas they increased shoot fresh weight in Tsp after 4 days at 5°C. As shown after the 3-week recovery at 20/17°C, the initial development of flower primordia was accelerated in Tsp, whereas it was not affected by the treatments in Ch. The chemicals differently affected the expression of ZCCT2 and that of several other genes related to freezing tolerance and initial development of flower primordia in Ch and Tsp after 4 d at 5°C. Conclusions Various redox-altering compounds and osmotica had differential effects on glutathione disulphide content and reduction potential, and consequently on the expression of the flowering repressor ZCCT2 in the winter wheat Ch and the spring wheat Tsp. We propose that the higher expression of ZCCT2 in Ch may be associated with activation of genes of cold acclimation and its lower

  16. Neisseria prophage repressor implicated in gonococcal pathogenesis.

    PubMed

    Daou, Nadine; Yu, Chunxiao; McClure, Ryan; Gudino, Cynthia; Reed, George W; Genco, Caroline A

    2013-10-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect and colonize multiple mucosal sites in both men and women. The ability to cope with different environmental conditions requires tight regulation of gene expression. In this study, we identified and characterized a gonococcal transcriptional regulatory protein (Neisseria phage repressor [Npr]) that was previously annotated as a putative gonococcal phage repressor protein. Npr was found to repress transcription of NGNG_00460 to NGNG_00463 (NGNG_00460-00463), an operon present within the phage locus NgoΦ4. Npr binding sites within the NGNG_00460-00463 promoter region were found to overlap the -10 and -35 promoter motifs. A gonococcal npr mutant demonstrated increased adherence to and invasion of human endocervical epithelial cells compared to a wild-type gonococcal strain. Likewise, the gonococcal npr mutant exhibited enhanced colonization in a gonococcal mouse model of mucosal infection. Analysis of the gonococcal npr mutant using RNA sequence (RNA-seq) analysis demonstrated that the Npr regulon is limited to the operon present within the phage locus. Collectively, our studies have defined a new gonococcal phage repressor protein that controls the transcription of genes implicated in gonococcal pathogenesis.

  17. Neisseria Prophage Repressor Implicated in Gonococcal Pathogenesis

    PubMed Central

    Daou, Nadine; Yu, Chunxiao; Mcclure, Ryan; Gudino, Cynthia; Reed, George W.

    2013-01-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect and colonize multiple mucosal sites in both men and women. The ability to cope with different environmental conditions requires tight regulation of gene expression. In this study, we identified and characterized a gonococcal transcriptional regulatory protein (Neisseria phage repressor [Npr]) that was previously annotated as a putative gonococcal phage repressor protein. Npr was found to repress transcription of NGNG_00460 to NGNG_00463 (NGNG_00460-00463), an operon present within the phage locus NgoΦ4. Npr binding sites within the NGNG_00460-00463 promoter region were found to overlap the −10 and −35 promoter motifs. A gonococcal npr mutant demonstrated increased adherence to and invasion of human endocervical epithelial cells compared to a wild-type gonococcal strain. Likewise, the gonococcal npr mutant exhibited enhanced colonization in a gonococcal mouse model of mucosal infection. Analysis of the gonococcal npr mutant using RNA sequence (RNA-seq) analysis demonstrated that the Npr regulon is limited to the operon present within the phage locus. Collectively, our studies have defined a new gonococcal phage repressor protein that controls the transcription of genes implicated in gonococcal pathogenesis. PMID:23876804

  18. A tale of two repressors.

    PubMed

    Lewis, Mitchell

    2011-05-27

    Few proteins have had such a strong impact on a field, as the lac repressor and λ repressor have had in Molecular Biology in bacteria. The genes required for lactose utilization are negatively regulated; the lac repressor binds to an upstream operator blocking the transcription of the enzymes necessary for lactose utilization. A similar switch regulates the virus life cycle; λ repressor binds to an operator site and blocks transcription of the phage genes necessary for lytic development. It is now 50 years since Jacob and Monod first proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. Jacob, F. & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356. This model provides a cogent depiction of how a set of genes can be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. A historical perspective that illustrates the role these two repressor molecules played and their contribution to our understanding of gene regulation is presented.

  19. The Arabidopsis RING-Type E3 Ligase TEAR1 Controls Leaf Development by Targeting the TIE1 Transcriptional Repressor for Degradation[OPEN

    PubMed Central

    Zhang, Jinzhe; Wei, Baoye; Yuan, Rongrong; Yu, Hao

    2017-01-01

    The developmental plasticity of leaf size and shape is important for leaf function and plant survival. However, the mechanisms by which plants form diverse leaves in response to environmental conditions are not well understood. Here, we identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) and found that it regulates leaf development by promoting the degradation of TCP INTERACTOR-CONTAINING EAR MOTIF PROTEIN1 (TIE1), an important repressor of CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are key for leaf development. TEAR1 contains a typical C3H2C3-type RING domain and has E3 ligase activity. We show that TEAR1 interacts with the TCP repressor TIE1, which is ubiquitinated in vivo and degraded by the 26S proteasome system. We demonstrate that TEAR1 is colocalized with TIE1 in nuclei and negatively regulates TIE1 protein levels. Overexpression of TEAR1 rescued leaf defects caused by TIE1 overexpression, whereas disruption of TEAR1 resulted in leaf phenotypes resembling those caused by TIE1 overexpression or TCP dysfunction. Deficiency in TEAR partially rescued the leaf defects of TCP4 overexpression line and enhanced the wavy leaf phenotypes of jaw-5D. We propose that TEAR1 positively regulates CIN-like TCP activity to promote leaf development by mediating the degradation of the TCP repressor TIE1. PMID:28100709

  20. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP

    PubMed Central

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-01-01

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type–dependent manner, with 4E-BP1 being a key player. PMID:27313212

  1. A genetic biosensor for identification of transcriptional repressors of target promoters.

    PubMed

    Wang, Weishan; Li, Xiao; Li, Yue; Li, Shanshan; Fan, Keqiang; Yang, Keqian

    2015-10-29

    Transcriptional repressors provide widespread biological significance in the regulation of gene expression. However, in prokaryotes, it is particularly difficult to find transcriptional repressors that recognize specific target promoters on genome-scale. To address this need, a genetic biosensor for identifying repressors of target promoters was developed in Escherichia coli from a de novo designed genetic circuit. This circuit can convert the negative input of repressors into positive output of reporters, thereby facilitating the selection and identification of repressors. After evaluating the sensitivity and bias, the biosensor was used to identify the repressors of scbA and aco promoters (PscbA and Paco), which control the transcription of signalling molecule synthase genes in Streptomyces coelicolor and Streptomyces avermitilis, respectively. Two previously unknown repressors of PscbA were identified from a library of TetR family regulators in S. coelicolor, and three novel repressors of Paco were identified from a genomic library of S. avermitilis. Further in vivo and in vitro experiments confirmed that these newly identified repressors attenuated the transcription of their target promoters by direct binding. Overall, the genetic biosensor developed here presents an innovative and powerful strategy that could be applied for identifying genome-wide unknown repressors of promoters in bacteria.

  2. Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

    PubMed

    Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

    2015-06-03

    Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.

  3. Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR.

    PubMed

    Uhde, Andreas; Brühl, Natalie; Goldbeck, Oliver; Matano, Christian; Gurow, Oksana; Rückert, Christian; Marin, Kay; Wendisch, Volker F; Krämer, Reinhard; Seibold, Gerd M

    2016-08-15

    Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the

  4. Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

    PubMed Central

    Uhde, Andreas; Brühl, Natalie; Goldbeck, Oliver; Matano, Christian; Gurow, Oksana; Rückert, Christian; Marin, Kay; Wendisch, Volker F.; Krämer, Reinhard

    2016-01-01

    ABSTRACT Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCE Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator

  5. miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer.

    PubMed

    Wen, S-Y; Lin, Y; Yu, Y-Q; Cao, S-J; Zhang, R; Yang, X-M; Li, J; Zhang, Y-L; Wang, Y-H; Ma, M-Z; Sun, W-W; Lou, X-L; Wang, J-H; Teng, Y-C; Zhang, Z-G

    2015-02-05

    Although significant advances have recently been made in the diagnosis and treatment of cervical carcinoma, the long-term survival rate for advanced cervical cancer remains low. Therefore, an urgent need exists to both uncover the molecular mechanisms and identify potential therapeutic targets for the treatment of cervical cancer. MicroRNAs (miRNAs) have important roles in cancer progression and could be used as either potential therapeutic agents or targets. miR-506 is a component of an X chromosome-linked miRNA cluster. The biological functions of miR-506 have not been well established. In this study, we found that miR-506 expression was downregulated in approximately 80% of the cervical cancer samples examined and inversely correlated with the expression of Ki-67, a marker of cell proliferation. Gain-of-function and loss-of-function studies in human cervical cancer, Caski and SiHa cells, demonstrated that miR-506 acts as a tumor suppressor by inhibiting cervical cancer growth in vitro and in vivo. Further studies showed that miR-506 induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of cervical cancer cell. We subsequently identified Gli3, a hedgehog pathway transcription factor, as a direct target of miR-506 in cervical cancer. Furthermore, Gli3 silencing recapitulated the effects of miR-506, and reintroduction of Gli3 abrogated miR-506-induced cell growth arrest and apoptosis. Taken together, we conclude that miR-506 exerts its anti-proliferative function by directly targeting Gli3. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of cervical cancer.

  6. RflM functions as a transcriptional repressor in the autogenous control of the Salmonella Flagellar master operon flhDC.

    PubMed

    Singer, Hanna M; Erhardt, Marc; Hughes, Kelly T

    2013-09-01

    Motility of bacteria like Salmonella enterica is a highly regulated process that responds to a variety of internal and external stimuli. A hierarchy of three promoter classes characterizes the Salmonella flagellar system, and the onset of flagellar gene expression depends on the oligomeric regulatory complex and class 1 gene product FlhD(4)C(2). The flhDC promoter is a target for a broad range of transcriptional regulators that bind within the flhDC promoter region and either negatively or positively regulate flhDC operon transcription. In this work, we demonstrate that the RflM protein is a key component of flhDC regulation. Transposon mutagenesis was performed to investigate a previously described autoinhibitory effect of the flagellar master regulatory complex FlhD(4)C(2). RflM is a LuxR homolog that functions as a flagellar class 1 transcriptional repressor. RflM was found to be the negative regulator of flhDC expression that is responsible for the formerly described autoinhibitory effect of the FlhD(4)C(2) complex on flhDC operon transcription (K. Kutsukake, Mol. Gen. Genet. 254:440-448, 1997). We conclude that upon commencement of flagellar gene expression, the FlhD(4)C(2) complex initiates a regulatory feedback loop by activating rflM gene expression. rflM encodes a transcriptional repressor, RflM, which fine-tunes flhDC expression levels.

  7. The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase- and E2F-associated bipartite repressor element.

    PubMed

    Polanowska, J; Fabbrizio, E; Le Cam, L; Trouche, D; Emiliani, S; Herrera, R; Sardet, C

    2001-07-12

    The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G(0) to move through the initial G(1)/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G(1) phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G(1)-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element.

  8. Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation.

    PubMed

    Calder, Elizabeth L; Tchieu, Jason; Steinbeck, Julius A; Tu, Edmund; Keros, Sotirios; Ying, Shui-Wang; Jaiswal, Manoj K; Cornacchia, Daniela; Goldstein, Peter A; Tabar, Viviane; Studer, Lorenz

    2015-08-19

    The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic

  9. Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation

    PubMed Central

    Calder, Elizabeth L.; Steinbeck, Julius A.; Tu, Edmund; Keros, Sotirios; Ying, Shui-Wang; Jaiswal, Manoj K.; Cornacchia, Daniela; Goldstein, Peter A.; Tabar, Viviane

    2015-01-01

    The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early

  10. A conserved lysine in the estrogen receptor DNA binding domain regulates ligand activation profiles at AP-1 sites, possibly by controlling interactions with a modulating repressor

    PubMed Central

    Uht, Rosalie M; Webb, Paul; Nguyen, Phuong; Price Jr, Richard H; Valentine, Cathleen; Favre, Helene; Kushner, Peter J

    2004-01-01

    Background Estrogen receptors alpha and beta (ERα and ERβ) differentially activate genes with AP-1 elements. ERα activates AP-1 targets via activation functions with estrogens (the AF-dependent pathway), whereas ERβ, and a short version of ERα (ERα DBD-LBD) activate only with anti-estrogens (AF-independent pathway). The DNA binding domain (DBD) plays an important role in both pathways, even though neither pathway requires ERE recognition. Results Mutations of a highly conserved DBD lysine (ERα.K206A/G), lead to super-activation of AP-1 through activation function dependent pathways, up to 200 fold. This super-activity can be elicited either through ER AFs 1 or 2, or that of a heterologous activation function (VP16). The homologous substitution in ERβ, K170A, or in ERα DBD-LBD leads to estrogen-dependent AP-1 activation and loss of the usually potent anti-estrogen effects. Each of numerous K206 substitutions in ERα, except K206R, eliminates anti-estrogen activation and this loss correlates perfectly with a loss of ability to titrate a repressive function from the RU486 bound progesterone receptor. Conclusion We conclude that ER DBDs contain a complex regulatory function that influences ligand activation profiles at AP-1. This function, which requires the integrity of the conserved lysine, both allows for activation at AP-1 with anti-estrogens (with ERβ and ERα DBD-LBD), and prevents ERα from becoming superactive at AP-1 with estrogens. We discuss the possibility that a repressor interaction with the DBD both mediates the AF-independent pathway and dampens the AF dependent pathway. Mutations in the conserved lysine might, by this model, disrupt the binding or function of the repressor. PMID:15132742

  11. Two essential FtsH proteases control the level of the Fur repressor during iron deficiency in the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Krynická, Vendula; Tichý, Martin; Krafl, Jaroslav; Yu, Jianfeng; Kaňa, Radek; Boehm, Marko; Nixon, Peter J; Komenda, Josef

    2014-11-01

    The cyanobacterium Synechocystis sp. PCC 6803 expresses four different FtsH protease subunits (FtsH1-4) that assemble into specific homo- and heterocomplexes. The FtsH2/FtsH3 complex is involved in photoprotection but the physiological roles of the other complexes, notably the essential FtsH1/FtsH3 complex, remain unclear. Here we show that the FtsH1 and FtsH3 proteases are involved in the acclimation of cells to iron deficiency. A mutant conditionally depleted in FtsH3 was unable to induce normal expression of the IsiA chlorophyll-protein and FutA1 iron transporter upon iron deficiency due to a block in transcription, which is regulated by the Fur transcriptional repressor. Levels of Fur declined in the WT and the FtsH2 null mutant upon iron depletion but not in the FtsH3 downregulated strain. A similar stabilizing effect on Fur was also observed in a mutant conditionally depleted in the FtsH1 subunit. Moreover, a mutant overexpressing FtsH1 showed reduced levels of Fur and enhanced accumulation of both IsiA and FutA1 even under iron sufficiency. Analysis of GFP-tagged derivatives and biochemical fractionation supported a common location for FtsH1 and FtsH3 in the cytoplasmic membrane. Overall we propose that degradation of the Fur repressor mediated by the FtsH1/FtsH3 heterocomplex is critical for acclimation to iron depletion.

  12. The Transcriptional Repressor TupA in Aspergillus niger Is Involved in Controlling Gene Expression Related to Cell Wall Biosynthesis, Development, and Nitrogen Source Availability

    PubMed Central

    Schachtschabel, Doreen; Arentshorst, Mark; Nitsche, Benjamin M.; Morris, Sam; Nielsen, Kristian F.; van den Hondel, Cees A. M. J. J.; Klis, Frans M.; Ram, Arthur F. J.

    2013-01-01

    The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the ΔtupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37°C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism. PMID:24205111

  13. Tet repressor-tetracycline interaction.

    PubMed

    Kaszycki, P; Guz, A; Drwiega, M; Wasylewski, Z

    1996-10-01

    Previous studies [Wasylewski et al. (1996), J. Protein Chem. 15, 45-58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7 x 10(9) M-1 sec-1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%). Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of

  14. Transcription repressor HANABA TARANU controls flower development by integrating the actions of multiple hormones, floral organ specification genes, and GATA3 family genes in Arabidopsis.

    PubMed

    Zhang, Xiaolan; Zhou, Yun; Ding, Lian; Wu, Zhigang; Liu, Renyi; Meyerowitz, Elliot M

    2013-01-01

    Plant inflorescence meristems and floral meristems possess specific boundary domains that result in proper floral organ separation and specification. HANABA TARANU (HAN) encodes a boundary-expressed GATA3-type transcription factor that regulates shoot meristem organization and flower development in Arabidopsis thaliana, but the underlying mechanism remains unclear. Through time-course microarray analyses following transient overexpression of HAN, we found that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ specification. Transient overexpression of HAN also represses the expression of HAN and three other GATA3 family genes, HANL2 (HAN-LIKE 2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED), and GNL (GNC-LIKE), forming a negative regulatory feedback loop. Genetic analysis indicates that HAN and the three GATA3 family genes coordinately regulate floral development, and their expression patterns are partially overlapping. HAN can homodimerize and heterodimerize with the three proteins encoded by these genes, and HAN directly binds to its own promoter and the GNC promoter in vivo. These findings, along with the fact that constitutive overexpression of HAN produces an even stronger phenotype than the loss-of-function mutation, support the hypothesis that HAN functions as a key repressor that regulates floral development via regulatory networks involving genes in the GATA3 family, along with genes involved in hormone action and floral organ specification.

  15. A repressor with similarities to prokaryotic and eukaryotic DNA helicases controls the assembly of the CAAT box binding complex at a photosynthesis gene promoter.

    PubMed

    Bezhani, S; Sherameti, I; Pfannschmidt, T; Oelmüller, R

    2001-06-29

    A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is subunit gamma of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmüller, R. (1999) J. Biol. Chem. 274, 36009-36014). We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region. Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons. In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpC expression levels. Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro, is significantly weaker than binding to the wild-type sequence. Again, the opposite results were obtained for the CBF. Thus, we conclude that the assembly of the CBF.DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region. The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure.

  16. Type I Repressors of P Element Mobility

    PubMed Central

    Gloor, G. B.; Preston, C. R.; Johnson-Schlitz, D. M.; Nassif, N. A.; Phillis, R. W.; Benz, W. K.; Robertson, H. M.; Engels, W. R.

    1993-01-01

    We describe here a family of P elements that we refer to as type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the canonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects wer very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3' boundary of a functional type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the type I repressors form a structurally distinct group that does not include more extensively deleted repressor elements such as the KP element described previously. PMID:8224830

  17. Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes.

    PubMed

    Veltmaat, Jacqueline M; Relaix, Frédéric; Le, Lendy T; Kratochwil, Klaus; Sala, Frédéric G; van Veelen, Wendy; Rice, Ritva; Spencer-Dene, Bradley; Mailleux, Arnaud A; Rice, David P; Thiery, Jean Paul; Bellusci, Saverio

    2006-06-01

    Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.

  18. Repressor logic modules assembled by rolling circle amplification platform to construct a set of logic gates

    PubMed Central

    Wei, Hua; Hu, Bo; Tang, Suming; Zhao, Guojie; Guan, Yifu

    2016-01-01

    Small molecule metabolites and their allosterically regulated repressors play an important role in many gene expression and metabolic disorder processes. These natural sensors, though valuable as good logic switches, have rarely been employed without transcription machinery in cells. Here, two pairs of repressors, which function in opposite ways, were cloned, purified and used to control DNA replication in rolling circle amplification (RCA) in vitro. By using metabolites and repressors as inputs, RCA signals as outputs, four basic logic modules were constructed successfully. To achieve various logic computations based on these basic modules, we designed series and parallel strategies of circular templates, which can further assemble these repressor modules in an RCA platform to realize twelve two-input Boolean logic gates and a three-input logic gate. The RCA-output and RCA-assembled platform was proved to be easy and flexible for complex logic processes and might have application potential in molecular computing and synthetic biology. PMID:27869177

  19. Metalloregulatory properties of the ArsD repressor.

    PubMed

    Chen, Y; Rosen, B P

    1997-05-30

    The plasmid-encoded arsenical resistance (ars) operon of plasmid R773 produces resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first two genes in the operon, arsR and arsD, were previously shown to encode trans-acting repressor proteins. ArsR controls the basal level of expression of the operon, while ArsD controls maximal expression. Thus, action of the two repressors form a homeostatic regulatory circuit that maintains the level of ars expression within a narrow range. In this study, we demonstrate that ArsD binds to the same site on the ars promoter element as ArsR but with 2 orders of magnitude lower affinity. The results of gel shift assays demonstrate that ArsD is released from the ars DNA promoter by phenylarsine oxide, sodium arsenite, and potassium antimonyl tartrate (in order of effectiveness), the same inducers to which ArsR responds. Using the quenching of intrinsic tryptophan fluorescence to measure the affinity of the repressor for inducers, apparent Kd values for Sb(III) and As(III) of 2 and 60 microM, respectively, were obtained. These results demonstrate that the arsR-arsD pair provide a sensitive mechanism for sensing a wide range of environmental heavy metals.

  20. Sumoylation regulates nuclear localization of repressor DREAM.

    PubMed

    Palczewska, Malgorzata; Casafont, Iñigo; Ghimire, Kedar; Rojas, Ana M; Valencia, Alfonso; Lafarga, Miguel; Mellström, Britt; Naranjo, Jose R

    2011-05-01

    DREAM is a Ca(2+)-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. 2010 Elsevier B.V. All rights reserved.

  1. Identification of a global repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that controls extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in soft-rotting Erwinia spp.

    PubMed

    Cui, Y; Chatterjee, A; Liu, Y; Dumenyo, C K; Chatterjee, A K

    1995-09-01

    The production of extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) is activated by the cell density (quorum)-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (HSL); plant signals; and aep genes during postexponential growth of Erwinia carotovora subsp. carotovora 71. Studies with mutants of E. carotovora subsp. carotovora 71 derepressed in exoenzyme production led to the identification of a negative regulator gene, rsmA (rsm, repressor of secondary metabolites). Nucleotide sequencing, transcript assays, and protein analysis established that a 183-bp open reading frame encodes the 6.8-kDa RsmA. rsmA has extensive homology with the csrA gene of Escherichia coli, which specifies a negative regulator of carbon storage. Moreover, the suppression of glycogen synthesis in E. coli by rsmA indicates that the Erwinia gene is functionally similar to csrA. Southern hybridizations revealed the presence of rsmA homologs in soft-rotting and non-soft-rotting Erwinia spp. and in other enterobacteria such as Enterobacter aerogenes, E. coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, and Yersinia pseudotuberculosis. rsmA suppresses production of Pel, Peh, Cel, and Prt, plant pathogenicity, and synthesis of HSL in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, and E. chrysanthemi. In the E. carotovora subsp. carotovora 71, rsmA reduces the levels of transcripts of hslI, a luxI homolog required for HSL biosynthesis. This specific effect and the previous finding that HSL is required for extracellular enzyme production and pathogenicity in soft-rotting Erwinia spp. support the hypothesis that rsmA controls these traits by modulating the levels of the cell density (quorum)-sensing signal.

  2. An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.

    PubMed

    Huynh, Mai Anh; Ikeuchi, Yoshiho; Netherton, Stuart; de la Torre-Ubieta, Luis; Kanadia, Rahul; Stegmüller, Judith; Cepko, Constance; Bonni, Shirin; Bonni, Azad

    2011-03-10

    Control of neuronal positioning is fundamental to normal brain development. However, the cell-intrinsic mechanisms that govern neuronal positioning remain to be elucidated. Here, we report that the spliced protein products of the transcriptional regulator SnoN, SnoN1 and SnoN2, harbor opposing functions in the coordinate regulation of neuronal branching and positioning. Knockdown of SnoN2 stimulates axon branching in primary neurons and impairs migration of granule neurons in the rat cerebellar cortex in vivo. By contrast, SnoN1 knockdown suppresses SnoN2 knockdown-induced neuronal branching and strikingly triggers excessive migration of granule neurons in the cerebellar cortex. We also find that SnoN1 forms a complex with the transcription factor FOXO1 that represses the X-linked lissencephaly gene encoding doublecortin (DCX). Accordingly, repression of DCX mediates the ability of SnoN1 to regulate branching in primary neurons and granule neuron migration in vivo. These data define an isoform-specific SnoN1-FOXO1 transcriptional complex that orchestrates neuronal branching and positioning in the brain with important implications for the study of developmental disorders of cognition and epilepsy. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon

    PubMed Central

    Rouquette-Loughlin, Corinne E.; Zalucki, Yaramah M.; Dhulipala, Vijaya L.; Balthazar, Jacqueline T.; Doyle, Raúl G.; Nicholas, Robert A.; Begum, Afrin A.; Raterman, Erica L.; Jerse, Ann E.

    2017-01-01

    ABSTRACT The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE, previously annotated gdhR in Neisseria meningitidis, is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA, which encodes an l-glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae, expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA, as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection. PMID:28400529

  4. Plant Pathogenic Bacteria Utilize Biofilm Growth-associated Repressor (BigR), a Novel Winged-helix Redox Switch, to Control Hydrogen Sulfide Detoxification under Hypoxia*

    PubMed Central

    Guimarães, Beatriz G.; Barbosa, Rosicler L.; Soprano, Adriana S.; Campos, Bruna M.; de Souza, Tiago A.; Tonoli, Celisa C. C.; Leme, Adriana F. P.; Murakami, Mario T.; Benedetti, Celso E.

    2011-01-01

    Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch. PMID:21632538

  5. Transcription factor co-repressors in cancer biology: roles and targeting.

    PubMed

    Battaglia, Sebastiano; Maguire, Orla; Campbell, Moray J

    2010-06-01

    Normal transcription displays a high degree of flexibility over the choice, timing and magnitude of mRNA expression levels that tend to oscillate and cycle. These processes allow for combinatorial actions, feedback control and fine-tuning. A central role has emerged for the transcriptional co-repressor proteins such as NCOR1, NCOR2/SMRT, CoREST and CTBPs, to control the actions of many transcriptional factors, in large part, by recruitment and activation of a range of chromatin remodeling enzymes. Thus, co-repressors and chromatin remodeling factors are recruited to transcription factors at specific promoter/enhancer regions and execute changes in the chromatin structure. The specificity of this recruitment is controlled in a spatial-temporal manner. By playing a central role in transcriptional control, as they move and target transcription factors, co-repressors act as a key driver in the epigenetic economy of the nucleus. Co-repressor functions are selectively distorted in malignancy, by both loss and gain of function and contribute to the generation of transcriptional rigidity. Features of transcriptional rigidity apparent in cancer cells include the distorted signaling of nuclear receptors and the WNTs/beta-catenin axis. Understanding and predicting the consequences of altered co-repressor expression patterns in cancer cells has diagnostic and prognostic significance, and also have the capacity to be targeted through selective epigenetic therapies.

  6. (68)Ga-labeled cyclic RGD dimers with Gly3 and PEG4 linkers: promising agents for tumor integrin alphavbeta3 PET imaging.

    PubMed

    Liu, Zhaofei; Niu, Gang; Shi, Jiyun; Liu, Shuanglong; Wang, Fan; Liu, Shuang; Chen, Xiaoyuan

    2009-06-01

    Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides have great potential for the early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response. (18)F-labeled RGD analogs ([(18)F]-AH111585 and [(18)F]Galacto-RGD) have been investigated in clinical trials for positron emission tomography (PET) imaging of integrin expression in cancer patients. To develop new RGD radiotracers with higher tumor accumulation, improved in vivo kinetics, easy availability and low cost, we developed two new RGD peptides and labeled them with generator-eluted (68)Ga (t(1/2) = 68 min) for PET imaging of integrin alpha(v)beta(3) expression in tumor xenograft models. The two new cyclic RGD dimers, E[PEG(4)-c(RGDfK)](2) (P(4)-RGD2, PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and E[Gly(3)-c(RGDfK)](2) (G(3)-RGD2, G(3) = Gly-Gly-Gly) were designed, synthesized and conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) for (68)Ga labeling. The microPET imaging and biodistribution of the (68)Ga labeled RGD tracers were investigated in integrin alpha(v)beta(3)-positive tumor xenografts. The new RGD dimers with the Gly(3) and PEG(4) linkers showed higher integrin alpha(v)beta(3) binding affinity than no-linker RGD dimer (RGD2). NOTA-G(3)-RGD2 and NOTA-P(4)-RGD2 could be labeled with (68)Ga within 30 min with higher purity (>98%) and specific activity (8.88-11.84 MBq/nmol). Both (68)Ga-NOTA-P(4)-RGD2 and (68)Ga-NOTA-G(3)-RGD2 exhibited significantly higher tumor uptake and tumor-to-normal tissue ratios than (68)Ga-NOTA-RGD2. Because of their high affinity, high specificity and excellent pharmacokinetic properties, further investigation of the two novel RGD dimers for clinical PET imaging of integrin alpha(v)beta(3) expression in cancer patients is warranted.

  7. A genome-wide association scan implicates DCHS2, RUNX2, GLI3, PAX1 and EDAR in human facial variation

    PubMed Central

    Adhikari, Kaustubh; Fuentes-Guajardo, Macarena; Quinto-Sánchez, Mirsha; Mendoza-Revilla, Javier; Camilo Chacón-Duque, Juan; Acuña-Alonzo, Victor; Jaramillo, Claudia; Arias, William; Lozano, Rodrigo Barquera; Pérez, Gastón Macín; Gómez-Valdés, Jorge; Villamil-Ramírez, Hugo; Hunemeier, Tábita; Ramallo, Virginia; Silva de Cerqueira, Caio C.; Hurtado, Malena; Villegas, Valeria; Granja, Vanessa; Gallo, Carla; Poletti, Giovanni; Schuler-Faccini, Lavinia; Salzano, Francisco M.; Bortolini, Maria- Cátira; Canizales-Quinteros, Samuel; Cheeseman, Michael; Rosique, Javier; Bedoya, Gabriel; Rothhammer, Francisco; Headon, Denis; González-José, Rolando; Balding, David; Ruiz-Linares, Andrés

    2016-01-01

    We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values<5 × 10−8) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar funtion. PMID:27193062

  8. A genome-wide association scan implicates DCHS2, RUNX2, GLI3, PAX1 and EDAR in human facial variation.

    PubMed

    Adhikari, Kaustubh; Fuentes-Guajardo, Macarena; Quinto-Sánchez, Mirsha; Mendoza-Revilla, Javier; Camilo Chacón-Duque, Juan; Acuña-Alonzo, Victor; Jaramillo, Claudia; Arias, William; Lozano, Rodrigo Barquera; Pérez, Gastón Macín; Gómez-Valdés, Jorge; Villamil-Ramírez, Hugo; Hunemeier, Tábita; Ramallo, Virginia; Silva de Cerqueira, Caio C; Hurtado, Malena; Villegas, Valeria; Granja, Vanessa; Gallo, Carla; Poletti, Giovanni; Schuler-Faccini, Lavinia; Salzano, Francisco M; Bortolini, Maria-Cátira; Canizales-Quinteros, Samuel; Cheeseman, Michael; Rosique, Javier; Bedoya, Gabriel; Rothhammer, Francisco; Headon, Denis; González-José, Rolando; Balding, David; Ruiz-Linares, Andrés

    2016-05-19

    We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values<5 × 10(-8)) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar funtion.

  9. Negative regulation of defence and stress genes by EAR-motif-containing repressors.

    PubMed

    Kazan, Kemal

    2006-03-01

    Although positive control or activation mechanism(s) involved in plant defence- and stress-related gene expression is relatively well studied, little is known about what keeps defensive armoury under control when not needed. Recent reports suggest that transcriptional repression of gene expression by EAR-motif-containing repressor proteins plays a key role in modulating plant defence and stress responses.

  10. Lac repressor: a genetic and nuclear magnetic resonance study of structure and function

    SciTech Connect

    Jarema, M.A.C.; Lu, P.; Miller, J.H.

    1980-10-01

    The prototype gene control system, the lac operon of E. coli, has recently also become the best chemically characterized system to date. The complete primary sequence of both the gene and the protein reponsible for the regulation of this operon, the repressor, is known, along with the DNA sequence of its site of action, the operator. The lac repressor is a tetrametic protein with four identical subunits of 360 amino acids each, giving a total molecular weight of 154,000. The lac operator sequence is about 25 to 30 base pairs long. With the wealth of information about the primary structure the next question is one of geometry. This leads to the application of either x-ray diffraction or nuclear magnetic resonance (NMR) methods, since these are the only approaches that yield information about the geometry and environment of specific groups and atoms in these molecules. Since we are interested in the interaction of repressor with a variety of small molecular weight inducers and anti-inducers, as well as the operator sequence in aqueous solution, we chose the NMR approach. As of this writing, no useful crystals of the lac repressor or the repressor and any of its ligands have been reported. Because of our extensive genetic work with this system, we have a unique advantage in taking this approach as well.

  11. Solitons and collapse in the λ-repressor protein.

    PubMed

    Krokhotin, Andrey; Lundgren, Martin; Niemi, Antti J

    2012-08-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding λ-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability, and folding pathways of the λ-repressor protein, which controls the transition from the lysogenic to the lytic state. We first investigate the supersecondary helix-loop helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schrödinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the λ-repressor protein. We introduce a coarse-grained energy function to model the backbone in terms of the C(α) atoms and the side chains in terms of the relative orientation of the C(β) atoms. We describe the folding dynamics in terms of relaxation dynamics and find that the folded configuration can be reached from a very generic initial configuration. We conclude that folding is dominated by the temporal ordering of soliton formation. In particular, the third soliton should appear before the first and second. Otherwise, the DNA binding turn does not acquire its correct structure. We confirm the stability of the folded configuration by repeated heating and cooling simulations.

  12. Solitons and collapse in the λ-repressor protein

    NASA Astrophysics Data System (ADS)

    Krokhotin, Andrey; Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding λ-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability, and folding pathways of the λ-repressor protein, which controls the transition from the lysogenic to the lytic state. We first investigate the supersecondary helix-loop helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schrödinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the λ-repressor protein. We introduce a coarse-grained energy function to model the backbone in terms of the Cα atoms and the side chains in terms of the relative orientation of the Cβ atoms. We describe the folding dynamics in terms of relaxation dynamics and find that the folded configuration can be reached from a very generic initial configuration. We conclude that folding is dominated by the temporal ordering of soliton formation. In particular, the third soliton should appear before the first and second. Otherwise, the DNA binding turn does not acquire its correct structure. We confirm the stability of the folded configuration by repeated heating and cooling simulations.

  13. Activators and repressors: A balancing act for X-inactivation.

    PubMed

    Goodrich, Leeanne; Panning, Barbara; Leung, Karen Nicole

    2016-08-01

    In early female embryos X-chromosome inactivation occurs concomitant with up regulation of the non-coding RNA, Xist, on the future inactive X-chromosome. Up regulation of Xist and coating of the future inactive X is sufficient to induce silencing. Therefore unlocking the mechanisms of X-chromosome inactivation requires thorough understanding of the transcriptional regulators, both activators and repressors, which control Xist. Mouse pluripotent embryonic stem cells, which have two active X chromosomes, provide a tractable ex vivo model system for studying X-chromosome inactivation, since this process is triggered by differentiation signals in these cultured cells. Yet there are significant discrepancies found between ex vivo analyses in mouse embryonic stem cells and in vivo studies of early embryos. In this review we elaborate on potential models of how Xist is up regulated on a single X chromosome in female cells and how ex vivo and in vivo analyses enlighten our understanding of the activators and repressors that control this non-coding RNA gene. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression

    NASA Astrophysics Data System (ADS)

    Lengyel, Iván M.; Morelli, Luis G.

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  15. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression.

    PubMed

    Lengyel, Iván M; Morelli, Luis G

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  16. Structure and function of Escherichia coli met repressor: similarities and contrasts with trp repressor.

    PubMed

    Phillips, S E; Stockley, P G

    1996-04-29

    Transcription of genes encoding enzymes for the biosynthesis of methionine and trytophan in Escherichia coli is regulated by the ligand-activated met and trp repressors. X-ray crystallographic studies show how these two small proteins, although similar in size and function, have totally different three-dimensional structures and specifically recognize their respective DNA operator sequences in different ways. A common feature is that both repressors bind as cooperative arrays to tandem repeats of 8 base-pair 'Met' or 'Trp boxes' respectively, and the consensus sequences share the rare tetranucleotide CTAG. A series of structural and functional studies have shown how the two repressors discriminate between their operators, using a combination of direct contacts between side chains and bases, and indirect sensing of conformational properties of the DNA.

  17. DND protein functions as a translation repressor during zebrafish embryogenesis.

    PubMed

    Kobayashi, Manami; Tani-Matsuhana, Saori; Ohkawa, Yasuka; Sakamoto, Hiroshi; Inoue, Kunio

    2017-03-04

    Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants.

    PubMed Central

    Klock, G; Unger, B; Gatz, C; Hillen, W; Altenbuchner, J; Schmid, K; Schmitt, R

    1985-01-01

    Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared. These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D). By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities. In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes. Tetracycline acted as an inducer. A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C. Repression of tetRA-linked galactokinase was ca. 80% in the presence of tetRA or tetRC, and ca. 50% in the presence of tetRB. Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants. Images PMID:3881391

  19. Loss of floral repressor function adapts rice to higher latitudes in Europe

    PubMed Central

    Gómez-Ariza, Jorge; Galbiati, Francesca; Goretti, Daniela; Brambilla, Vittoria; Shrestha, Roshi; Pappolla, Andrea; Courtois, Brigitte; Fornara, Fabio

    2015-01-01

    The capacity to discriminate variations in day length allows plants to align flowering with the most favourable season of the year. This capacity has been altered by artificial selection when cultivated varieties became adapted to environments different from those of initial domestication. Rice flowering is promoted by short days when HEADING DATE 1 (Hd1) and EARLY HEADING DATE 1 (Ehd1) induce the expression of florigenic proteins encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Repressors of flowering antagonize such induction under long days, maintaining vegetative growth and delaying flowering. To what extent artificial selection of long day repressor loci has contributed to expand rice cultivation to Europe is currently unclear. This study demonstrates that European varieties activate both Hd3a and RFT1 expression regardless of day length and their induction is caused by loss-of-function mutations at major long day floral repressors. However, their contribution to flowering time control varies between locations. Pyramiding of mutations is frequently observed in European germplasm, but single mutations are sufficient to adapt rice to flower at higher latitudes. Expression of Ehd1 is increased in varieties showing reduced or null Hd1 expression under natural long days, as well as in single hd1 mutants in isogenic backgrounds. These data indicate that loss of repressor genes has been a key strategy to expand rice cultivation to Europe, and that Ehd1 is a central node integrating floral repressive signals. PMID:25732533

  20. The role of repressor kinetics in relief of transcriptional interference between convergent promoters

    PubMed Central

    Hao, Nan; Palmer, Adam C.; Ahlgren-Berg, Alexandra; Shearwin, Keith E.; Dodd, Ian B.

    2016-01-01

    Transcriptional interference (TI), where transcription from a promoter is inhibited by the activity of other promoters in its vicinity on the same DNA, enables transcription factors to regulate a target promoter indirectly, inducing or relieving TI by controlling the interfering promoter. For convergent promoters, stochastic simulations indicate that relief of TI can be inhibited if the repressor at the interfering promoter has slow binding kinetics, making it either sensitive to frequent dislodgement by elongating RNA polymerases (RNAPs) from the target promoter, or able to be a strong roadblock to these RNAPs. In vivo measurements of relief of TI by CI or Cro repressors in the bacteriophage λ PR–PRE system show strong relief of TI and a lack of dislodgement and roadblocking effects, indicative of rapid CI and Cro binding kinetics. However, repression of the same λ promoter by a catalytically dead CRISPR Cas9 protein gave either compromised or no relief of TI depending on the orientation at which it binds DNA, consistent with dCas9 being a slow kinetics repressor. This analysis shows how the intrinsic properties of a repressor can be evolutionarily tuned to set the magnitude of relief of TI. PMID:27378773

  1. Weak operator binding enhances simulated Lac repressor-mediated DNA looping.

    PubMed

    Colasanti, Andrew V; Grosner, Michael A; Perez, Pamela J; Clauvelin, Nicolas; Lu, Xiang-Jun; Olson, Wilma K

    2013-12-01

    The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding.

  2. A tale of two repressors – a historical perspective

    PubMed Central

    Lewis, Mitchell

    2011-01-01

    Few proteins have had such a strong impact on a field as the lac repressor and λ repressor have had in Molecular Biology In bacteria, the genes required for lactose utilization are negatively regulated; the lac repressor binds to an upstream operator blocking transcription of the enzymes necessary for lactose utilization. A similar switch regulates the virus life cycle; λ repressor binds to an operator site and blocks transcription of the phage genes necessary for lytic development. It is now 50 years since Jacob and Monod first proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks1. This model provides a cogent depiction of how a set of genes can be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. A historical perspective is presented that illustrates the role these two repressor molecules played and their contribution to our understanding of gene regulation. PMID:21392509

  3. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  4. Tn10 tet operator mutations affecting Tet repressor recognition.

    PubMed Central

    Wissmann, A; Meier, I; Wray, L V; Geissendörfer, M; Hillen, W

    1986-01-01

    The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions. PMID:3086838

  5. Broadly expressed repressors integrate patterning across orthogonal axes in embryos.

    PubMed

    Koromila, Theodora; Stathopoulos, Angelike

    2017-07-18

    The role of spatially localized repressors in supporting embryonic patterning is well appreciated, but, alternatively, the role ubiquitously expressed repressors play in this process is not well understood. We investigated the function of two broadly expressed repressors, Runt (Run) and Suppressor of Hairless [Su(H)], in patterning the Drosophila embryo. Previous studies have shown that Run and Su(H) regulate gene expression along anterior-posterior (AP) or dorsal-ventral (DV) axes, respectively, by spatially limiting activator action, but here we characterize a different role. Our data show that broadly expressed repressors silence particular enhancers within cis-regulatory systems, blocking their expression throughout the embryo fully but transiently, and, in this manner, regulate spatiotemporal outputs along both axes. Our results suggest that Run and Su(H) regulate the temporal action of enhancers and are not dedicated regulators of one axis but, instead, act coordinately to pattern both axes, AP and DV.

  6. Repressor Dimerization in the Zebrafish Somitogenesis Clock

    PubMed Central

    Cinquin, Olivier

    2007-01-01

    The oscillations of the somitogenesis clock are linked to the fundamental process of vertebrate embryo segmentation, yet little is known about their generation. In zebrafish, it has been proposed that Her proteins repress the transcription of their own mRNA. However, in its simplest form, this model is incompatible with the fact that morpholino knockdown of Her proteins can impair expression of their mRNA. Simple self-repression models also do not account for the spatiotemporal pattern of gene expression, with waves of gene expression shrinking as they propagate. Here we study computationally the networks generated by the wealth of dimerization possibilities amongst transcriptional repressors in the zebrafish somitogenesis clock. These networks can reproduce knockdown phenotypes, and strongly suggest the existence of a Her1–Her7 heterodimer, so far untested experimentally. The networks are the first reported to reproduce the spatiotemporal pattern of the zebrafish somitogenesis clock; they shed new light on the role of Her13.2, the only known link between the somitogenesis clock and positional information in the paraxial mesoderm. The networks can also account for perturbations of the clock by manipulation of FGF signaling. Achieving an understanding of the interplay between clock oscillations and positional information is a crucial first step in the investigation of the segmentation mechanism. PMID:17305423

  7. Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

    PubMed Central

    Bullwinkle, Tammy J.; Samorodnitsky, Daniel; Rosati, Rayna C.; Koudelka, Gerald B.

    2012-01-01

    We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein. PMID:22509323

  8. The hive bee to forager transition in honeybee colonies: the double repressor hypothesis.

    PubMed

    Amdam, Gro Vang; Omholt, Stig W

    2003-08-21

    In summer, the honeybee (Apis mellifera) worker population consists of two temporal castes, a hive bee group performing a multitude of tasks including nursing inside the nest, and a forager group specialized on collecting nectar, pollen, water and propolis. Elucidation of the regulatory mechanisms responsible for the hive bee to forager transition holds a prominent position within present day sociobiology. Here we suggest a new explanation dubbed the "double repressor hypothesis" aimed to account for the substantial amount of empirical data in this field. This is the first time where both the regular transition and starvation-induced precocious transition are explained within the same regulatory framework. We suggest that the transition is under regulatory control by an internal and an external repressor of the allatoregulatory central nervous system, where these two repressors modulate a positive regulatory feedback loop involving juvenile hormone (JH) and the lipoprotein vitellogenin. The concepts of age-neutrality, fixed and variable response thresholds and reinforcement are integral parts of our explanation, and in addition they are given explicit physiological content. The hypothesis is represented by a differential equations model at the level of the individual bee, and by a discrete individual-based colony model. The two models generate predictions in accordance with empirical data concerning the cumulative probability of becoming a forager, mean age at onset of foraging, reversal of foragers, time window of reversal, relationship between JH titre and onset of foraging, relative representations of genotypic groups, and effects of forager depletion and confinement.

  9. Engineering alternate cooperative-communications in the lactose repressor protein scaffold.

    PubMed

    Meyer, Sarai; Ramot, Roee; Kishore Inampudi, Krishna; Luo, Beibei; Lin, Chenyu; Amere, Swathi; Wilson, Corey J

    2013-06-01

    To expand our understanding of the hallmarks of allosteric control we used directed-evolution to engineer alternate cooperative communication in the lactose repressor protein (LacI) scaffold. Starting with an I(s) type LacI mutant D88A (i.e. a LacI variant that is insensitive to the exogenous ligand isopropyl-β-d-thiogalactoside (IPTG) and remains bound to operator DNA, + or -IPTG) we used error-prone polymerase chain reaction to introduce compensatory mutations to restore modulated DNA binding function to the allosterically 'dead' I(s)(D88A) background. Five variants were generated, three variants (C4, C32 and C80) with wild-type like function and two co-repressor variants (C101 and C140) that are functionally inverted. To better resolve the residues that define new allosteric networks in the LacI variants, we conducted mutational tolerance analysis via saturation mutagenesis at each of the evolved positions to assess sensitivity to mutation--a hallmark of allosteric residues. To better understand the physicochemical bases of alternate allosteric function, variant LacI(C80) was characterized to assess IPTG ligand binding at equilibrium, kinetically using stopped-flow, and via isothermal titration calorimetry. These data suggest that the conferred modulated DNA binding function observed for LacI(C80), while thermodynamically similar to wild-type LacI, is mechanistically different from the wild-type repressor, suggesting a new allosteric network and communication route.

  10. DWARF 53 acts as a repressor of strigolactone signalling in rice

    NASA Astrophysics Data System (ADS)

    Jiang, Liang; Liu, Xue; Xiong, Guosheng; Liu, Huihui; Chen, Fulu; Wang, Lei; Meng, Xiangbing; Liu, Guifu; Yu, Hong; Yuan, Yundong; Yi, Wei; Zhao, Lihua; Ma, Honglei; He, Yuanzheng; Wu, Zhongshan; Melcher, Karsten; Qian, Qian; Xu, H. Eric; Wang, Yonghong; Li, Jiayang

    2013-12-01

    Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCFD3 ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCFD3 ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

  11. Dynamic equilibrium on DNA defines transcriptional regulation of a multidrug binding transcriptional repressor, LmrR.

    PubMed

    Takeuchi, Koh; Imai, Misaki; Shimada, Ichio

    2017-03-21

    LmrR is a multidrug binding transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. Promiscuous compound ligations reduce the affinity of LmrR for the lmrCD operator by several fold to release the transcriptional repression; however, the affinity reduction is orders of magnitude smaller than that of typical transcriptional repressors. Here, we found that the transcriptional regulation of LmrR is achieved through an equilibrium between the operator-bound and non-specific DNA-adsorption states in vivo. The effective dissociation constant of LmrR for the lmrCD operator under the equilibrium is close to the endogenous concentration of LmrR, which allows a substantial reduction of LmrR occupancy upon compound ligations. Therefore, LmrR represents a dynamic type of transcriptional regulation of prokaryotic multidrug resistance systems, where the small affinity reduction induced by compounds is coupled to the functional relocalization of the repressor on the genomic DNA via nonspecific DNA adsorption.

  12. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

    PubMed

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-08-03

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages.

  13. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro

    PubMed Central

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. PMID:27527206

  14. Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification.

    PubMed Central

    Oehmichen, R; Klock, G; Altschmied, L; Hillen, W

    1984-01-01

    Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene. Images Fig. 4. Fig. 5. Fig. 6. PMID:6325175

  15. SPOROCYTELESS is a novel embryophyte-specific transcription repressor that interacts with TPL and TCP proteins in Arabidopsis.

    PubMed

    Chen, Guang-Hui; Sun, Jia-Ying; Liu, Man; Liu, Jie; Yang, Wei-Cai

    2014-12-20

    Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS (SPL). A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins (SPEARs). Consistently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or heterodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS (TPL) co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants.

  16. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  17. Nuclear hormone receptor co-repressors: Structure and function

    PubMed Central

    Watson, Peter J.; Fairall, Louise; Schwabe, John W.R.

    2012-01-01

    Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered “hub proteins” that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined of co-repressor interaction regions in complex with their interacting partners. These have yielded considerable insight into the mechanism of assembly of these complexes, the structural basis for the specificity of the interactions and also open opportunities for targeting these interactions therapeutically. PMID:21925568

  18. Cooperative folding units of escherichia coli tryptophan repressor.

    PubMed Central

    Wallqvist, A; Lavoie, T A; Chanatry, J A; Covell, D G; Carey, J

    1999-01-01

    A previously published computational procedure was used to identify cooperative folding units within tryptophan repressor. The theoretical results predict the existence of distinct stable substructures in the protein chain for the monomer and the dimer. The predictions were compared with experimental data on structure and folding of the repressor and its proteolytic fragments and show excellent agreement for the dimeric form of the protein. The results suggest that the monomer, the structure of which is currently unknown, is likely to have a structure different from the one it has within the context of the highly intertwined dimer. Application of this method to the repressor monomer represents an extension of the computations into the realm of evaluating hypothetical structures such as those produced by threading. PMID:10465773

  19. NINJA connects the co-repressor TOPLESS to jasmonate signalling

    PubMed Central

    Pauwels, Laurens; Barbero, Gemma Fernández; Geerinck, Jan; Tilleman, Sofie; Grunewald, Wim; Pérez, Amparo Cuellar; Chico, José Manuel; Bossche, Robin Vanden; Sewell, Jared; Gil, Eduardo; García-Casado, Gloria; Witters, Erwin; Inzé, Dirk; Long, Jeff A.; De Jaeger, Geert; Solano, Roberto; Goossens, Alain

    2010-01-01

    Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes1–5. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. Upon JA-Ile perception, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated6,7. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL)8 and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel INteractor of JAZ (NINJA). NINJA acts as a transcriptional repressor of which the activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress- and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants. PMID:20360743

  20. NINJA connects the co-repressor TOPLESS to jasmonate signalling.

    PubMed

    Pauwels, Laurens; Barbero, Gemma Fernández; Geerinck, Jan; Tilleman, Sofie; Grunewald, Wim; Pérez, Amparo Cuéllar; Chico, José Manuel; Bossche, Robin Vanden; Sewell, Jared; Gil, Eduardo; García-Casado, Gloria; Witters, Erwin; Inzé, Dirk; Long, Jeff A; De Jaeger, Geert; Solano, Roberto; Goossens, Alain

    2010-04-01

    Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

  1. Transcription of Two Adjacent Carbohydrate Utilization Gene Clusters in Bifidobacterium breve UCC2003 Is Controlled by LacI- and Repressor Open Reading Frame Kinase (ROK)-Type Regulators

    PubMed Central

    O'Connell, Kerry Joan; O'Connell Motherway, Mary; Liedtke, Andrea; Fitzgerald, Gerald F.; Ross, R. Paul; Stanton, Catherine; Zomer, Aldert

    2014-01-01

    Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control. PMID:24705323

  2. Mapping DNA-Lac repressor interaction with ultra-fast optical tweezers

    NASA Astrophysics Data System (ADS)

    Monico, Carina; Tempestini, Alessia; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2015-03-01

    The lac operon is a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with different DNA constructs. Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences and transient interactions with nonspecific sequences.

  3. Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer

    NASA Astrophysics Data System (ADS)

    Lewis, Mitchell; Chang, Geoffrey; Horton, Nancy C.; Kercher, Michele A.; Pace, Helen C.; Schumacher, Maria A.; Brennan, Richard G.; Lu, Ponzy

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

  4. Drosophila CK2 phosphorylates Deadpan, a member of the HES family of basic-helix-loop-helix (bHLH) repressors.

    PubMed

    Karandikar, Umesh C; Shaffer, Jonathan; Bishop, Clifton P; Bidwai, Ashok P

    2005-06-01

    In Drosophila, protein kinase CK2 regulates a diverse array of developmental processes. One of these is cell-fate specification (neurogenesis) wherein CK2 regulates basic-helix-loop-helix (bHLH) repressors encoded by the Enhancer of Split Complex (E(spl)C). Specifically, CK2 phosphorylates and activates repressor functions of E(spl)M8 during eye development. In this study we describe the interaction of CK2 with an E(spl)-related bHLH repressor, Deadpan (Dpn). Unlike E(spl)-repressors which are expressed in cells destined for a non-neural cell fate, Dpn is expressed in the neuronal cells and is thought to control the activity of proneural genes. Dpn also regulates sex-determination by repressing sxl, the primary gene involved in sex differentiation. We demonstrate that Dpn is weakly phosphorylated by monomeric CK2alpha, whereas it is robustly phosphorylated by the embryo-holoenzyme, suggesting a positive role for CK2beta. The weak phosphorylation by CK2alpha is markedly stimulated by the activator polylysine to levels comparable to those with the holoenzyme. In addition, pull down assays indicate a direct interaction between Dpn and CK2. This is the first demonstration that Dpn is a partner and target of CK2, and raises the possibility that its repressor functions might also be regulated by phosphorylation.

  5. Hexokinase 2 Is an Intracellular Glucose Sensor of Yeast Cells That Maintains the Structure and Activity of Mig1 Protein Repressor Complex.

    PubMed

    Vega, Montserrat; Riera, Alberto; Fernández-Cid, Alejandra; Herrero, Pilar; Moreno, Fernando

    2016-04-01

    Hexokinase 2 (Hxk2) fromSaccharomyces cerevisiaeis a bi-functional enzyme, being both a catalyst in the cytosol and an important regulator of the glucose repression signal in the nucleus. Despite considerable recent progress, little is known about the regulatory mechanism that controls nuclear Hxk2 association with theSUC2promoter chromatin and how this association is necessary forSUC2gene repression. Our data indicate that in theSUC2promoter context, Hxk2 functions through a variety of structurally unrelated factors, mainly the DNA-binding Mig1 and Mig2 repressors and the regulatory Snf1 and Reg1 factors. Hxk2 sustains the repressor complex architecture maintaining transcriptional repression at theSUC2gene. Using chromatin immunoprecipitation assays, we discovered that the Hxk2 in its open configuration, at low glucose conditions, leaves the repressor complex that induces its dissociation and promotesSUC2gene expression. In high glucose conditions, Hxk2 adopts a close conformation that promotes Hxk2 binding to the Mig1 protein and the reassembly of theSUC2repressor complex. Additional findings highlight the possibility that Hxk2 constitutes an intracellular glucose sensor that operates by changing its conformation in response to cytoplasmic glucose levels that regulate its interaction with Mig1 and thus its recruitment to the repressor complex of theSUC2promoter. Thus, our data indicate that Hxk2 is more intimately involved in gene regulation than previously thought.

  6. SHORT VEGETATIVE PHASE Up-Regulates TEMPRANILLO2 Floral Repressor at Low Ambient Temperatures1[OPEN

    PubMed Central

    Marín-González, Esther; Matías-Hernández, Luis; Aguilar-Jaramillo, Andrea E.; Lee, Jeong Hwan; Ahn, Ji Hoon; Suárez-López, Paula; Pelaz, Soraya

    2015-01-01

    Plants integrate day length and ambient temperature to determine the optimal timing for developmental transitions. In Arabidopsis (Arabidopsis thaliana), the floral integrator FLOWERING LOCUS T (FT) and its closest homolog TWIN SISTER OF FT promote flowering in response to their activator CONSTANS under long-day inductive conditions. Low ambient temperature (16°C) delays flowering, even under inductive photoperiods, through repression of FT, revealing the importance of floral repressors acting at low temperatures. Previously, we have reported that the floral repressors TEMPRANILLO (TEM; TEM1 and TEM2) control flowering time through direct regulation of FT at 22°C. Here, we show that tem mutants are less sensitive than the wild type to changes in ambient growth temperature, indicating that TEM genes may play a role in floral repression at 16°C. Moreover, we have found that TEM2 directly represses the expression of FT and TWIN SISTER OF FT at 16°C. In addition, the floral repressor SHORT VEGETATIVE PHASE (SVP) directly regulates TEM2 but not TEM1 expression at 16°C. Flowering time analyses of svp tem mutants indicate that TEM may act in the same genetic pathway as SVP to repress flowering at 22°C but that SVP and TEM are partially independent at 16°C. Thus, TEM2 partially mediates the temperature-dependent function of SVP at low temperatures. Taken together, our results indicate that TEM genes are also able to repress flowering at low ambient temperatures under inductive long-day conditions. PMID:26243615

  7. Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ

    SciTech Connect

    Augustus, Anne; Reardon, Patrick; Heller, William T; Spicer, Leonard D.

    2006-01-01

    The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.

  8. Perspective on unraveling the versatility of 'co-repressor' complexes.

    PubMed

    Baymaz, H Irem; Karemaker, Ino D; Vermeulen, Michiel

    2015-08-01

    A multitude of post-translational modifications take place on histones, one of the best studied being acetylation on lysine residues, which is generally associated with gene activation. During the last decades, several so-called co-repressor protein complexes that carry out the reverse process, histone deacetylation, have been identified and characterized, such as the Sin3, N-CoR/SMRT and NuRD complexes. Although a repressive role for these complexes in regulating gene expression is well established, accumulating evidence also points to a role in gene activation. Here, we argue that integration of various state-of-the-art technologies, addressing different aspects of transcriptional regulation, is essential to unravel this apparent biological versatility of 'co-repressor' complexes.

  9. Chemical modification of arginine residues in the lactose repressor

    SciTech Connect

    Whitson, P.A.; Matthews, K.S.

    1987-10-06

    The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino aced analysis or incorporation of /sup 14/C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approx. 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.

  10. The lac repressor displays facilitated diffusion in living cells.

    PubMed

    Hammar, Petter; Leroy, Prune; Mahmutovic, Anel; Marklund, Erik G; Berg, Otto G; Elf, Johan

    2012-06-22

    Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO(1) operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

  11. Engineering a root-specific, repressor-operator gene complex.

    PubMed

    Kim, Tehryung; Balish, Rebecca S; Heaton, Andrew C P; McKinney, Elizabeth C; Dhankher, Om Parkash; Meagher, Richard B

    2005-11-01

    Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.

  12. A single amino acid converts a repressor to an activator of flowering

    PubMed Central

    Hanzawa, Yoshie; Money, Tracy; Bradley, Desmond

    2005-01-01

    Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue. PMID:15894619

  13. A single amino acid converts a repressor to an activator of flowering.

    PubMed

    Hanzawa, Yoshie; Money, Tracy; Bradley, Desmond

    2005-05-24

    Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.

  14. Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

    PubMed

    Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

    2013-09-17

    Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

  15. Genomic mining of prokaryotic repressors for orthogonal logic gates.

    PubMed

    Stanton, Brynne C; Nielsen, Alec A K; Tamsir, Alvin; Clancy, Kevin; Peterson, Todd; Voigt, Christopher A

    2014-02-01

    Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10(54) circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

  16. Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates

    PubMed Central

    Stanton, Brynne C.; Nielsen, Alec A.K.; Tamsir, Alvin; Clancy, Kevin; Peterson, Todd; Voigt, Christopher A.

    2014-01-01

    Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply “part mining” to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits. PMID:24316737

  17. DNA supercoiling: A regulatory signal for the λ repressor

    PubMed Central

    Ding, Yue; Manzo, Carlo; Fulcrand, Geraldine; Leng, Fenfei; Dunlap, David; Finzi, Laura

    2014-01-01

    Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, protein-mediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from −15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density. PMID:25319264

  18. Transcriptional repressor DREAM regulates trigeminal noxious perception.

    PubMed

    Benedet, Tomaso; Gonzalez, Paz; Oliveros, Juan C; Dopazo, Jose M; Ghimire, Kedar; Palczewska, Malgorzata; Mellstrom, Britt; Naranjo, Jose R

    2017-05-01

    Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain". © 2016 International Society for Neurochemistry.

  19. Subspecialization of R2R3-MYB Repressors for Anthocyanin and Proanthocyanidin Regulation in Forage Legumes

    PubMed Central

    Albert, Nick W.

    2015-01-01

    The synthesis of anthocyanin pigments and proanthocyanidins (condensed tannins) is regulated by MYB-bHLH-WDR (MBW) transcription factor complexes in all angiosperms studied to date. Tr-MYB133 and Tr-MYB134 were isolated from Trifolium repens and encode R2R3-MYBs that antagonize the activity of MBW activation complexes. These two genes are conserved in other legume species, and form two sub-clades within the larger anthocyanin/proanthocyanidin clade of MYB repressors. However, unlike petunia and Arabidopsis, these R2R3-MYB repressors do not prevent ectopic accumulation of anthocyanins or proanthocyanidins. Instead, they are expressed when anthocyanins or proanthocyanidins are being synthesized, and provide feedback regulation to MBW complexes. This feedback occurs because Tr-MYB133 and Tr-MYB134 are themselves regulated by MBW complexes. Tr-MYB133 is regulated by MBW complexes containing anthocyanin-related R2R3-MYB proteins (Tr-RED LEAF), while Tr-MYB134 is regulated by complexes containing the proanthocyanidin R2R3-MYBs (Tr-MYB14). Other features of the MBW gene regulation networks are also conserved within legumes, including the ability for the anthocyanin MBW complexes to activate the expression of the AN1/TT8 clade bHLH factor. The regulation of Tr-MYB133 and Tr-MYB134 by distinct, pathway-specific MBW complexes has resulted in subspecialization for controlling anthocyanin or proanthocyanidin synthesis. PMID:26779194

  20. Epigenetic repressor-like genes are differentially regulated during grapevine (Vitis vinifera L.) development.

    PubMed

    Almada, Rubén; Cabrera, Nuri; Casaretto, José A; Peña-Cortés, Hugo; Ruiz-Lara, Simón; González Villanueva, Enrique

    2011-10-01

    Grapevine sexual reproduction involves a seasonal separation between inflorescence primordia (flowering induction) and flower development. We hypothesized that a repression mechanism implicating epigenetic changes could play a role in the seasonal separation of these two developmental processes in grapevine. Therefore, the expression of five grapevine genes with homology to the Arabidopsis epigenetic repressor genes FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and SWINGER (SWN) was analyzed during the development of buds and vegetative and reproductive organs. During bud development, the putative grapevine epigenetic repressor genes VvCLF, VvEMF2, VvMSI1, VvSWN and VvFIE are mainly expressed in latent buds at the flowering induction period, but also detected during bud burst and inflorescence/flower development. The overlapping expression patterns of grapevine PcG-like genes in buds suggest that chromatin remodeling mechanisms could be operating during grapevine bud development for controlling processes such as seasonal flowering, dormancy and bud burst. Furthermore, the expression of grapevine PcG-like genes was also detected in fruits and vegetative organs, suggesting that epigenetic changes could be at the basis of the regulation of various proliferation-differentiation cell transitions that occur during grapevine development.

  1. Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression.

    PubMed

    Tanaka, Yuri; Oshima, Yoshimi; Yamamura, Tomomichi; Sugiyama, Masao; Mitsuda, Nobutaka; Ohtsubo, Norihiro; Ohme-Takagi, Masaru; Terakawa, Teruhiko

    2013-01-01

    Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.

  2. The Transcriptional Repressor dMnt Is a Regulator of Growth in Drosophila melanogaster†

    PubMed Central

    Loo, Lenora W. M.; Secombe, Julie; Little, John T.; Carlos, Leni-Sue; Yost, Cynthia; Cheng, Pei-Feng; Flynn, Erin M.; Edgar, Bruce A.; Eisenman, Robert N.

    2005-01-01

    The Myc-Max-Mad/Mnt network of transcription factors has been implicated in oncogenesis and the regulation of proliferation in vertebrate cells. The identification of Myc and Max homologs in Drosophila melanogaster has demonstrated a critical role for dMyc in cell growth control. In this report, we identify and characterize the third member of this network, dMnt, the sole fly homolog of the mammalian Mnt and Mad family of transcriptional repressors. dMnt possesses two regions characteristic of Mad and Mnt proteins: a basic helix-loop-helix-zipper domain, through which it dimerizes with dMax to form a sequence-specific DNA binding complex, and a Sin-interacting domain, which mediates interaction with the dSin3 corepressor. Using the upstream activation sequence/GAL4 system, we show that expression of dMnt results in an inhibition of cellular growth and proliferation. Furthermore, we have generated a dMnt null allele, which results in flies with larger cells, increased weight, and decreased life span compared to wild-type flies. Our results demonstrate that dMnt is a transcriptional repressor that regulates D. melanogaster body size. PMID:16055719

  3. A novel repressor of nif and glnA expression in the methanogenic archaeon Methanococcus maripaludis.

    PubMed

    Lie, Thomas J; Leigh, John A

    2003-01-01

    Nitrogen assimilation in the methanogenic archaeon Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic 'nitrogen operator' repressor binding sequence. Here we report the isolation of the nitrogen repressor, NrpR, from M. maripaludis using DNA affinity purification. Deletion of the nrpR gene resulted in loss of nitrogen operator binding activity in cell extracts and loss of repression of nif (nitrogen-fixation) and glnA (glutamine synthetase) gene expression in vivo. Genetic complementation of the nrpR mutation restored all functions. NrpR contained a putative N-terminal winged helix-turn-helix motif followed by two mutually homologous domains of unknown function. Comparison of the migration of NrpR in gel-filtration chromatography with its subunit molecular weight (60 kDa) suggested that NrpR was a tetramer. Several lines of evidence suggested that the level of NrpR itself is not regulated, and the binding affinity of NrpR to the nitrogen operator is controlled by an unknown mechanism. Homologues of NrpR were found only in certain species in the kingdom Euryarchaeota. Full length homologues were found in Methanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus, and homologues lacking one or more of the three polypeptide domains were found in Archaeoglobus fulgidus, Methanopyrus kandleri, Methanosarcina acetivorans, and Methanosarcina mazei. NrpR represents a new family of regulators unique to the Euryarchaeota.

  4. Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

    SciTech Connect

    Goffinont, S.; Davidkova, M.

    2009-08-21

    The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro {gamma}-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucine residues of the tetramerization domain.

  5. Inhibition of the transcriptional repressor complex Bcl-6/BCoR induces endothelial sprouting but does not promote tumor growth

    PubMed Central

    Buchberger, Elisabeth; Payrhuber, Dietmar; Harchi, Miriam El; Zagrapan, Branislav; Scheuba, Katharina; Zommer, Anna; Bugyik, Edina; Dome, Balazs; Kral, Julia Barbara; Schrottmaier, Waltraud Cornelia; Schabbauer, Gernot; Petzelbauer, Peter; Gröger, Marion; Bilban, Martin; Brostjan, Christine

    2017-01-01

    The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors. PMID:27880939

  6. Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

    PubMed

    Hadjighassem, Mahmoud R; Austin, Mark C; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A; Albert, Paul R

    2009-08-01

    Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

  7. Enhanced generation of myeloid lineages in hematopoietic differentiation from embryonic stem cells by silencing transcriptional repressor Twist-2.

    PubMed

    Sharabi, Andrew B; Lee, Sung-Hyung; Goodell, Margaret A; Huang, Xue F; Chen, Si-Yi

    2009-12-01

    The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1(+) and F4/80(+) cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor.

  8. An ORF from Bacillus licheniformis encodes a putative DNA repressor.

    PubMed

    Naval, J; Aguilar, D; Serra, X; Pérez-Pons, J A; Piñol, J; Lloberas, J; Querol, E

    2000-01-01

    The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported. It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome. Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins. A maxicells assay shows that the encoded polypeptide is expressed. A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

  9. Protein Under Pressure: Molecular Dynamics Simulation of the Arc Repressor

    SciTech Connect

    Trzesniak, Daniel Rodrigo F.; Lins, Roberto D.; van Gunsteren, Wilfred F.

    2006-10-01

    Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogenbonding pattern of the intermolecular -sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the -sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.

  10. Protein under pressure: molecular dynamics simulation of the arc repressor.

    PubMed

    Trzesniak, Daniel; Lins, Roberto D; van Gunsteren, Wilfred F

    2006-10-01

    Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.

  11. Inhibition of cell proliferation by the Mad1 transcriptional repressor.

    PubMed Central

    Roussel, M F; Ashmun, R A; Sherr, C J; Eisenman, R N; Ayer, D E

    1996-01-01

    Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor. PMID:8649388

  12. Novel repressor regulates insulin sensitivity through interaction with Foxo1

    PubMed Central

    Nakae, Jun; Cao, Yongheng; Hakuno, Fumihiko; Takemori, Hiroshi; Kawano, Yoshinaga; Sekioka, Risa; Abe, Takaya; Kiyonari, Hiroshi; Tanaka, Toshiya; Sakai, Juro; Takahashi, Shin-Ichiro; Itoh, Hiroshi

    2012-01-01

    Forkhead box-containing protein o (Foxo) 1 is a key transcription factor in insulin and glucose metabolism. We identified a Foxo1-CoRepressor (FCoR) protein in mouse adipose tissue that inhibits Foxo1's activity by enhancing acetylation via impairment of the interaction between Foxo1 and the deacetylase Sirt1 and via direct acetylation. FCoR is phosphorylated at Threonine 93 by catalytic subunit of protein kinase A and is translocated into nucleus, making it possible to bind to Foxo1 in both cytosol and nucleus. Knockdown of FCoR in 3T3-F442A cells enhanced expression of Foxo target and inhibited adipocyte differentiation. Overexpression of FCoR in white adipose tissue decreased expression of Foxo-target genes and adipocyte size and increased insulin sensitivity in Leprdb/db mice and in mice fed a high-fat diet. In contrast, Fcor knockout mice were lean, glucose intolerant, and had decreased insulin sensitivity that was accompanied by increased expression levels of Foxo-target genes and enlarged adipocytes. Taken together, these data suggest that FCoR is a novel repressor that regulates insulin sensitivity and energy metabolism in adipose tissue by acting to fine-tune Foxo1 activity. PMID:22510882

  13. Modular construction of mammalian gene circuits using TALE transcriptional repressors

    PubMed Central

    Liao, Weixi; Li, Zhihua; Weiss, Ron; Xie, Zhen

    2014-01-01

    An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator-like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches employing feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation. PMID:25643171

  14. Plastic downregulation of the transcriptional repressor BCL6 during maturation of human dendritic cells

    SciTech Connect

    Pantano, Serafino . E-mail: serafino.pantano@unil.ch; Jarrossay, David; Saccani, Simona; Bosisio, Daniela; Natoli, Gioacchino

    2006-05-01

    Dendritic cell (DC) maturation links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. Here, we describe an as yet unrecognized modulator of human DC maturation, the transcriptional repressor BCL6. We found that both myeloid and plasmacytoid DCs constitutively express BCL6, which is rapidly downregulated following maturation triggered by selected stimuli. Both in unstimulated and maturing DCs, control of BCL6 protein levels reflects the convergence of several mechanisms regulating BCL6 stability, mRNA transcription and nuclear export. By regulating the induction of several genes implicated in the immune response, including inflammatory cytokines, chemokines and survival genes, BCL6 may represent a pivotal modulator of the afferent branch of the immune response.

  15. Neuronal Transcriptional Repressor REST Suppresses an Atoh7-Independent Program for Initiating Retinal Ganglion Cell Development

    PubMed Central

    Mao, Chai-An; Tsai, Wen-Wei; Cho, Jang-Hyeon; Pan, Ping; Barton, Michelle Craig; Klein, William H.

    2010-01-01

    As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs. PMID:20969844

  16. Arousal Level in Repressors and Sensitizers as a Function of Response Context

    ERIC Educational Resources Information Center

    Stein, Steven H.

    1971-01-01

    Repressors and sensitizers were given "noncontextual" and "contextual" tasks, with galvanic skin response as a measure of arousal. Results from the noncontextual task showed that repressors had lower arousal levels than sensitizers during perception and verbal report, but higher during free association. Findings were reversed, however, in the…

  17. Arousal Level in Repressors and Sensitizers as a Function of Response Context

    ERIC Educational Resources Information Center

    Stein, Steven H.

    1971-01-01

    Repressors and sensitizers were given "noncontextual" and "contextual" tasks, with galvanic skin response as a measure of arousal. Results from the noncontextual task showed that repressors had lower arousal levels than sensitizers during perception and verbal report, but higher during free association. Findings were reversed, however, in the…

  18. Continuous Associative Responding to Threatening and Nonthreatening Stimuli by Repressors and Sensitizers

    ERIC Educational Resources Information Center

    Haney, Jack N.

    1974-01-01

    The focus of this study is on the approach-avoidance reactions durning mediational activity of repressors and sensitizers. It was hypothesized that repressors would exhibit (a) disruption and early termination of chained free association, (b) a redundancy in their association repertoires, and (c) a lesser degree of sexual content in their…

  19. The valosin-containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload.

    PubMed

    Zhou, Ning; Ma, Ben; Stoll, Shaunrick; Hays, Tristan T; Qiu, Hongyu

    2017-10-01

    Hypertension-induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin-containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4-month male spontaneously hypertensive rats (SHRs) vs. age-matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild-type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin-containing protein VCP expression was also observed to be time- and dose-dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac-specific overexpression of VCP showed a significant repression in TAC-induced LVH vs. litter-matched WT controls upon 2-week TAC. TAC-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. The NF-kappa B-like site in the TNF-alpha repressor element is essential for its repressor function.

    PubMed

    Fong, C L; Mark, D F

    1995-07-26

    We have previously identified a repressor element in the TNF-alpha promoter (-280 to -172) by deletion analysis. When this 108 bp repressor element was placed in front of a heterologous promoter containing an NF-kappa B binding site, less repression was observed. When this element was dimerized and placed in front of the positive element (-125 to -102) of the TNF-alpha promoter, instead of repression, activation was observed. There is an NF-kappa B-like site in the 108 bp repressor region (-211 to -202) and our gel retardation analysis showed that this site and a known NF-kappa B binding site both could compete for one of the specific protein complexes formed on the 108 bp probe. To test the functionality of this NF-kappa B-like site, we mutagenized the critical GGGG sequence to ATCC. Contrary to our prediction, such a mutation blocked the repressor function of the 108 bp element. This suggests that the NF-kappa B-like site is an essential sequence for the repressor function of the 108 bp repressor element.

  1. Transcriptional regulation of the sucrase gene of Staphylococcus xylosus by the repressor ScrR.

    PubMed Central

    Gering, M; Brückner, R

    1996-01-01

    In Staphylococcus xylosus, scrB is one of two genes necessary for sucrose utilization. It encodes a sucrase that hydrolyzes intracellular sucrose-6-phosphate generated by the uptake of sucrose via the sucrose-specific enzyme II of the phosphotransferase system, the gene product of scrA. ScrB sucrase activity is inducible by the presence of sucrose in the culture medium. Primer extension experiments demonstrated that the observed regulation is achieved at the level of scrB transcription initiation. The protein mediating sucrose-specific regulation of scrB was found to be encoded immediately upstream of the sucrase gene. The nucleotide sequence of the regulatory gene scrR comprises an open reading frame that specifies a protein of 35.8 kDa. This protein exhibits similarity to transcriptional regulators of the GalR-LacI family. Inactivation of the scrR reading frame in the genome of S. xylosus led to the constitutive expression of scrB at a high level, identifying ScrR as a repressor of transcription. Sucrose-specific regulation of scrB was also lost upon deletion of 4 bp of a palindromic sequence (OB) covering positions +6 to +21 downstream of the scrB transcriptional start site. These results suggested a direct interaction of the ScrR repressor and the operator OB. Accordingly, a fusion protein consisting of the maltose-binding protein of Escherichia coli and the ScrR protein was able to interact with an scrB promoter fragment in gel mobility shift experiments but failed to bind an scrB fragment carrying the 4-bp deletion derivative of OB. An scrR promoter fragment, which dose not contain a sequence resembling OB, was not shifted by the fusion protein. This result corroborates scrR primer extension analyses showing that transcription of the repressor gene itself is not regulated. Therefore, the sucrase gene operator OB is the target sequence through which the ScrR protein exerts its negative effect on transcription initiation. In the promoter region of scrA, the

  2. The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi.

    PubMed

    Maruyama, Yosuke; Yamoto, Natsuko; Suzuki, Yuya; Chiba, Yukako; Yamazaki, Ken-ichi; Sato, Takeo; Yamaguchi, Junji

    2013-12-01

    Complex plant defenses that include the hypersensitive response (HR) are mediated by plant hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene. We previously isolated the Arabidopsis DEAR1 (DREB AND EAR MOTIF PROTEIN 1) regulator and showed that its overexpression DEAR1 (DEAR1ox) resulted in a dwarf phenotype and lesion-like cell death, accompanied by elevated expression of PR (PATHOGENESIS-RELATED) genes. Here, we show that transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) has enhanced resistance to the necrotrophic fungus Botrytis cinerea (B. cinerea). This result indicates that DEAR1 represses negative regulators of plant defense responses, including transcriptional repressors belonging to the ERF (ETHYLEN RESPONSE FACTOR) family. Knockout mutants of ERF9 (erf9), which were down-regulated in DEAR1ox plants, showed transcriptional promotion of PDF1.2 (PATHOGEN-INDUCIBLE PLANT DEFENSIN) genes, which serve as positive markers for the ethylene/jasmonic acid (JA) signaling pathway and provide enhanced resistance to B. cinerea. Biochemical assays demonstrated that the ERF9 in capable of binding to the GCC box, a cis-element contained in the promoters of the PDF1.2 gene that possesses trans-repression activity. Moreover, infection with B. cinerea resulted in the promotion of the PDF1.2 expression, coinciding with suppression of the ERF9 gene under the control of the DEAR1 gene. These results indicate that the transcriptional repressor ERF9 participates in plant defense mechanisms against necrotic fungi mediated by the DEAR1-dependent ethylene/JA signaling pathway.

  3. DNA sequence requirements for the activation of 434 P(RM) transcription by 434 repressor.

    PubMed

    Xu, J; Koudelka, G B

    2000-10-01

    A dimer of the 434 repressor bound at O(R)2 activated transcription initiation from P(RM) by contacting RNA polymerase. Although DNA-binding site mutations at either end of O(R)2 decreased the ability of the repressor to activate P(RM) transcription, mutations proximal to the promoter had a greater effect on transcription activation. Orienting a repressor subunit bearing the altered specificity Gln-28 --> Ala mutation to the halfsite of O(R)2 proximal to the P(RM) promoter decreased the repressor's ability to activate transcription initiation at 434 P(RM) to a much greater extent than if this subunit was placed in the O(R)2 half-site distal to P(RM). In addition to showing that the downstream (promoter proximal) subunit of the O(R)2-bound 434 repressor functions in activating 434 P(RM), the results indicated that DNA sequence-dependent conformational changes alter the efficiency with which the repressor activates P(RM) transcription. These unexpected findings highlight the importance of the structure of the repressor-DNA interface in activating transcription from P(RM).

  4. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer

    SciTech Connect

    Lewis, M.; Chang, G.; Horton, N.C.

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor a product of the lacl gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-B-D-1thiogalactoside (IPTG) and the lac repressor complexed with a 21 base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and the repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quarternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites in the genomic DNA. 76 refs., 11 figs., 1 tab.

  5. The Ime2 protein kinase enhances the disassociation of the Sum1 repressor from middle meiotic promoters.

    PubMed

    Ahmed, Noreen T; Bungard, David; Shin, Marcus E; Moore, Michael; Winter, Edward

    2009-08-01

    Meiotic development in Saccharomyces cerevisiae (sporulation) is controlled by the sequential transcription of temporally distinct sets of meiosis-specific genes. The induction of middle genes controls exit from meiotic prophase, the completion of the nuclear divisions, and spore formation. Middle promoters are controlled through DNA elements termed middle sporulation elements (MSEs) that are bound by the Sum1 repressor during vegetative growth and by the Ndt80 activator during meiosis. It has been proposed that the induction of middle promoters is controlled by competition between Ndt80 and Sum1 for MSE occupancy. Here, we show that the Sum1 repressor can be removed from middle promoters in meiotic cells independent of Ndt80 expression. This process requires the phosphorylation of Sum1 by the meiosis-specific cyclin-dependent kinase-like kinase Ime2. The deletion of HST1, which encodes a Sir2 paralog that interacts with Sum1, bypasses the requirement for this phosphorylation. These findings suggest that in the presence of Ndt80, Sum1 may be displaced from MSEs through a competition-based mechanism but that in the absence of Ndt80, Sum1 is removed from chromatin in a separate pathway requiring the phosphorylation of Sum1 by Ime2 and the inhibition of Hst1.

  6. A transcriptional repressor of the ERF family confers drought tolerance to rice and regulates genes preferentially located on chromosome 11.

    PubMed

    Joo, Joungsu; Choi, Hae Jong; Lee, Youn Hab; Kim, Yeon-Ki; Song, Sang Ik

    2013-07-01

    Plant-specific ethylene response factors (ERFs) play important roles in abiotic and biotic stress responses in plants. Using a transgenic approach, we identified two rice ERF genes, OsERF4a and OsERF10a, which conferred drought stress tolerance. In particular, OsERF4a contains a conserved ERF-associated amphiphilic repression (EAR) motif in its C-terminal region that has been shown to function as a transcriptional repression domain. Expression profiling of transgenic rice plants over-expressing OsERF4a using either a constitutively active or an ABA-inducible promoter identified 45 down-regulated and 79 up-regulated genes in common. The increased stress tolerance by over-expression of the EAR domain-containing protein OsERF4a could result from suppression of a repressor of the defense response. Expression of the putative silent information regulator 2 (Sir2) repressor protein was repressed, and expression of several stress-response genes were induced by OsERF4a over-expression. The Sir2 and 7 out of 9 genes that were down-regulated by OsERF4a over-expression were induced by high salinity and drought treatments in non-transgenic control plants. Genes that were down- and up-regulated by OsERF4a over-expression were highly biased toward chromosome 11. Rice chromosome 11 has several large clusters of disease-resistance and defense-response genes. Taken together, our results suggest that OsERF4a is a positive regulator of shoot growth and water-stress tolerance in rice during early growth stages. We propose that OsERF4a could work by suppressing a repressor of the defense responses and/or by controlling the expression of a large number of genes located on chromosome 11.

  7. Analysis of the tet repressor-operator interactions using the uracil-DNA glycosylase footprinting system

    SciTech Connect

    Devchand, P.R.; McGhee, J.D.; Sande, J.H. Van De

    1994-12-31

    The tet repressor regulated expression of the Tn-10-encoded tetracycline resistance determinant in a tetracycline-dependent manner. In the absence of tetracycline, the tet repressor binds as a dimer to the 19-base-pair palindromic tet operator sequence. Amino acid homologies and genetic studies with trans-dominant mutants suggest that sequence-specific recognition of the tet operator involves the extensively studied helix-turn-helix motif. We have used the uracil-DNA glycosylase (UDG) footprinting systems to identify thymine contacts in the tet operator that are essential for the formation of tet repressor-operator complexes.

  8. PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies

    SciTech Connect

    Fleischer, Sandra; Wiemann, Stefan; Will, Hans; Hofmann, Thomas G. . E-mail: t.hofmann@dkfz.de

    2006-04-01

    Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krueppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C{sub 2}H{sub 2}-ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1{beta}/KRAB-associated protein 1 (TIF-1{beta}/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1{beta} from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs.

  9. Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration.

    PubMed Central

    Baluch, J; Sussman, R

    1978-01-01

    Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation. PMID:353300

  10. Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor

    SciTech Connect

    Peterson, K.R.; Ossanna, N.; Mount, D.W.

    1988-04-01

    LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).

  11. REST is a hypoxia-responsive transcriptional repressor

    PubMed Central

    Cavadas, Miguel A. S.; Mesnieres, Marion; Crifo, Bianca; Manresa, Mario C.; Selfridge, Andrew C.; Keogh, Ciara E.; Fabian, Zsolt; Scholz, Carsten C.; Nolan, Karen A.; Rocha, Liliane M. A.; Tambuwala, Murtaza M.; Brown, Stuart; Wdowicz, Anita; Corbett, Danielle; Murphy, Keith J.; Godson, Catherine; Cummins, Eoin P.; Taylor, Cormac T.; Cheong, Alex

    2016-01-01

    Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia. PMID:27531581

  12. Akt phosphorylates Tal1 oncoprotein and inhibits its repressor activity.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-06-01

    The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

  13. HTLV-1 p30II: selective repressor of gene expression.

    PubMed

    Green, Patrick L

    2004-11-24

    Human T-lymphotropic virus type-1 (HTLV-1) is a complex retrovirus that causes adult T-cell leukemia/lymphoma (ATL) and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 pX ORF II encodes two proteins, p13II and p30II whose roles are beginning to be defined in the virus life cycle. Previous studies indicate the importance of these viral proteins in the ability of the virus to maintain viral loads and persist in an animal model of HTLV-1 infection. Intriguing new studies indicate that p30II is a multifunctional regulator that differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein (CBP)/p300 and specifically binds and represses tax/rex mRNA nuclear export. A new study characterized the role of p30II in regulation of cellular gene expression using comprehensive human gene arrays. Interestingly, p30II is an overall repressor of cellular gene expression, while selectively favoring the expression of regulatory gene pathways important to T lymphocytes. These new findings suggest that HTLV-1, which is associated with lymphoproliferative diseases, uses p30II to selectively repress cellular and viral gene expression to favor the survival of cellular targets ultimately resulting in leukemogenesis.

  14. Novel INHAT repressor (NIR) is required for early lymphocyte development.

    PubMed

    Ma, Chi A; Pusso, Antonia; Wu, Liming; Zhao, Yongge; Hoffmann, Victoria; Notarangelo, Luigi D; Fowlkes, B J; Jain, Ashish

    2014-09-23

    Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4(+)CD8(+) thymocytes, whereas bone-marrow-derived B cells were arrested at the B220(+)CD19(-) pro-B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220(+)CD19(+) pro-B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.

  15. Heat-induced fibrillation of BclXL apoptotic repressor.

    PubMed

    Bhat, Vikas; Olenick, Max B; Schuchardt, Brett J; Mikles, David C; Deegan, Brian J; McDonald, Caleb B; Seldeen, Kenneth L; Kurouski, Dmitry; Faridi, Mohd Hafeez; Shareef, Mohammed M; Gupta, Vineet; Lednev, Igor K; Farooq, Amjad

    2013-09-01

    The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease.

  16. RREB-1 is a transcriptional repressor of HLA-G.

    PubMed

    Flajollet, Sébastien; Poras, Isabelle; Carosella, Edgardo D; Moreau, Philippe

    2009-12-01

    The nonclassical HLA-G is a molecule specifically involved in immune tolerance with highly restricted tissue distribution in healthy conditions. Yet it is overexpressed in numerous tumors and in allografts with better acceptance. Major mechanisms involved in regulation of HLA-G transcription are still poorly described. Thus, to characterize these mechanisms we have developed a specific proteomic approach to identify proteins that bind differentially to the HLA-G gene promoter by promoter pull-down assay followed by spectrometry mass analysis. Among specific binding factors, we focused on RREB-1, a ras-responsive element binding protein 1. We demonstrated that RREB-1 represses HLA-G transcriptional activity and binds three ras response elements within the HLA-G promoter. RREB-1 protein, specifically in HLA-G-negative cells, interacts with subunits of CtBP complex implicated in chromatin remodeling. This demonstration is the first of a repressor factor of HLA-G transcriptional activity taking part in HLA-G repression by epigenetic mechanisms.

  17. Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation.

    PubMed

    Soprano, Adriana Santos; Giuseppe, Priscila Oliveira de; Shimo, Hugo Massayoshi; Lima, Tatiani Brenelli; Batista, Fernanda Aparecida Heleno; Righetto, Germanna Lima; Pereira, José Geraldo de Carvalho; Granato, Daniela Campos; Nascimento, Andrey Fabricio Ziem; Gozzo, Fabio Cesar; de Oliveira, Paulo Sérgio Lopes; Figueira, Ana Carolina Migliorini; Smetana, Juliana Helena Costa; Paes Leme, Adriana Franco; Murakami, Mario Tyago; Benedetti, Celso Eduardo

    2017-09-05

    MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding

    PubMed Central

    Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

    2015-01-01

    The RpoS/σS sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σS-dependent control, that of a repressor. Negative regulation by σS has been proposed to result largely from competition between σS and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σS binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σS protein proficient for EσS complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σS requires its binding to DNA. Although the mechanisms of repression by σS are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by EσS. PMID:25578965

  19. A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia

    PubMed Central

    Kasajima, Ichiro; Sasaki, Katsutomo

    2016-01-01

    ABSTRACT The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3′-hydroxylase (F3′H) and Flavonoid 3′5′-hydroxylase (F3′5′H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis. PMID:27089475

  20. Anaphase promoting complex-dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae.

    PubMed

    Ostapenko, Denis; Solomon, Mark J

    2011-07-01

    The anaphase-promoting complex/cyclosome (APC/C) is an essential ubiquitin ligase that targets cell cycle proteins for proteasome-mediated degradation in mitosis and G1. The APC regulates a number of cell cycle processes, including spindle assembly, mitotic exit, and cytokinesis, but the full range of its functions is still unknown. To better understand cellular pathways controlled by the APC, we performed a proteomic screen to identify additional APC substrates. We analyzed cell cycle-regulated proteins whose expression peaked during the period when other APC substrates were expressed. Subsequent analysis identified several proteins, including the transcriptional repressors Nrm1 and Yhp1, as authentic APC substrates. We found that APC(Cdh1) targeted Nrm1 and Yhp1 for degradation in early G1 through Destruction-box motifs and that the degradation of these repressors coincided with transcriptional activation of MBF and Mcm1 target genes, respectively. In addition, Nrm1 was stabilized by phosphorylation, most likely by the budding yeast cyclin-dependent protein kinase, Cdc28. We found that expression of stabilized forms of Nrm1 and Yhp1 resulted in reduced cell fitness, due at least in part to incomplete activation of G1-specific genes. Therefore, in addition to its known functions, APC-mediated targeting of Nrm1 and Yhp1 coordinates transcription of multiple genes in G1 with other cell cycle events.

  1. Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis

    SciTech Connect

    Procházková, Kateřina; Čermáková, Kateřina; Pachl, Petr; Sieglová, Irena; Fábry, Milan; Otwinowski, Zbyszek; Řezáčová, Pavlína

    2012-02-01

    The crystal structure of the effector-binding domain of the transcriptional repressor AraR from B. subtilis in complex with the effector molecule (l-arabinose) was determined at 2.2 Å resolution. A detailed analysis of the crystal identified a dimer organization that is distinctive from that of other members of the GalR/LacI family. In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K{sub d} value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.

  2. Arc-repressor dimerization on DNA: folding rate enhancement by colocalization.

    PubMed

    Marcovitz, Amir; Levy, Yaakov

    2009-05-20

    Multimeric proteins are ubiquitous in many cellular processes that require high levels of regulation. Eukaryotic gene expression is often regulated by a mechanism of combinatorial control that involves the binding of dimeric transcription factors to DNA together with the coordinated activity of additional proteins. In this study, we investigated the dimerization of the Arc-repressor on DNA with the aim of achieving microscopic insight into the possible advantages of interacting with DNA as a complex rather than as a monomeric single-domain protein. We used a computational coarse-grained model in which the protein dynamics was governed by native interactions and protein-DNA interactions were dictated by electrostatic forces. Inspired by previous experimental work that showed an enhanced refolding rate for the Arc-repressor in the presence of DNA and other polyanions, we focused on the mechanism and kinetics of the assembly of Arc monomers in the presence of single- (ssDNA) and double-stranded DNA (dsDNA) molecules in a low-salt concentration environment. The electrostatic interactions that attract the protein to the dsDNA were shown to be fundamental in colocalizing the unfolded Arc chains and in accelerating refolding. Arc monomers bind the dsDNA efficiently and nonspecifically, and search for each other via one-dimensional diffusion. The fastest folding of Arc is observed for DNA of 30 bp. Longer DNA is significantly less efficient in accelerating the Arc refolding rate, since the two subunits search distinct regions of the one-dimensional DNA and are therefore much less colocalized. The probability that the two unfolded chains will meet on 200 bp DNA is similar to that in the bulk. The colocalization of Arc subunits on ssDNA results in much faster folding compared to that obtained on dsDNA of the same length. Differences in the rate of Arc refolding, cooperativity, and the structure of its transition state ensemble introduced by ssDNA and dsDNA molecules

  3. Genome-wide characterization of JASMONATE-ZIM DOMAIN transcription repressors in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yukun; Qiao, Linyi; Bai, Jianfang; Wang, Peng; Duan, Wenjing; Yuan, Shaohua; Yuan, Guoliang; Zhang, Fengting; Zhang, Liping; Zhao, Changping

    2017-02-13

    The JASMONATE-ZIM DOMAIN (JAZ) repressor family proteins are jasmonate co-receptors and transcriptional repressor in jasmonic acid (JA) signaling pathway, and they play important roles in regulating the growth and development of plants. Recently, more and more researches on JAZ gene family are reported in many plants. Although the genome sequencing of common wheat (Triticum aestivum L.) and its relatives is complete, our knowledge about this gene family remains vacant. Fourteen JAZ genes were identified in the wheat genome. Structural analysis revealed that the TaJAZ proteins in wheat were as conserved as those in other plants, but had structural characteristics. By phylogenetic analysis, all JAZ proteins from wheat and other plants were clustered into 11 sub-groups (G1-G11), and TaJAZ proteins shared a high degree of similarity with some JAZ proteins from Aegliops tauschii, Brachypodium distachyon and Oryza sativa. The Ka/Ks ratios of TaJAZ genes ranged from 0.0016 to 0.6973, suggesting that the TaJAZ family had undergone purifying selection in wheat. Gene expression patterns obtained by quantitative real-time PCR (qRT-PCR) revealed differential temporal and spatial regulation of TaJAZ genes under multifarious abiotic stress treatments of high salinity, drought, cold and phytohormone. Among these, TaJAZ7, 8 and 12 were specifically expressed in the anther tissues of the thermosensitive genic male sterile (TGMS) wheat line BS366 and normal control wheat line Jing411. Compared with the gene expression patterns in the normal wheat line Jing411, TaJAZ7, 8 and 12 had different expression patterns in abnormally dehiscent anthers of BS366 at the heading stage 6, suggesting that specific up- or down-regulation of these genes might be associated with the abnormal anther dehiscence in TGMS wheat line. This study analyzed the size and composition of the JAZ gene family in wheat, and investigated stress responsive and differential tissue-specific expression profiles of each

  4. Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.

    PubMed

    Goffinont, Stephane; Villette, Sandrine; Spotheim-Maurizot, Melanie

    2012-06-01

    The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.

  5. Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus.

    PubMed

    Voss, Ty C; Demarco, Ignacio A; Booker, Cynthia F; Day, Richard N

    2005-08-01

    The co-repressor proteins SMRT and NCoR concentrate in specific subnuclear compartments and function with DNA-binding factors to inhibit transcription. To provide detailed mechanistic understanding of these activities, this study tested the hypothesis that functional interactions with transcription factors, such as the pituitary-gland-specific Pit-1 homeodomain protein, direct the subnuclear organization and activity of co-repressor complexes. Both SMRT and NCoR repressed Pit-1-dependent transcription, and NCoR was co-immunoprecipitated with Pit-1. Immunofluorescence experiments confirmed that endogenous NCoR is concentrated in small focal bodies and that incremental increases in fluorescent-protein-tagged NCoR expression lead to progressive increases in the size of these structures. In pituitary cells, the endogenous NCoR localized with endogenous Pit-1 and the co-expression of a fluorescent-protein-labeled Pit-1 redistributed both NCoR and SMRT into diffuse nucleoplasmic compartments that also contained histone deacetylase and chromatin. Automated image-analysis methods were applied to cell populations to characterize the reorganization of co-repressor proteins by Pit-1 and mutation analysis showed that Pit-1 DNA-binding activity was necessary for the reorganization of co-repressor proteins. These data support the hypothesis that spherical foci serve as co-repressor storage compartments, whereas Pit-1/co-repressor complexes interact with target genes in more widely dispersed subnuclear domains. The redistribution of co-repressor complexes by Pit-1 might represent an important mechanism by which transcription factors direct changes in cell-specific gene expression.

  6. Functional analysis of NsrR, a nitric oxide sensing Rrf2 repressor in Neisseria gonorrhoeae

    PubMed Central

    Isabella, Vincent M.; Lapek, John D.; Kennedy, Edward M.; Clark, Virginia L.

    2008-01-01

    Nitric oxide has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analyzed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA, and nsrR upstream regions with a Kd of 7 nM, 19 nM, and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97, or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in coordination of a NO-sensitive Fe-S center required for DNA binding. PMID:19007408

  7. Mutations in the TGF-β repressor SKI cause Shprintzen-Goldberg syndrome with aortic aneurysm.

    PubMed

    Doyle, Alexander J; Doyle, Jefferson J; Bessling, Seneca L; Maragh, Samantha; Lindsay, Mark E; Schepers, Dorien; Gillis, Elisabeth; Mortier, Geert; Homfray, Tessa; Sauls, Kimberly; Norris, Russell A; Huso, Nicholas D; Leahy, Dan; Mohr, David W; Caulfield, Mark J; Scott, Alan F; Destrée, Anne; Hennekam, Raoul C; Arn, Pamela H; Curry, Cynthia J; Van Laer, Lut; McCallion, Andrew S; Loeys, Bart L; Dietz, Harry C

    2012-11-01

    Elevated transforming growth factor (TGF)-β signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). However, the location and character of many of the causal mutations in LDS intuitively imply diminished TGF-β signaling. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm. We identified causative variation in ten individuals with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity. Cultured dermal fibroblasts from affected individuals showed enhanced activation of TGF-β signaling cascades and higher expression of TGF-β-responsive genes relative to control cells. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in humans with SGS. These data support the conclusions that increased TGF-β signaling is the mechanism underlying SGS and that high signaling contributes to multiple syndromic presentations of aortic aneurysm.

  8. Apoptosis repressor with a CARD domain (ARC) restrains Bax-mediated pathogenesis in dystrophic skeletal muscle.

    PubMed

    Davis, Jennifer; Kwong, Jennifer Q; Kitsis, Richard N; Molkentin, Jeffery D

    2013-01-01

    Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3 (-/-) Sgcd (-/-) double targeted mice. Mechanistically, Nol3 (-/-) Sgcd (-/-) mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.

  9. The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages

    PubMed Central

    Koe, Chwee Tat; Li, Song; Rossi, Fabrizio; Wong, Jack Jing Lin; Wang, Yan; Zhang, Zhizhuo; Chen, Keng; Aw, Sherry Shiying; Richardson, Helena E; Robson, Paul; Sung, Wing-Kin; Yu, Fengwei; Gonzalez, Cayetano; Wang, Hongyan

    2014-01-01

    The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. Here, we demonstrate that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. We show that multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts. DOI: http://dx.doi.org/10.7554/eLife.01906.001 PMID:24618901

  10. NIR is a novel INHAT repressor that modulates the transcriptional activity of p53

    PubMed Central

    Hublitz, Philip; Kunowska, Natalia; Mayer, Ulrich P.; Müller, Judith M.; Heyne, Kristina; Yin, Na; Fritzsche, Claudia; Poli, Cecilia; Miguet, Laurent; Schupp, Ingo W.; van Grunsven, Leo A.; Potiers, Noëlle; van Dorsselaer, Alain; Metzger, Eric; Roemer, Klaus; Schüle, Roland

    2005-01-01

    Most transcriptional repression pathways depend on the targeted deacetylation of histone tails. In this report, we characterize NIR, a novel transcriptional corepressor with inhibitor of histone acetyltransferase (INHAT) activity. NIR (Novel INHAT Repressor) is ubiquitously expressed throughout embryonic development and adulthood. NIR is a potent transcriptional corepressor that is not blocked by histone deacetylase inhibitors and is capable of silencing both basal and activator-driven transcription. NIR directly binds to nucleosomes and core histones and prevents acetylation by histone acetyltransferases, thus acting as a bona fide INHAT. Using a tandem affinity purification approach, we identified the tumor suppressor p53 as a NIR-interacting partner. Association of p53 and NIR was verified in vitro and in vivo. Upon recruitment by p53, NIR represses transcription of both p53-dependent reporters and endogenous target genes. Knock-down of NIR by RNA interference significantly enhances histone acetylation at p53-regulated promoters. Moreover, p53-dependent apoptosis is robustly increased upon depletion of NIR. In summary, our findings describe NIR as a novel INHAT that plays an important role in the control of p53 function. PMID:16322561

  11. New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

    PubMed

    Dinu, Sorin; Damian, Maria; Badell, Edgar; Dragomirescu, Cristiana Cerasella; Guiso, Nicole

    2014-10-01

    Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The Capicua repressor--a general sensor of RTK signaling in development and disease.

    PubMed

    Jiménez, Gerardo; Shvartsman, Stanislav Y; Paroush, Ze'ev

    2012-03-15

    Receptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes.

  13. DSF nuclear receptor acts as a repressor in culture and in vivo.

    PubMed

    Pitman, Jeffrey L; Tsai, Chih-Cheng; Edeen, Philip T; Finley, Kim D; Evans, Ronald M; McKeown, Michael

    2002-05-15

    Loss-of-function mutations affecting the dissatisfaction (DSF) nuclear receptor alter both sexual behavior and the sex-specific nervous system in Drosophila. As a step toward understanding the way DSF controls development and function of the nervous system, we have analyzed the regulatory activities of the DSF protein. DSF prefers an atypical DNA half site, AAGTCA. Wild-type DSF, but not the point mutant DSF(7), monomerically binds and represses reporter constructs bearing this site. DSF also contains an atypically long, 356-amino-acid hinge separating its DNA-binding domain (DBD) and ligand-binding domain (LBD). The hinge contains at least two functions: a region that drastically lowers DNA-binding efficiency in vitro, and an amino-terminal repressive domain. The DBD and LBD of DSF, along with major portions of the hinge, are highly conserved in other insects. Ectopic expression of DSF in Drosophila imaginal discs results in developmental disruptions in disc-derived tissues, disruptions which are largely suppressed when DSF is fused to the VP16 activation domain, consistent with a repressive role for DSF. Finally, when tethered to DNA, DSF's hinge and LBD regions act as strong transcriptional repressors in multiple larval and pupal tissues, including many DSF-expressing tissues. These results suggest DSF can repress transcription in vivo, that repression is largely responsible for its ectopic expression phenotypes, and that repression may be a key component of normal DSF function.

  14. Sulfur deficiency–induced repressor proteins optimize glucosinolate biosynthesis in plants

    PubMed Central

    Aarabi, Fayezeh; Kusajima, Miyuki; Tohge, Takayuki; Konishi, Tomokazu; Gigolashvili, Tamara; Takamune, Makiko; Sasazaki, Yoko; Watanabe, Mutsumi; Nakashita, Hideo; Fernie, Alisdair R.; Saito, Kazuki; Takahashi, Hideki; Hubberten, Hans-Michael; Hoefgen, Rainer; Maruyama-Nakashita, Akiko

    2016-01-01

    Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (−S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links –S to GSL biosynthesis has remained understudied. We report here the identification of the –S marker genes sulfur deficiency induced 1 (SDI1) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under –S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of –S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions. PMID:27730214

  15. The Capicua repressor – a general sensor of RTK signaling in development and disease

    PubMed Central

    Jiménez, Gerardo; Shvartsman, Stanislav Y.; Paroush, Ze'ev

    2012-01-01

    Receptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes. PMID:22526417

  16. Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

    NASA Astrophysics Data System (ADS)

    Monico, Carina; Capitanio, Marco; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.

    2013-06-01

    We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the kinetics of both short- and long-lived interactions of LacI along the DNA template (from ˜100 μs to tens of seconds), as well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other single-molecule methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.

  17. The general transcriptional repressor Tup1 is required for dimorphism and virulence in a fungal plant pathogen.

    PubMed

    Elías-Villalobos, Alberto; Fernández-Álvarez, Alfonso; Ibeas, José I

    2011-09-01

    A critical step in the life cycle of many fungal pathogens is the transition between yeast-like growth and the formation of filamentous structures, a process known as dimorphism. This morphological shift, typically triggered by multiple environmental signals, is tightly controlled by complex genetic pathways to ensure successful pathogenic development. In animal pathogenic fungi, one of the best known regulators of dimorphism is the general transcriptional repressor, Tup1. However, the role of Tup1 in fungal dimorphism is completely unknown in plant pathogens. Here we show that Tup1 plays a key role in orchestrating the yeast to hypha transition in the maize pathogen Ustilago maydis. Deletion of the tup1 gene causes a drastic reduction in the mating and filamentation capacity of the fungus, in turn leading to a reduced virulence phenotype. In U. maydis, these processes are controlled by the a and b mating-type loci, whose expression depends on the Prf1 transcription factor. Interestingly, Δtup1 strains show a critical reduction in the expression of prf1 and that of Prf1 target genes at both loci. Moreover, we observed that Tup1 appears to regulate Prf1 activity by controlling the expression of the prf1 transcriptional activators, rop1 and hap2. Additionally, we describe a putative novel prf1 repressor, named Pac2, which seems to be an important target of Tup1 in the control of dimorphism and virulence. Furthermore, we show that Tup1 is required for full pathogenic development since tup1 deletion mutants are unable to complete the sexual cycle. Our findings establish Tup1 as a key factor coordinating dimorphism in the phytopathogen U. maydis and support a conserved role for Tup1 in the control of hypha-specific genes among animal and plant fungal pathogens.

  18. Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1.

    PubMed

    Bandhu, Amitava; Ganguly, Tridib; Jana, Biswanath; Mondal, Rajkrishna; Sau, Subrata

    2010-05-18

    Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.

  19. Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.

    PubMed

    Darling, P J; Holt, J M; Ackers, G K

    2000-09-22

    The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity. Copyright 2000 Academic Press.

  20. Human Freud-2/CC2D1B: a novel repressor of post-synaptic 5-HT1A receptor expression

    PubMed Central

    Hadjighassem, Mahmoud R.; Austin, Mark C.; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A.; Albert, Paul R.

    2014-01-01

    Background Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene (HTR1A), a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Methods Freud-2 distribution was examined using Northern and Western blot, RT-PCR, immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay and Western blot. Results Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus, but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5′ or 3′ human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of post-synaptic 5-HT1A receptor-positive cells. Furthermore siRNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Conclusion Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons. PMID:19423080

  1. Loss of the co-repressor GPS2 sensitizes macrophage activation upon metabolic stress induced by obesity and type 2 diabetes.

    PubMed

    Fan, Rongrong; Toubal, Amine; Goñi, Saioa; Drareni, Karima; Huang, Zhiqiang; Alzaid, Fawaz; Ballaire, Raphaelle; Ancel, Patricia; Liang, Ning; Damdimopoulos, Anastasios; Hainault, Isabelle; Soprani, Antoine; Aron-Wisnewsky, Judith; Foufelle, Fabienne; Lawrence, Toby; Gautier, Jean-Francois; Venteclef, Nicolas; Treuter, Eckardt

    2016-07-01

    Humans with obesity differ in their susceptibility to developing insulin resistance and type 2 diabetes (T2D). This variation may relate to the extent of adipose tissue (AT) inflammation that develops as their obesity progresses. The state of macrophage activation has a central role in determining the degree of AT inflammation and thus its dysfunction, and these states are driven by epigenomic alterations linked to gene expression. The underlying mechanisms that regulate these alterations, however, are poorly defined. Here we demonstrate that a co-repressor complex containing G protein pathway suppressor 2 (GPS2) crucially controls the macrophage epigenome during activation by metabolic stress. The study of AT from humans with and without obesity revealed correlations between reduced GPS2 expression in macrophages, elevated systemic and AT inflammation, and diabetic status. The causality of this relationship was confirmed by using macrophage-specific Gps2-knockout (KO) mice, in which inappropriate co-repressor complex function caused enhancer activation, pro-inflammatory gene expression and hypersensitivity toward metabolic-stress signals. By contrast, transplantation of GPS2-overexpressing bone marrow into two mouse models of obesity (ob/ob and diet-induced obesity) reduced inflammation and improved insulin sensitivity. Thus, our data reveal a potentially reversible disease mechanism that links co-repressor-dependent epigenomic alterations in macrophages to AT inflammation and the development of T2D.

  2. Characterization of the gene cluster involved in allantoate catabolism and its transcriptional regulation by the RpiR-type repressor HpxU in Klebsiella pneumoniae.

    PubMed

    Guzmán, Karla; Campos, Evangelina; Aguilera, Laura; Toloza, Lorena; Giménez, Rosa; Aguilar, Juan; Baldoma, Laura; Badia, Josefa

    2013-09-01

    Bacteria, fungi, and plants have metabolic pathways for the utilization of nitrogen present in purine bases. In Klebsiella pneumoniae, the genes responsible for the assimilation of purine ring nitrogen are distributed in three separated clusters. We characterized the gene cluster involved in the metabolism of allantoate (genes KPN_01761 to KPN_01771). The functional assignments of HpxK, as an allantoate amidohydrolase, and of HpxU, as a regulator involved in the control of allantoate metabolism, were assessed experimentally. Gene hpxU encodes a repressor of the RpiR family that mediates the regulation of this system by allantoate. In this study, the binding of HpxU to the hpxF promoter and to the hpxU-hpxW intergenic region containing the divergent promoter for these genes was evidenced by electrophoretic mobility shift assays. Allantoate released the HpxU repressor from its target operators whereas other purine intermediate metabolites, such as allantoin and oxamate, failed to induce complex dissociation. Sequence alignment of the four HpxU identified operators identified TGAA-N8-TTCA as the consensus motif recognized by the HpxU repressor.

  3. Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions.

    PubMed

    Richards, David H; Meyer, Sarai; Wilson, Corey J

    2017-01-20

    The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.

  4. Visualization of trp repressor and its complexes with DNA by atomic force microscopy.

    PubMed Central

    Margeat, E; Le Grimellec, C; Royer, C A

    1998-01-01

    We used tapping mode atomic force microscopy to visualize the protein/protein and the protein/DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. In the presence of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mica surface. We observed the expected decrease in specificity of TR for its operators with increasing protein concentration (1-5 nM). This loss of DNA-binding specificity is accompanied by the formation of large protein assemblies of varying sizes on the mica surface, consistent with the known tendency of the repressor to oligomerize in solution. When the co-repressor is omitted, no repressor molecules are seen, either on the plasmid fragments or free on the mica surface, probably because of the formation of larger aggregates that are removed from the surface upon washing. All these findings support a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor. PMID:9826594

  5. Crystal Structure of the lamda Repressor and a Model for Pairwise Cooperative Operator Binding

    SciTech Connect

    Stayrook,S.; Jaru-Ampornpan, P.; Ni, J.; Hochschild, A.; Lewis, M.

    2008-01-01

    Bacteriophage {lambda} has for many years been a model system for understanding mechanisms of gene regulation1. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the chromosome that are separated by about 2,400 base pairs (bp)2, 3. A hallmark of the system is the pairwise cooperativity of repressor binding4. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

  6. A novel GDNF-inducible gene, BMZF3, encodes a transcriptional repressor associated with KAP-1

    SciTech Connect

    Suzuki, Chikage; Murakumo, Yoshiki Kawase, Yukari; Sato, Tomoko; Morinaga, Takatoshi; Fukuda, Naoyuki; Enomoto, Atsushi; Ichihara, Masatoshi; Takahashi, Masahide

    2008-02-01

    The Krueppel-associated box (KRAB)-containing zinc finger proteins (ZFPs) comprise the largest family of zinc finger transcription factors that function as transcriptional repressors. In the study of glial cell line-derived neurotrophic factor (GDNF)-RET signaling, we have identified bone marrow zinc finger 3 (BMZF3), encoding a KRAB-ZFP, as a GDNF-inducible gene by differential display analysis. The expression of BMZF3 transcripts in the human neuroblastoma cell line TGW increased 1 h after GDNF stimulation, as determined by Northern blotting and quantitative reverse-transcriptase polymerase chain reaction. The BMZF3 possesses transcriptional repressor activity in the KRAB domain. BMZF3 interacts with a co-repressor protein, KRAB-associated protein 1 (KAP-1), through the KRAB domain and siRNA-mediated knockdown of KAP-1 abolished the transcriptional repressor activity of BMZF3, indicating that KAP-1 is necessary for BMZF3 function. Furthermore, siRNA-mediated silencing of BMZF3 inhibited cell proliferation. These findings suggest that BMZF3 is a transcriptional repressor induced by GDNF that plays a role in cell proliferation.

  7. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

    PubMed

    Ohta, M; Matsui, K; Hiratsu, K; Shinshi, H; Ohme-Takagi, M

    2001-08-01

    We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

  8. In Vitro Repression of Transcription of the Trytophan Operon by trp Repressor

    PubMed Central

    Shimizu, Yoshiko; Shimizu, Nobuyoshi; Hayashi, Masaki

    1973-01-01

    The in vitro repression of transcription of the tryptophan operon by the trp repressor from Escherichia coli was studied. By measuring the inhibitory effect for trp-specific RNA synthesis in an in vitro transcription system directed by DNA of trp-transducing phage, we have detected and concentrated a trp repressor in an eluate of a Φ80 ptED native DNA-cellulose column. The repression of transcription of trp operon required the addition of L-tryptophan in the system, and when several tryptophan analogues were added, the repression or derepression was similar to that observed in vivo. The repressor fraction was separated from the majority of tryptophanyl-tRNA synthetase activity by Bio-gel P60 column chromatography. PMID:4579009

  9. Neither Two-State nor Three-State: Dimerization of Lambda Cro Repressor.

    PubMed

    Yao, John; Wang, Jin

    2015-06-04

    Lambda Cro repressor is one of the best studied dimeric transcription factors. However, there has still been an unsettled debate for decades about whether it is a two-state dimer or three-state dimer. We provide a new mechanism model that can reconcile these seemingly conflicting (mutually exclusive) experimental results. From simulations with all-atom structure-based model, we observe that the dimerization process of Lambda Cro repressor starts from one folded monomer with one unfolded monomer. Intrasubunit folding and intersubunit binding are partially coupled, in a fly casting manner.

  10. Deuterium exchange of operator 8CH groups as a Raman probe of repressor recognition: interactions of wild-type and mutant lambda repressors with operator OL1.

    PubMed

    Reilly, K E; Becka, R; Thomas, G J

    1992-03-31

    The rate of deuterium exchange of a purine 8CH group in DNA is highly sensitive to both macromolecular secondary structure and intermolecular interactions which restrict solvent access to the major groove [Lamba, O.P., Becka, R., & Thomas, G.J., Jr. (1990) Biopolymers 29, 1465-1477]. We have exploited the sensitivity of the 8CH----8CD reaction to probe DNA recognition by the helix-turn-helix (HTH) motif of phage lambda cI repressor. We find that purine exchanges in the 19-base-pair OL1 operator are strongly and specifically restricted by binding of the HTH N-terminal domain of the repressor fragment (RF) comprising residues 1-102. The kinetics indicate large-scale obstruction of solvent access to operator 7N-8C purine sites. Interpretation of the exchange kinetics using a simple model suggests that only 7 purine residues (5 of 10 adenines and 2 of 9 guanines) remain unrestricted with respect to 8CH exchange in complexes of OL1 with the wild-type repressor. On the other hand, the 8CH exchange profile for the complex of OL1 with the Tyr88----Cys mutant repressor indicates that 9 purines (7 adenines and 2 guanines) are exchangeable. These results suggest important differences in major groove recognition in the two complexes. The proposed 8CH labeling profiles are consistent with molecular models of related complexes determined by X-ray crystallography [Jordan, S.R., & Pabo, C.O. (1988) Science 242, 893-899] and indicate that the structures observed in the crystal are largely maintained in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor.

    PubMed

    Lee, G; Wu, J; Luu, P; Ghazal, P; Flores, O

    1996-03-19

    Transcriptional repression is an important component of regulatory networks that govern gene expression. In this report, we have characterized the mechanisms by which the immediate early protein 2 (IE2 or IE86), a master transcriptional regulator of human cytomegalovirus, down-regulates its own expression. In vitro transcription and DNA binding experiments demonstrate that IE2 blocks specifically the association of RNA polymerase II with the preinitiation complex. Although, to our knowledge, this is the first report to describe a eukaryotic transcriptional repressor that selectively impedes RNA polymerase II recruitment, we present data that suggest that this type of repression might be widely used in the control of transcription by RNA polymerase II.

  12. Energetic methods to study bifunctional biotin operon repressor.

    PubMed

    Beckett, D

    1998-01-01

    measurements. The results of quantitative studies of the biotin regulatory system can be interpreted in the context of the biological function of the system. The biotin holoenzyme ligases are a class of enzymes found across the evolutionary spectrum. Only a subset of these enzymes, including BirA, also function as transcriptional repressors. The tight binding of the allosteric effector may be understood in light of the bifunctional nature of the BirA-bio-5'-AMP complex. It is possible that the unusually high thermodynamic and kinetic stability of the complex ensures that the most probable state of the protein in vivo is the adenylate-bound form. This complex, not the unliganded protein, is active in both enzymatic transfer of biotin and site-specific DNA binding. This ensures that on depletion of the intracellular pool of apoBCCP, BirA-bio-5'-AMP accumulates and binds to bioO to repress transcription of the biotin biosynthesis operon. The intracellular demand for and synthesis of biotin are, consequently, tightly coupled in the system. The dimerization that accompanies adenylate binding to BirA appears to be significant for site-specific binding of the protein to bioO. Functionally, the simultaneous binding of the two monomers to the two operator half-sites, regardless of the kinetic mechanism by which it occurs, ensures coordinate regulation of transcription initiation from both biotin operon promoters. The multifaceted approach utilized in studies of the biotin regulatory system can serve as a model for studies of any complex transcriptional regulatory system. It is critical in elucidating the functional energetics of any of these systems that the assembly first be dissected into the constituent interactions and that each of these interactions be studied in isolation. This is not only critical for understanding the physicochemical properties of each individual contributing interaction, but is also a necessary precursor to studies of thermodynamic linkage in the system. (AB

  13. A novel MAs(III)-selective ArsR transcriptional repressor.

    PubMed

    Chen, Jian; Nadar, Venkadesh Sarkarai; Rosen, Barry P

    2017-09-01

    Microbial expression of genes for resistance to heavy metals and metalloids is usually transcriptionally regulated by the toxic ions themselves. Arsenic is a ubiquitous, naturally occurring toxic metalloid widely distributed in soil and groundwater. Microbes biotransform both arsenate (As(V)) and arsenite (As(III)) into more toxic methylated metabolites methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)). Environmental arsenic is sensed by members of the ArsR/SmtB family. The arsR gene is autoregulated and is typically part of an operon that contains other ars genes involved in arsenic detoxification. To date every identified ArsR is regulated by inorganic As(III). Here we described a novel ArsR from Shewanella putrefaciens selective for MAs(III). SpArsR orthologs control expression of two MAs(III) resistance genes, arsP that encodes the ArsP MAs(III) efflux permease, and arsH encoding the ArsH MAs(III) oxidase. SpArsR has two conserved cysteine residues, Cys101 and Cys102. Mutation of either resulted in loss of MAs(III) binding, indicating that they form an MAs(III) binding site. SpArsR can be converted into an As(III)-responsive repressor by introduction of an additional cysteine that allows for three-coordinate As(III) binding. Our results indicate that SpArsR evolved selectivity for MAs(III) over As(III) in order to control expression of genes for MAs(III) detoxification. © 2017 John Wiley & Sons Ltd.

  14. Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry.

    PubMed

    Su, Baofeng; Shang, Mei; Li, Chao; Perera, Dayan A; Pinkert, Carl A; Irwin, Michael H; Peatman, Eric; Grewe, Peter; Patil, Jawahar G; Dunham, Rex A

    2015-04-01

    Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output

  15. Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

    PubMed

    Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

    1999-08-01

    Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain

  16. Whi7 is an unstable cell-cycle repressor of the Start transcriptional program.

    PubMed

    Gomar-Alba, Mercè; Méndez, Ester; Quilis, Inma; Bañó, M Carmen; Igual, J Carlos

    2017-08-24

    Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCF(Grr1) ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.The commitment of cells to a new cycle of division involves inactivation of the Start transcriptional repressor Whi5. Here the authors show that the sequence related protein Whi7 associates to G1/S gene promoters in late G1 and collaborates with Whi5 in Start repression.

  17. Diverse roles of Groucho/Tup1 co-repressors in plant growth and development.

    PubMed

    Lee, Joanne E; Golz, John F

    2012-01-01

    Transcriptional regulation involves coordinated and often complex interactions between activators and repressors that together dictate the temporal and spatial activity of target genes. While the study of developmental regulation has often focused on positively acting transcription factors, it is becoming increasingly clear that transcriptional repression is a key regulatory mechanism underpinning many developmental processes in both plants and animals. In this review, we focus on the plant Groucho (Gro)/Tup1-like co-repressors and discuss their roles in establishing the apical-basal axis of the developing embryo, maintaining the stem cell population in the shoot apex and determining floral organ identity.  As well as being developmental regulators, recent studies have shown that these co-repressors play a central role in regulating auxin and jasmonate signalling pathways and are also linked to the regulation of pectin structure in the seed coat. These latest findings point to the Gro/Tup1-like co-repressors playing a much broad role in plant growth and development than previously thought; an observation that underlines the central importance of transcriptional repression in plant gene regulation.

  18. Effects of Task Familiarity on Stress Responses of Repressors and Sensitizers

    ERIC Educational Resources Information Center

    Pagano, Don F.

    1973-01-01

    R.S. Lazarus's theory of coping was used to investigate appraisal and reappraisal of threat in repressors and sensitizers. Two indexes of stress, self-report ratings of affect and palmar skin conductance, were measured prior to performance on a reaction time task, after one-third of the task was completed and after two-thirds of the task was…

  19. Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

    NASA Astrophysics Data System (ADS)

    D'Aquino, J. Alejandro; Ringe, Dagmar

    2006-08-01

    The diphtheria toxin repressor, DtxR, is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear (1 - 3). Calorimetric techniques have demonstrated that while binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10-7, binding site 2 (primary) is a low affinity binding site with a binding constant of 6.3 × 10-4. These two binding sites act independently and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here and the previously reported DtxR(H79A) (4) has allowed us to propose a mechanism of metal ion activation for DtxR.

  20. Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

    PubMed Central

    Labow, M A; Baim, S B; Shenk, T; Levine, A J

    1990-01-01

    A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture. Images PMID:2162473

  1. The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding.

    PubMed

    Streaker, Emily D; Gupta, Aditi; Beckett, Dorothy

    2002-12-03

    The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP. Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization. The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex. It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface. In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form. Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics. However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

  2. Redox signaling regulates transcriptional activity of the Ca2+-dependent repressor DREAM.

    PubMed

    Rivas, Marcos; Aurrekoetxea, Koldo; Mellström, Britt; Naranjo, José R

    2011-04-01

    DREAM/KChIP3 (Downstream Regulatory Element Antagonist Modulator) is a multifunctional Ca(2+)-binding protein that acts in the nucleus as a Ca(2+)-dependent transcriptional repressor. Binding to DNA and repressor activity of DREAM is regulated by Ca(2+), specific post-translational modifications as well as by protein-protein interactions with several nucleoproteins. Here, using the yeast two-hybrid assay, we characterized the interaction of DREAM with peroxiredoxin 3 (Prdx3), an antioxidant enzyme that uses the thioredoxin system as electron donor. Importantly, the DREAM/Prdx3 interaction is Ca(2+) dependent and is blocked by DTT. Coexpression of Prdx3 enhances DREAM binding to DRE sites and its repressor activity in vivo. Two cysteine residues in the N-terminal domain of DREAM are responsible for the redox modulation of its activity. Double Cys to Ser substitution results in a mutant DREAM with stronger repressor activity. Finally, we show that transient DREAM knockdown sensitizes PC12 cells to H(2)O(2)-induced oxidative stress, suggesting a protective role for DREAM against oxidative damage.

  3. CRES-T, an effective gene silencing system utilizing chimeric repressors.

    PubMed

    Mitsuda, Nobutaka; Matsui, Kyoko; Ikeda, Miho; Nakata, Masaru; Oshima, Yoshimi; Nagatoshi, Yukari; Ohme-Takagi, Masaru

    2011-01-01

    Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.

  4. The Translational Repressor 4E-BP1 Contributes to Diabetes-Induced Visual Dysfunction

    PubMed Central

    Miller, William P.; Mihailescu, Maria L.; Yang, Chen; Barber, Alistair J.; Kimball, Scot R.; Jefferson, Leonard S.; Dennis, Michael D.

    2016-01-01

    Purpose The translational repressor 4E-BP1 interacts with the mRNA cap-binding protein eIF4E and thereby promotes cap-independent translation of mRNAs encoding proteins that contribute to diabetic retinopathy. Interaction of 4E-BP1 with eIF4E is enhanced in the retina of diabetic rodents, at least in part, as a result of elevated 4E-BP1 protein expression. In the present study, we examined the role of 4E-BP1 in diabetes-induced visual dysfunction, as well as the mechanism whereby hyperglycemia promotes 4E-BP1 expression. Methods Nondiabetic and diabetic wild-type and 4E-BP1/2 knockout mice were evaluated for visual function using a virtual optomotor test (Optomotry). Retinas were harvested from nondiabetic and type 1 diabetic mice and analyzed for protein abundance and posttranslational modifications. Similar analyses were performed on cells in culture exposed to hyperglycemic conditions or an O-GlcNAcase inhibitor (Thiamet G [TMG]). Results Diabetes-induced visual dysfunction was delayed in mice deficient of 4E-BP1/2 as compared to controls. 4E-BP1 protein expression was enhanced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in retinal cells in culture. A similar elevation in 4E-BP1 expression was observed with TMG. The rate of 4E-BP1 degradation was significantly prolonged by either hyperglycemic conditions or TMG. A PEST motif in the C-terminus of 4E-BP1 regulated polyubiquitination, turnover, and binding of an E3 ubiquitin ligase complex containing CUL3. Conclusions The findings support a model whereby elevated 4E-BP1 expression observed in the retina of diabetic rodents is the result of O-GlcNAcylation of 4E-BP1 within its PEST motif. PMID:26998719

  5. The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub*

    PubMed Central

    Kwong, Pak N.; Chambers, Michael; Vashisht, Ajay A.; Turki-Judeh, Wiam; Yau, Tak Yu; Wohlschlegel, James A.; Courey, Albert J.

    2015-01-01

    Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro. PMID:26483546

  6. Genes regulated by the Escherichia coli SOS repressor LexA exhibit heterogenous expression

    PubMed Central

    2010-01-01

    Background Phenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level. Results The investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells. Conclusion LexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogenous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes. PMID:21070632

  7. Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

    PubMed

    Garza de Leon, Federico; Sellars, Laura; Stracy, Mathew; Busby, Stephen J W; Kapanidis, Achillefs N

    2017-04-11

    Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (∼40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials

    PubMed Central

    Fernandez-Escamilla, Ana Maria; Fernandez-Ballester, Gregorio; Morel, Bertrand; Casares-Atienza, Salvador; Ramos, Juan Luis

    2015-01-01

    A disturbing phenomenon in contemporary medicine is the prevalence of multidrug-resistant pathogenic bacteria. Efflux pumps contribute strongly to this antimicrobial drug resistance, which leads to the subsequent failure of clinical treatments. The TtgR protein of Pseudomonas putida is a HTH-type transcriptional repressor that controls expression of the TtgABC efflux pump, which is the main contributor to resistance against several antimicrobials and toxic compounds in this microbe. One of the main strategies to modulate the bacterial resistance is the rational modification of the ligand binding target site. We report the design and characterization of four mutants-TtgRS77A, TtgRE78A, TtgRN110A and TtgRH114A - at the active ligand binding site. The biophysical characterization of the mutants, in the presence and in the absence of different antimicrobials, revealed that TtgRN110A is the variant with highest thermal stability, under any of the experimental conditions tested. EMSA experiments also showed a different dissociation pattern from the operator for TtgRN110A, in the presence of several antimicrobials, making it a key residue in the TtgR protein repression mechanism of the TtgABC efflux pump. We found that TtgRE78A stability is the most affected upon effector binding. We also probe that one mutation at the C-terminal half of helix-α4, TtgRS77A, provokes a severe protein structure distortion, demonstrating the important role of this residue in the overall protein structure and on the ligand binding site. The data provide new information and deepen the understanding of the TtgR-effector binding mechanism and consequently the TtgABC efflux pump regulation mechanism in Pseudomonas putida. PMID:26422008

  9. The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

    PubMed Central

    Jennings, Barbara H.

    2014-01-01

    Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation

  10. Conversion of the LIN-1 ETS protein of Caenorhabditis elegans from a SUMOylated transcriptional repressor to a phosphorylated transcriptional activator.

    PubMed

    Leight, Elizabeth R; Murphy, John T; Fantz, Douglas A; Pepin, Danielle; Schneider, Daniel L; Ratliff, Thomas M; Mohammad, Duaa H; Herman, Michael A; Kornfeld, Kerry

    2015-03-01

    The LIN-1 ETS transcription factor plays a pivotal role in controlling cell fate decisions during development of the Caenorhabditis elegans vulva. Prior to activation of the RTK/Ras/ERK-signaling pathway, LIN-1 functions as a SUMOylated transcriptional repressor that inhibits vulval cell fate. Here we demonstrate using the yeast two-hybrid system that SUMOylation of LIN-1 mediates interactions with a protein predicted to be involved in transcriptional repression: the RAD-26 Mi-2β/CHD4 component of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies indicated that rad-26 functions to inhibit vulval cell fates in worms. Using the yeast two-hybrid system, we showed that the EGL-27/MTA1 component of the NuRD complex binds the carboxy-terminus of LIN-1 independently of LIN-1 SUMOylation. EGL-27 also binds UBC-9, an enzyme involved in SUMOylation, and MEP-1, a zinc-finger protein previously shown to bind LIN-1. Genetic studies indicate that egl-27 inhibits vulval cell fates in worms. These results suggest that LIN-1 recruits multiple proteins that repress transcription via both the SUMOylated amino-terminus and the unSUMOylated carboxy-terminus. Assays in cultured cells showed that the carboxy-terminus of LIN-1 was converted to a potent transcriptional activator in response to active ERK. We propose a model in which LIN-1 recruits multiple transcriptional repressors to inhibit the 1° vulval cell fate, and phosphorylation by ERK converts LIN-1 to a transcriptional activator that promotes the 1° vulval cell fate.

  11. C1 repressor-mediated DNA looping is involved in C1 autoregulation of bacteriophage P1.

    PubMed

    Heinzel, T; Lurz, R; Dobrinski, B; Velleman, M; Schuster, H

    1994-12-16

    C1 repressor is required to repress the lytic functions of a P1 prophage in vivo. Transcription of the c1 gene is autoregulated via the C1-controlled operator Op99a,b which overlaps the promoter of the c1 gene. It is negatively affected by Lxc corepressor and the DNA region upstream of c1, which contains the additional operators Op99c, d, and e. We have explored these effects by constructing a set of lacZ reporter plasmids with Op99a,b and varying parts of the upstream DNA region. Transcription levels were measured in vivo with a two-plasmid system containing the lacZ reporter and a c1+ lxc+ or c1+ lxc- plasmid. Compared to the C1+Lxc-repressed lacZ reporter with all operators present, the basal level of beta-galactosidase activity increases successively when (i) upstream operators were deleted or inactivated, (ii) Lxc corepressor was removed, and (iii) C1 and Lxc were absent. By that means a 2 x 2 x 15-fold stepwise increase in enzyme activity was found. Using electron microscopy to visualize the interaction of C1 repressor with the operators in vitro, looped DNA molecules were observed. Although all operators can participate in C1-mediated DNA looping, loops between Op99a,b and Op99d occurred predominantly. Lxc is not required but increases drastically the frequency of loop formation. The results indicate that C1-mediated DNA looping may be a second element besides Lxc for fine-tuning the autoregulation of c1 transcription.

  12. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

    PubMed

    Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

  13. CC-type glutaredoxins recruit the transcriptional co-repressor TOPLESS to TGA-dependent target promoters in Arabidopsis thaliana.

    PubMed

    Uhrig, Joachim F; Huang, Li-Jun; Barghahn, Sina; Willmer, Moritz; Thurow, Corinna; Gatz, Christiane

    2017-02-01

    Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs in Arabidopsis thaliana) for which the biochemical functions are yet unknown. ROXYs and CC-type GRXs from rice and maize physically and genetically interact with bZIP transcription factors of the TGA family to control developmental and stress-associated processes. Here we demonstrate that ROXYs interact with transcriptional co-repressors of the TOPLESS (TPL) family which are related to Tup1 in fungi and Groucho/TLE in animals. In ROXYs, the functionally important conserved A(L/I)W(L/V) motif at the very C terminus mediates the interaction with TPL. A ternary TGA2/ROXY19/TPL complex is formed when all three proteins are co-expressed in yeast. Loss-of-function evidence for the role of TPL in ROXY19-mediated repression was hampered by the redundancy of the five members of the TPL gene family and developmental defects of higher order tpl mutants. As an alternative strategy, we ectopically expressed known TPL-interacting proteins in order to out-compete the amount of available TPL in transiently transformed protoplasts. Indeed, ROXY19-mediated transcriptional repression was strongly alleviated by this approach. Our data suggest a yet unrecognized function of GRXs acting as adapter proteins for the assembly of transcriptional repressor complexes on TGA-regulated target promoters. Copyright © 2016. Published by Elsevier B.V.

  14. The Groucho co-repressor is primarily recruited to local target sites in active chromatin to attenuate transcription.

    PubMed

    Kaul, Aamna; Schuster, Eugene; Jennings, Barbara H

    2014-08-01

    Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active" chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and

  15. Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor

    PubMed Central

    Kim, Minsik; Kim, Hee Jung; Son, Sang Hyeon; Yoon, Hye Jin; Lim, Youngbin; Lee, Jong Woo; Seok, Yeong-Jae; Jin, Kyeong Sik; Yu, Yeon Gyu; Kim, Seong Keun; Ryu, Sangryeol; Lee, Hyung Ho

    2016-01-01

    DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92–198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant. PMID:27099293

  16. A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements.

    PubMed

    Metruccio, Matteo M E; Pigozzi, Eva; Roncarati, Davide; Berlanda Scorza, Francesco; Norais, Nathalie; Hill, Stuart A; Scarlato, Vincenzo; Delany, Isabel

    2009-12-01

    Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

  17. A Flowering Locus C Homolog Is a Vernalization-Regulated Repressor in Brachypodium and Is Cold Regulated in Wheat1[OPEN

    PubMed Central

    Sharma, Neha; Ruelens, Philip; D'hauw, Mariëlla; Maggen, Thomas; Dochy, Niklas; Kaufmann, Kerstin; Rohde, Antje

    2017-01-01

    Winter cereals require prolonged cold to transition from vegetative to reproductive development. This process, referred to as vernalization, has been extensively studied in Arabidopsis (Arabidopsis thaliana). In Arabidopsis, a key flowering repressor called FLOWERING LOCUS C (FLC) quantitatively controls the vernalization requirement. By contrast, in cereals, the vernalization response is mainly regulated by the VERNALIZATION genes, VRN1 and VRN2. Here, we characterize ODDSOC2, a recently identified FLC ortholog in monocots, knowing that it belongs to the FLC lineage. By studying its expression in a diverse set of Brachypodium accessions, we find that it is a good predictor of the vernalization requirement. Analyses of transgenics demonstrated that BdODDSOC2 functions as a vernalization-regulated flowering repressor. In most Brachypodium accessions BdODDSOC2 is down-regulated by cold, and in one of the winter accessions in which this down-regulation was evident, BdODDSOC2 responded to cold before BdVRN1. When stably down-regulated, the mechanism is associated with spreading H3K27me3 modifications at the BdODDSOC2 chromatin. Finally, homoeolog-specific gene expression analyses identify TaAGL33 and its splice variant TaAGL22 as the FLC orthologs in wheat (Triticum aestivum) behaving most similar to Brachypodium ODDSOC2. Overall, our study suggests that ODDSOC2 is not only phylogenetically related to FLC in eudicots but also functions as a flowering repressor in the vernalization pathway of Brachypodium and likely other temperate grasses. These insights could prove useful in breeding efforts to refine the vernalization requirement of temperate cereals and adapt varieties to changing climates. PMID:28034954

  18. Transcriptional repressor DREAM regulates T-lymphocyte proliferation and cytokine gene expression

    PubMed Central

    Savignac, Magali; Pintado, Belen; Gutierrez-Adan, Alfonso; Palczewska, Malgorzata; Mellström, Britt; Naranjo, Jose R

    2005-01-01

    Downstream Regulatory Element Antagonist Modulator (DREAM) is a Ca2+-dependent transcriptional repressor expressed in the brain, thyroid gland and thymus. Here, we analyzed the function of DREAM and the related protein KChIP-2 in the immune system using transgenic (tg) mice expressing a cross-dominant active mutant (EFmDREAM) for DREAM and KChIPs Ca2+-dependent transcriptional derepression. EFmDREAM tg mice showed reduced T-cell proliferation. Tg T cells exhibited decreased interleukin (IL)-2, -4 and interferon (IFN)γ production after polyclonal activation and following antigen-specific response. Chromatin immunoprecipitation and transfection assays showed that DREAM binds to and represses transcription from these cytokine promoters. Importantly, specific transient knockdown of DREAM or KChIP-2 induced basal expression of IL-2 and IFNγ in wild-type splenocytes. These data propose DREAM and KChIP-2 as Ca2+-dependent repressors of the immune response. PMID:16177826

  19. Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms*

    PubMed Central

    Ogden, Stacey K.; Casso, David J.; Ascano, Manuel; Yore, Mark M.; Kornberg, Thomas B.; Robbins, David J.

    2013-01-01

    The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci. PMID:16423832

  20. Lac repressor: Crystallization of intact tetramer and its complexes with inducer and operator DNA

    SciTech Connect

    Pace, H.C.; Lu, P. ); Lewis, M. Smith Kline and French Labs., King of Prussia, PA )

    1990-03-01

    The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer. The crystals without DNA diffract to better than 3.5 {angstrom}. They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 {angstrom}, b = 75.6 {angstrom}, and c = 161.2 {angstrom}, with {alpha} = {gamma} = 90{degree} and {beta} = 125.5{degree}. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 {angstrom}. These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl {beta}-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.

  1. Inducible protein expression in Drosophila Schneider 2 cells using the lac operator-repressor system.

    PubMed

    Wakiyama, Motoaki; Muramatsu, Reiko; Kaitsu, Yoko; Ikeda, Mariko; Yokoyama, Shigeyuki

    2011-12-01

    Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO(4) or CdCl(2) as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator-repressor regulatory system functioned in chromosomally integrated cell lines.

  2. A novel repressor domain is required for maximal growth inhibition by the IRF-1 tumor suppressor.

    PubMed

    Eckert, Mirjam; Meek, Sarah E M; Ball, Kathryn L

    2006-08-11

    Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumor suppressor that can regulate gene expression in a manner requiring either its sequence specific DNA binding activity or its ability to bind the p300 coactivator. We show that IRF-1-mediated growth inhibition is dependent on the integrity of a C-terminal transcriptional enhancer domain. An enhancer subdomain (amino acids 301-325) that differentially regulates IRF-1 activity has been identified and this region mediates the repression of Cdk2. The repressor domain encompasses an LXXLL coregulator signature motif and mutations or deletions within this region completely uncouple transcriptional activation from repression. The loss of growth suppressor activity when the Cdk2-repressor domain of IRF-1 is mutated implicates repression as a determinant of its maximal growth inhibitory potential. The data link IRF-1 regulatory domains to its growth inhibitory activity and provide information about how differential gene regulation may contribute to IRF-1 tumor suppressor activity.

  3. The non-JAZ TIFY protein TIFY8 from Arabidopsis thaliana is a transcriptional repressor.

    PubMed

    Cuéllar Pérez, Amparo; Nagels Durand, Astrid; Vanden Bossche, Robin; De Clercq, Rebecca; Persiau, Geert; Van Wees, Saskia C M; Pieterse, Corné M J; Gevaert, Kris; De Jaeger, Geert; Goossens, Alain; Pauwels, Laurens

    2014-01-01

    Jasmonate (JA) signalling is mediated by the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins, which are degraded upon JA perception to release downstream responses. The ZIM protein domain is characteristic of the larger TIFY protein family. It is currently unknown if the atypical member TIFY8 is involved in JA signalling. Here we show that the TIFY8 ZIM domain is functional and mediated interaction with PEAPOD proteins and NINJA. TIFY8 interacted with TOPLESS through NINJA and accordingly acted as a transcriptional repressor. TIFY8 expression was inversely correlated with JAZ expression during development and after infection with Pseudomonas syringae. Nevertheless, transgenic lines with altered TIFY8 expression did not show changes in JA sensitivity. Despite the functional ZIM domain, no interaction with JAZ proteins could be found. In contrast, TIFY8 was found in protein complexes involved in regulation of dephosphorylation, deubiquitination and O-linked N-acetylglucosamine modification suggesting an important role in nuclear signal transduction.

  4. The Non-JAZ TIFY Protein TIFY8 from Arabidopsis thaliana Is a Transcriptional Repressor

    PubMed Central

    Cuéllar Pérez, Amparo; Nagels Durand, Astrid; Vanden Bossche, Robin; De Clercq, Rebecca; Persiau, Geert; Van Wees, Saskia C. M.; Pieterse, Corné M. J.; Gevaert, Kris; De Jaeger, Geert; Goossens, Alain; Pauwels, Laurens

    2014-01-01

    Jasmonate (JA) signalling is mediated by the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins, which are degraded upon JA perception to release downstream responses. The ZIM protein domain is characteristic of the larger TIFY protein family. It is currently unknown if the atypical member TIFY8 is involved in JA signalling. Here we show that the TIFY8 ZIM domain is functional and mediated interaction with PEAPOD proteins and NINJA. TIFY8 interacted with TOPLESS through NINJA and accordingly acted as a transcriptional repressor. TIFY8 expression was inversely correlated with JAZ expression during development and after infection with Pseudomonas syringae. Nevertheless, transgenic lines with altered TIFY8 expression did not show changes in JA sensitivity. Despite the functional ZIM domain, no interaction with JAZ proteins could be found. In contrast, TIFY8 was found in protein complexes involved in regulation of dephosphorylation, deubiquitination and O-linked N-acetylglucosamine modification suggesting an important role in nuclear signal transduction. PMID:24416306

  5. Suppression of the biosynthesis of proanthocyanidin in Arabidopsis by a chimeric PAP1 repressor.

    PubMed

    Matsui, Kyoko; Tanaka, Hideo; Ohme-Takagi, Masaru

    2004-11-01

    Flavonoids are secondary metabolites that are specific to higher plants. PAP1, a member of the family of MYB domain transcription factors in Arabidopsis, is a positive regulator of the biosynthesis of anthocyanin. We show here that a chimeric PAP1 repressor, in which the EAR-motif repression domain from SUPERMAN was fused to PAP1, suppressed the expression of four flavonoid biosynthetic genes, namely CHS, DFR, LDOX, and BAN, in siliques, and inhibited the accumulation of proanthocyanidin, even in the presence of an endogenous positive regulator, such as TT2. This suppression resulted in the formation of light yellow seeds in 35S::PAP1SRDX transgenic plants. Our results indicate that PAP1 has the potential ability to regulate the biosynthesis not only of anthocyanin but also of proanthocyanidin. Our gene silencing system, using chimeric repressors, appears to be a useful tool for the manipulation of the biosynthesis of secondary metabolites in plants.

  6. Situational Discrimination in Repressor-type and Sensitizer-type Approval Seekers and the Birth Order by Subject Sex Interaction

    ERIC Educational Resources Information Center

    Becker, Gilbert

    1970-01-01

    Five experiments are reported. One conclusion in that repressor-type high need-for-approval subjects made the discrimination and permitted less favorable self-description, but sensitizer-type high need-for-approval subjects did not. (DB)

  7. Situational Discrimination in Repressor-type and Sensitizer-type Approval Seekers and the Birth Order by Subject Sex Interaction

    ERIC Educational Resources Information Center

    Becker, Gilbert

    1970-01-01

    Five experiments are reported. One conclusion in that repressor-type high need-for-approval subjects made the discrimination and permitted less favorable self-description, but sensitizer-type high need-for-approval subjects did not. (DB)

  8. Competition studies with repressors and activators of viral enhancer function in F9 mouse embryonal carcinoma cells.

    PubMed Central

    Sleigh, M J; Lockett, T J; Kelly, J; Lewy, D

    1987-01-01

    DNA competition studies have been used to investigate the presence of a repressor of viral enhancer function in F9 mouse embryonal carcinoma cells. The complete polyoma virus enhancer region, cotransfected into F9 cells with the SV40 promoter/enhancer attached to a chloramphenicol acetyl transferase marker gene, induced a small increase in pSV2CAT expression. This can be explained by preferential but weak binding by polyoma sequences of a molecule repressing pSV2CAT transcription. Repressor activity substantially disappeared when the cells were induced to differentiate by retinoic acid. Repressor binding was localised to one half of the polyoma enhancer, but was lost on further fragmentation of this region. It appears that multiple sequence elements may be required for repressor binding and that these are at least partially separable from the complement of elements binding enhancer activating molecules. PMID:3035489

  9. All Tcf HMG box transcription factors interact with Groucho-related co-repressors

    PubMed Central

    Brantjes, Helen; Roose, Jeroen; van de Wetering, Marc; Clevers, Hans

    2001-01-01

    Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, β-catenin translocates to the nucleus, interacts with Tcf (1–3) and thus activates transcription of target genes (4,5). Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The ‘long’ Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for Drosophila Groucho, we show that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription. PMID:11266540

  10. Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

    SciTech Connect

    Hagiwara, Hiroki; Saito, Fumiaki; Masaki, Toshihiro; Ikeda, Miki; Nakamura-Ohkuma, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. Black-Right-Pointing-Pointer TSA enhances the expression of myosin heavy chain without affecting DAPC expression. Black-Right-Pointing-Pointer TSA enhances the expression of the early MRFs, Myf5 and MEF2, and suppresses the late MRF, myogenin, after 24 h treatment. Black-Right-Pointing-Pointer TSA enhances the expression of the myogenic repressors, Ids, which inhibit myogenic differentiation. Black-Right-Pointing-Pointer TSA promotes myogenesis by coordinating the expression of MRFs and myogenic repressors. -- Abstract: Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

  11. The transcriptional repressor ARR1-SRDX suppresses pleiotropic cytokinin activities in Arabidopsis.

    PubMed

    Heyl, Alexander; Ramireddy, Eswar; Brenner, Wolfram G; Riefler, Michael; Allemeersch, Joke; Schmülling, Thomas

    2008-07-01

    The signal transduction of the phytohormone cytokinin is mediated by a multistep histidine-to-aspartate phosphorelay system. One component of this system are B-type response regulators, transcription factors mediating at least part of the response to cytokinin. In planta functional analysis of this family is hampered by the high level of functional redundancy of its 11 members. We generated a dominant repressor version of the Arabidopsis (Arabidopsis thaliana) response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology in order to study the extent of the contribution of B-type response regulators to cytokinin activities. In a protoplast test system, ARR1-SRDX suppressed ARR6:beta-glucuronidase reporter gene activation by different B-type ARRs. 35S:ARR1-SRDX transgenic Arabidopsis plants showed phenotypic changes reminiscent of plants with a reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system, and larger seeds. Several bioassays showed that 35S:ARR1-SRDX plants have an increased resistance toward cytokinin. The rapid induction of a large part of the cytokinin response genes was dampened. The transcript levels of more than 500 genes were more than 2.5-fold reduced in 35S:ARR1-SRDX transgenic seedlings, suggesting a broad function of B-type ARRs. Collectively, the suppression of pleiotropic cytokinin activities by a dominant repressor version of a B-type ARR indicates that this protein family is involved in mediating most, if not all, of the cytokinin activities in Arabidopsis. In addition, a role for B-type ARRs in mediating cross talk with other pathways is supported by the resistance of 35S:ARR1-SRDX seeds to phytochrome B-mediated inhibition of germination by far-red light. This study demonstrates the usefulness of chimeric repressor silencing technology to overcome redundancy in transcription factor families for functional studies.

  12. Dimethylnitrosamine-demethylase: molecular size-dependence of repression by polynuclear hydrocarbons. Nonhydrocarbon repressors.

    PubMed

    Arcos, J C; Valle, R T; Bryant, G M; Buu-Hoi, N P; Argus, M F

    1976-01-01

    Studies with 58 polynuclear aromatic hydrocarbons have shown that to repress demethylation of dimethylnitrosamine (DMN) in rat liver, the hydrocarbons must satisfy specific requirements of molecular geometry regarding size, shape, and coplanarity. Expressing the molecular size of these planar compounds by the two-dimensional area occupied, the size for maximal repressor activity ranges between about 85 and 150 A2. In addition to being within the correct molecular size range the hydrocarbons must have an elongated-rather than compact-molecular shape; circularly shaped and/or highly symmetrical hydrocarbons, such as coronene, triphenylene, ovalene, and tetrabenzonaphthalene, have very low activity or are inactive, in spite of being in the optimum size range. Coplanarity of the molecule is a critical requirement; thus, the potent carcinogen, 9,10-dimethyl-1,2-benzanthracene, is inactive as repressor of DMN-demethylase synthesis. Two exceptions, fluoranthene and benzol[ghi] fluoranthene, showed significant induction of DMN-demethylase. The molecular size distribution of hydrocarbons that repress the DMN-demethylase shows a mirror-image relationship with respect to the earlier reported molecular size requirement for indcution of azo dye N-demethylase. Compounds other than hydrocarbons also show the mirror-image relationship in the sense that pregnenolene-16alpha-carbonitrile, alpha- and beta-naphthoflavone, and Aroclor 1254 (known to be inducers of various mixed-function oxidases) are strong repressors of DMN-demethylase. Aminoacetonitrile, a strong inhibitor of carcinogenesis by DMN, is also a potent repressor of DMN-demethylase. The enzyme is inhibited by pretreatment of the animals with cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450. Pregnenolone-16alpha-carbonitrile and 3-methylcholanthrene, despite their similarity of action on DMN-demethylase, have different effects on azo reductase, which is repressed by the former and induced by the latter

  13. A Novel Repressor of Estrogen-Regulated Genes for Breast Cancer Growth Suppression

    DTIC Science & Technology

    1998-08-01

    Meyer, W., Vissing, H., Thiesen, H-J., and Rauscher , F.J. III (1994) Kruppel-associated Boxes are Potent Transcriptional Repressor Domains. Proc. Nat’l...400. 31.Wrenn, C. K., and Katzenellenbogen, B. S. ( 1993 ) Structure-Function Analysis of the Hormone Binding Domain of the Human Estrogen Receptor by...and Katzenellenbogen, B.S. ( 1993 ) Potent Dominant Negative Mutants of the Human Estrogen Receptor. J. Biol. Chem. 268,14026-14032. 33. Mattick, S

  14. Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

    PubMed

    Le Cam, L; Polanowska, J; Fabbrizio, E; Olivier, M; Philips, A; Ng Eaton, E; Classon, M; Geng, Y; Sardet, C

    1999-04-01

    Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1-specific genes in G0/G1.

  15. Identification and characterization of a novel repressor of beta-interferon gene expression.

    PubMed

    Keller, A D; Maniatis, T

    1991-05-01

    We have identified and characterized a novel repressor of human beta-interferon (beta-IFN) gene expression. This protein, designated PRDI-BF1, binds specifically to the PRDI element of the beta-IFN gene promoter and is distinct from previously reported proteins that bind to this sequence. PRDI-BF1 is an 88-kD protein containing five zinc-finger motifs. Cotransfection experiments in cultured mammalian cells revealed that PRDI-BF1 is a potent repressor of PRDI-dependent transcription. PRDI-BF1 blocks virus induction of the intact beta-IFN gene promoter and of synthetic promoters containing multiple PRDI sites. PRDI-BF1 can also block the SV40 enhancer when PRDI sites are located between the enhancer and the promoter. This repression is highly dependent on the location of the PRDI sites, however, indicating that PRDI-BF1 cannot act at a distance. On the basis of the properties of PRDI-BF1 and the observation that PRDI-BF1 mRNA accumulation is virus inducible, we propose that PRDI-BF1 may act as a postinduction repressor of the beta-IFN gene by displacing positive regulatory proteins from the PRDI site of the promoter.

  16. Co-repressor activity of scaffold attachment factor B1 requires sumoylation

    SciTech Connect

    Garee, Jason P.; Meyer, Rene; Oesterreich, Steffi

    2011-05-20

    Highlights: {yields} SAFB1 is sumoylated to two lysine residues K231 and K294. {yields} SAFB1 sumoylation is regulated by PIAS1 and SENP1. {yields} Sumoylation of SAFB1 regulates its transcriptional repressor activity. {yields} Mutation of sumoylation sites leads to decreased SAFB1 binding to HDAC3. -- Abstract: Sumoylation is an emerging modification associated with a variety of cellular processes including the regulation of transcriptional activities of nuclear receptors and their coregulators. As SUMO modifications are often associated with transcriptional repression, we examined if sumoylation was involved in modulation of the transcriptional repressive activity of scaffold attachment factor B1. Here we show that SAFB1 is modified by both the SUMO1 and SUMO2/3 family of proteins, on lysine's K231 and K294. Further, we demonstrate that SAFB1 can interact with PIAS1, a SUMO E3 ligase which mediates SAFB1 sumoylation. Additionally, SENP1 was identified as the enzyme desumoylating SAFB1. Mutation of the SAFB1 sumoylation sites lead to a loss of transcriptional repression, at least in part due to decreased interaction with HDAC3, a known transcriptional repressor and SAFB1 binding partner. In summary, the transcriptional repressor SAFB1 is modified by both SUMO1 and SUMO2/3, and this modification is necessary for its full repressive activity.

  17. A chimeric NST repressor has the potential to improve glucose productivity from plant cell walls.

    PubMed

    Iwase, Akira; Hideno, Akihiro; Watanabe, Keiji; Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-07-15

    Bioethanol might be produced more economically and with less ecological impact (with reduced exploitation of food crops) if we could increase the production of glucose from the cellulosic materials in plant cell walls. However, plant cell walls are relatively resistant to enzymatic and physicochemical hydrolysis and, therefore, it is necessary to develop methods for reducing such resistance. Changes in plant cell wall materials, by genetic engineering, that render them more easily hydrolyzable to glucose might be a valuable approach to this problem. We showed previously that, in Arabidopsis, NAC secondary wall thickening-promoting factor1 (NST1) and NST3 are key regulators of secondary wall formation. We report here that transgenic Arabidopsis plants that expressed a chimeric repressor derived from NST1 produced cell wall materials that were twice as susceptible to both enzymatic and physicochemical hydrolysis as those from wild-type plants. The yields of glucose from both fresh and dry biomass were increased in the chimeric repressor lines. Use of the NST1 chimeric repressor might enhance production of glucose from plant cell walls, by changing the nature of the cell walls themselves.

  18. High-resolution specificity from DNA sequencing highlights alternative modes of Lac repressor binding.

    PubMed

    Zuo, Zheng; Stormo, Gary D

    2014-11-01

    Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressor-operator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection.

  19. CRTR-1, a developmentally regulated transcriptional repressor related to the CP2 family of transcription factors.

    PubMed

    Rodda, S; Sharma, S; Scherer, M; Chapman, G; Rathjen, P

    2001-02-02

    CP2-related proteins comprise a family of DNA-binding transcription factors that are generally activators of transcription and expressed ubiquitously. We reported a differential display polymerase chain reaction fragment, Psc2, which was expressed in a regulated fashion in mouse pluripotent cells in vitro and in vivo. Here, we report further characterization of the Psc2 cDNA and function. The Psc2 cDNA contained an open reading frame homologous to CP2 family proteins. Regions implicated in DNA binding and oligomeric complex formation, but not transcription activation, were conserved. Psc2 expression in vivo during embryogenesis and in the adult mouse demonstrated tight spatial and temporal regulation, with the highest levels of expression in the epithelial lining of distal convoluted tubules in embryonic and adult kidneys. Functional analysis demonstrated that PSC2 repressed transcription 2.5-15-fold when bound to a heterologous promoter in ES, 293T, and COS-1 cells. The N-terminal 52 amino acids of PSC2 were shown to be necessary and sufficient for this activity and did not share obvious homology with reported repressor motifs. These results represent the first report of a CP2 family member that is expressed in a developmentally regulated fashion in vivo and that acts as a direct repressor of transcription. Accordingly, the protein has been named CP2-Related Transcriptional Repressor-1 (CRTR-1).

  20. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    PubMed

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2016-12-20

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (eg. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated Conserved Sequence-guided Repressor Inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. This article is protected by copyright. All rights reserved.

  1. Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave differently).

    PubMed

    Plumbridge, J

    2001-07-01

    NagC and Mlc are paralogous transcriptional repressors in E.coli. Unexpectedly they possess almost identical amino acid sequences in their helix-turn-helix (H-T-H), DNA binding motif and they bind to very similar consensus operator targets. Binding to each others sites can be demonstrated in vitro but no cross regulation can be detected in vivo with physiological amounts of the two proteins. Although both proteins are involved in regulating the expression of PTS genes, the characteristics of their repression and induction are very different. NagC is a dual-function, activator-repressor which co-ordinates the metabolism of the amino sugars, N-acetylglucosamine (GlcNAc) and glucosamine, by repressing the divergent nagE-BA operons and by activating the glmUS operon. Repression (and activation) by NagC requires that NagC binds simultaneously to two operators, thus forming a DNA loop. This chelation effect allows use of lower affinity sites which would not individually bind the repressor. The specific inducer for NagC is GlcNAc-6-P, the product of GlcNAc transport by the PTS and a key compound in amino sugar metabolism. Mlc represses several genes implicated in the uptake of glucose; ptsG, ptsHI and manXYZ, and malT, the activator of the mal regulon. Glucose behaves like the inducer but growth on glucose only produces an overall increase in expression for ptsG and ptsHI. All Mlc repressed genes are also controlled by cAMP/CAP, so that glucose affects their transcription in two opposing ways: increasing expression by acting as the inducer for Mlc but decreasing expression by lowering the cAMP/CAP concentration. The Mlc protein is not directly responsive to glucose per se but to the activity status of the PTS. Displacement of Mlc from its binding sites occurs during growth on glucose and other PTS sugars and involves sequestration of the repressor to membranes by binding to dephosphorylated PtsG.

  2. Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

    PubMed Central

    Ross, J B; Senear, D F; Waxman, E; Kombo, B B; Rusinova, E; Huang, Y T; Laws, W R; Hasselbacher, C A

    1992-01-01

    We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems. PMID:1465434

  3. Tob1 is a constitutively expressed repressor of liver regeneration

    PubMed Central

    Ho, Karen J.; Do, Nhue L.; Otu, Hasan H.; Dib, Martin J.; Ren, Xianghui; Enjyoji, Keiichi; Robson, Simon C.; Terwilliger, Ernest F.

    2010-01-01

    How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclin-dependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins. PMID:20513747

  4. HDAC9 is an epigenetic repressor of kidney angiotensinogen establishing a sex difference.

    PubMed

    Bourgeois, Camille T; Satou, Ryousuke; Prieto, Minolfa C

    2017-01-01

    Sexual difference has been shown in the pathogenesis of chronic kidney disease induced by hypertension. Females are protected from hypertension and related end-organ damage. Augmentation of renal proximal tubular angiotensinogen (AGT) expression can promote intrarenal angiotensin formation and the development of associated hypertension and kidney injury. Female rodents exhibit lower intrarenal AGT levels than males under normal conditions, suggesting that the suppressed intrarenal AGT production by programmed mechanisms in females may provide protection from these diseases. This study was performed to examine whether epigenetic mechanisms serve as repressors of AGT. Male and female Sprague Dawley rats were used to investigate sex differences of systemic, hepatic, and intrarenal AGT levels. All histone deacetylase (HDAC) mRNA levels in the kidneys were determined using a PCR array. HDAC9 protein expression in the kidneys and cultured renal proximal tubular cells (PTC) was analyzed by Western blot analysis and immunohistochemistry. The effects of HDAC9 on AGT expression were evaluated by using an inhibitor and siRNA. ChIP assay was performed to investigate the interaction between the AGT promoter and HDAC9. Plasma and liver AGT levels did not show differences between male and female Sprague-Dawley rats. In contrast, females exhibited lower AGT levels than males in the renal cortex and urine. In the absence of supplemented sex hormones, primary cultured renal cortical cells isolated from female rats sustained lower AGT levels than those from males, suggesting that the kidneys have a unique mechanism of AGT regulation controlled by epigenetic factors rather than sex hormones. HDAC9 mRNA and protein levels were higher in the renal cortex of female rats versus male rats (7.09 ± 0.88, ratio to male) while other HDACs did not exhibit a sex difference. HDAC9 expression was localized in PTC which are the primary source of intrarenal AGT. Importantly, HDAC9 knockdown

  5. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  6. Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

    PubMed Central

    Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

    2014-01-01

    Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

  7. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    PubMed

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. Copyright © 2011 Wiley Periodicals, Inc.

  8. Human SLFN5 is a transcriptional co-repressor of STAT1-mediated interferon responses and promotes the malignant phenotype in glioblastoma.

    PubMed

    Arslan, A D; Sassano, A; Saleiro, D; Lisowski, P; Kosciuczuk, E M; Fischietti, M; Eckerdt, F; Fish, E N; Platanias, L C

    2017-07-03

    We provide evidence that the IFN-regulated member of the Schlafen (SLFN) family of proteins, SLFN5, promotes the malignant phenotype in glioblastoma multiforme (GBM). Our studies indicate that SLFN5 expression promotes motility and invasiveness of GBM cells, and that high levels of SLFN5 expression correlate with high-grade gliomas and shorter overall survival in patients suffering from GBM. In efforts to uncover the mechanism by which SLFN5 promotes GBM tumorigenesis, we found that this protein is a transcriptional co-repressor of STAT1. Type-I IFN treatment triggers the interaction of STAT1 with SLFN5, and the resulting complex negatively controls STAT1-mediated gene transcription via interferon stimulated response elements. Thus, SLFN5 is both an IFN-stimulated response gene and a repressor of IFN-gene transcription, suggesting the existence of a negative-feedback regulatory loop that may account for suppression of antitumor immune responses in glioblastoma.Oncogene advance online publication, 3 July 2017; doi:10.1038/onc.2017.205.

  9. The chimeric repressor version of an Ethylene Response Factor (ERF) family member, Sl-ERF.B3, shows contrasting effects on tomato fruit ripening.

    PubMed

    Liu, Mingchun; Diretto, Gianfranco; Pirrello, Julien; Roustan, Jean-Paul; Li, Zhengguo; Giuliano, Giovanni; Regad, Farid; Bouzayen, Mondher

    2014-07-01

    Fruit ripening involves a complex interplay between ethylene and ripening-associated transcriptional regulators. Ethylene Response Factors (ERFs) are downstream components of ethylene signaling, known to regulate the expression of ethylene-responsive genes. Although fruit ripening is an ethylene-regulated process, the role of ERFs remains poorly understood. The role of Sl-ERF.B3 in tomato (Solanum lycopersicum) fruit maturation and ripening is addressed here using a chimeric dominant repressor version (ERF.B3-SRDX). Over-expression of ERF.B3-SRDX results in a dramatic delay of the onset of ripening, enhanced climacteric ethylene production and fruit softening, and reduced pigment accumulation. Consistently, genes involved in ethylene biosynthesis and in softening are up-regulated and those of carotenoid biosynthesis are down-regulated. Moreover, the expression of ripening regulators, such as RIN, NOR, CNR and HB-1, is stimulated in ERF.B3-SRDX dominant repressor fruits and the expression pattern of a number of ERFs is severely altered. The data suggest the existence of a complex network enabling interconnection between ERF genes which may account for the pleiotropic alterations in fruit maturation and ripening. Overall, the study sheds new light on the role of Sl-ERF.B3 in the transcriptional network controlling the ripening process and uncovers a means towards uncoupling some of the main ripening-associated processes. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  10. GLIS3, a novel member of the GLIS subfamily of Krüppel-like zinc finger proteins with repressor and activation functions.

    PubMed

    Kim, Yong-Sik; Nakanishi, Gen; Lewandoski, Mark; Jetten, Anton M

    2003-10-01

    In this study, we describe the identification and characterization of a novel transcription factor GLI-similar 3 (GLIS3). GLIS3 is an 83.8 kDa nuclear protein containing five C2H2-type Krüppel-like zinc finger motifs that exhibit 93% identity with those of GLIS1, however, little homology exists outside their zinc finger domains. GLIS3 can function as a repressor and activator of transcription. Deletion mutant analysis determined that the N- and C-termini are required for optimal transcriptional activity. GLIS3 binds to the GLI-RE consensus sequence and is able to enhance GLI-RE-dependent transcription. GLIS3(DeltaC496), a dominant-negative mutant, inhibits transcriptional activation by GLIS3 and GLI1. Whole mount in situ hybridization on mouse embryos from stage E6.5 through E14.5 demonstrated that GLIS3 is expressed in specific regions in developing kidney and testis and in a highly dynamic pattern during neurulation. From E11.5 through E12.5 GLIS3 was strongly expressed in the interdigital regions, which are fated to undergo apoptosis. The temporal and spatial pattern of GLIS3 expression observed during embryonic development suggests that it may play a critical role in the regulation of a variety of cellular processes during development. Both the repressor and activation functions of GLIS3 may be involved in this control.

  11. Yet1p-Yet3p interacts with Scs2p-Opi1p to regulate ER localization of the Opi1p repressor.

    PubMed

    Wilson, Joshua D; Thompson, Sarah L; Barlowe, Charles

    2011-05-01

    Lipid sensing mechanisms at the endoplasmic reticulum (ER) coordinate an array of biosynthetic pathways. A major phospholipid regulatory circuit in yeast is controlled by Scs2p, an ER membrane protein that binds the transcriptional repressor protein Opi1p. Cells grown in the absence of inositol sequester Scs2p-Opi1p at the ER and derepress target genes including INO1. We recently reported that Yet1p and Yet3p, the yeast homologues of BAP29 and BAP31, are required for normal growth in the absence of inositol. Here we show that the Yet1p-Yet3p complex acts in derepression of INO1 through physical association with Scs2p-Opi1p. Yet complex binding to Scs2p-Opi1p was enhanced by inositol starvation, although the interaction between Scs2p and Opi1p was not influenced by YET1 or YET3 deletion. Interestingly, live-cell imaging analysis indicated that Opi1p does not efficiently relocalize to the ER during inositol starvation in yet3Δ cells. Together our data demonstrate that a physical association between the Yet complex and Scs2p-Opi1p is required for proper localization of the Opi1p repressor to ER membranes and subsequent INO1 derepression.

  12. bHLH003, bHLH013 and bHLH017 Are New Targets of JAZ Repressors Negatively Regulating JA Responses

    PubMed Central

    Fonseca, Sandra; Fernández-Calvo, Patricia; Fernández, Guillermo M.; Díez-Díaz, Monica; Gimenez-Ibanez, Selena; López-Vidriero, Irene; Godoy, Marta; Fernández-Barbero, Gemma; Van Leene, Jelle; De Jaeger, Geert; Franco-Zorrilla, José Manuel; Solano, Roberto

    2014-01-01

    Cell reprogramming in response to jasmonates requires a tight control of transcription that is achieved by the activity of JA-related transcription factors (TFs). Among them, MYC2, MYC3 and MYC4 have been described as activators of JA responses. Here we characterized the function of bHLH003, bHLH013 and bHLH017 that conform a phylogenetic clade closely related to MYC2, MYC3 and MYC4. We found that these bHLHs form homo- and heterodimers and also interact with JAZ repressors in vitro and in vivo. Phenotypic analysis of JA-regulated processes, including root and rosette growth, anthocyanin accumulation, chlorophyll loss and resistance to Pseudomonas syringae, on mutants and overexpression lines, suggested that these bHLHs are repressors of JA responses. bHLH003, bHLH013 and bHLH017 are mainly nuclear proteins and bind DNA with similar specificity to that of MYC2, MYC3 and MYC4, but lack a conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Moreover, expression of bHLH017 is induced by JA and depends on MYC2, suggesting a negative feed-back regulation of the activity of positive JA-related TFs. Our results suggest that the competition between positive and negative TFs determines the output of JA-dependent transcriptional activation. PMID:24465948

  13. Mutations in the transcriptional repressor REST predispose to Wilms tumor.

    PubMed

    Mahamdallie, Shazia S; Hanks, Sandra; Karlin, Kristen L; Zachariou, Anna; Perdeaux, Elizabeth R; Ruark, Elise; Shaw, Chad A; Renwick, Alexander; Ramsay, Emma; Yost, Shawn; Elliott, Anna; Birch, Jillian; Capra, Michael; Gray, Juliet; Hale, Juliet; Kingston, Judith; Levitt, Gill; McLean, Thomas; Sheridan, Eamonn; Renwick, Anthony; Seal, Sheila; Stiller, Charles; Sebire, Neil; Westbrook, Thomas F; Rahman, Nazneen

    2015-12-01

    Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor.

  14. Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification.

    PubMed

    Amalraj, Amritha; Luang, Sukanya; Kumar, Manoj Yadav; Sornaraj, Pradeep; Eini, Omid; Kovalchuk, Nataliya; Bazanova, Natalia; Li, Yuan; Yang, Nannan; Eliby, Serik; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2016-02-01

    Plants respond to abiotic stresses by changes in gene regulation, including stress-inducible expression of transcriptional activators and repressors. One of the best characterized families of drought-related transcription factors are dehydration-responsive element binding (DREB) proteins, known as C-repeat binding factors (CBF). The wheat DREB/CBF gene TaRAP2.1L was isolated from drought-affected tissues using a dehydration-responsive element (DRE) as bait in a yeast one-hybrid screen. TaRAP2.1L is induced by elevated abscisic acid, drought and cold. A C-terminal ethylene responsive factor-associated amphiphilic repression (EAR) motif, known to be responsible for active repression of target genes, was identified in the TaRAP2.1L protein. It was found that TaRAP2.1L has a unique selectivity of DNA-binding, which differs from that of DREB activators. This binding selectivity remains unchanged in a TaRAP2.1L variant with an inactivated EAR motif (TaRAP2.1Lmut). To study the role of the TaRAP2.1L repressor activity associated with the EAR motif in planta, transgenic wheat overexpressing native or mutated TaRAP2.1L was generated. Overexpression of TaRAP2.1L under constitutive and stress-inducible promoters in transgenic wheat and barley led to dwarfism and decreased frost tolerance. By contrast, constitutive overexpression of the TaRAP2.1Lmut gene had little or no negative influence on wheat development or grain yield. Transgenic lines with the TaRAP2.1Lmut transgene had an enhanced ability to survive frost and drought. The improved stress tolerance is attributed to up-regulation of several stress-related genes known to be downstream genes of DREB/CBF activators.

  15. Slow assembly and disassembly of lambda Cro repressor dimers.

    PubMed

    Jia, Haifeng; Satumba, W John; Bidwell, Gene L; Mossing, Michael C

    2005-07-29

    Dimers of Cro are required to recognize operator DNA and repress transcription, but dimerization is weak compared to DNA binding. Fluorophore-conjugated, single-cysteine variants of Cro have been used to investigate the equilibria and kinetics of dimer assembly. Equilibrium distributions of mixed dimers, monitored by fluorescence resonance energy transfer (FRET), confirm that labeled variants have equilibrium dimer dissociation constants in the micromolar concentration range. Subunit exchange experiments yield first order rate constants for dimer dissociation that range from 0.02 s(-1) to 0.04 s(-1). Association rate constants calculated from the ratios of dissociation equilibrium and rate constants range from 0.7x10(4) M(-1) s(-1) to 3x10(4) M(-1) s(-1), depending on the site of the fluorescent label. At nanomolar concentrations of subunits, assembly can be driven by addition of DNA. The bimolecular association rate constants measured under these conditions are not dramatically enhanced, ranging from 7x10(4) M(-1) s(-1) to 9x10(4) M(-1) s(-1). The association rate is second order in protein but independent of DNA concentration between 10 nM and 200 nM. The association of subunits under native conditions is more than four orders of magnitude slower than the fast assembly phase measured previously in refolding experiments, and is unaffected by peptidyl-prolyl isomerases. Stabilization of the folded structure of the protein by residue substitution in Cro F58W or reduced temperature increases the ratio of dimers to monomers and decreases the rate of subunit exchange. These data suggest that native monomers have compact structures with substantial barriers to unfolding and that unfolded or partially folded monomers are the preferred substrates for dimer assembly. Cro binding in vivo may be under kinetic rather than thermodynamic control. The slow assembly of Cro dimers demonstrated here provides a new perspective on the lysis/lysogeny switch of bacteriophage lambda.

  16. Regulatory interactions among three members of the vertebrate aryl hydrocarbon receptor family: AHR repressor, AHR1, and AHR2.

    PubMed

    Karchner, Sibel I; Franks, Diana G; Powell, Wade H; Hahn, Mark E

    2002-03-01

    The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds occur via the aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix-Per-ARNT-Sim homology (bHLH-PAS) protein superfamily. A single AHR gene has been identified in mammals, whereas many fish species, including the Atlantic killifish (Fundulus heteroclitus) possess two distinct AHR genes (AHR1 and a novel form, AHR2). A mouse bHLH-PAS protein closely related to AHR and designated AHR repressor (AHRR) is induced by 3-methylcholanthrene and represses the transcriptional activity of the AHR. To determine whether AHRR is the mammalian ortholog of fish AHR2 and to investigate the mechanisms by which AHRR regulates AHR function, we cloned an AHRR ortholog in F. heteroclitus with high sequence identity to the mouse and human AHRRs. Killifish AHRR encodes a 680-residue protein with a predicted molecular mass of 75.2 kDa. We show that in vitro expressed AHRR proteins from human, mouse, and killifish all fail to bind [(3)H]TCDD or [(3)H]beta-naphthoflavone. In transient transfection experiments using a luciferase reporter gene under control of AHR response elements, killifish AHRR inhibited the TCDD-dependent transactivation function of both AHR1 and AHR2. AHRR mRNA is widely expressed in killifish tissues and is inducible by TCDD or polychlorinated biphenyls, but its expression is not altered in a population of fish exhibiting genetic resistance to these compounds. The F. heteroclitus AHRR promoter contains three putative AHR response elements. Both AHR1 and AHR2 activated transcription of luciferase driven by the AHRR promoter, and AHRR could repress its own promoter. Thus, AHRR is an evolutionarily conserved, TCDD-inducible repressor of AHR1 and AHR2 function. Phylogenetic analysis shows that AHRR, AHR1, and AHR2 are distinct genes, members of an AHR gene family; these three vertebrate AHR-like genes descended from a single invertebrate AHR.

  17. The Filamentous Growth MAPK Pathway Responds to Glucose Starvation Through the Mig1/2 Transcriptional Repressors in Saccharomyces cerevisiae

    PubMed Central

    Karunanithi, Sheelarani; Cullen, Paul J.

    2012-01-01

    In the budding yeast S. cerevisiae, nutrient limitation induces a MAPK pathway that regulates filamentous growth and biofilm/mat formation. How nutrient levels feed into the regulation of the filamentous growth pathway is not entirely clear. We characterized a newly identified MAPK regulatory protein of the filamentous growth pathway, Opy2. A two-hybrid screen with the cytosolic domain of Opy2 uncovered new interacting partners including a transcriptional repressor that functions in the AMPK pathway, Mig1, and its close functional homolog, Mig2. Mig1 and Mig2 coregulated the filamentous growth pathway in response to glucose limitation, as did the AMP kinase Snf1. In addition to associating with Opy2, Mig1 and Mig2 interacted with other regulators of the filamentous growth pathway including the cytosolic domain of the signaling mucin Msb2, the MAP kinase kinase Ste7, and the MAP kinase Kss1. As for Opy2, Mig1 overproduction dampened the pheromone response pathway, which implicates Mig1 and Opy2 as potential regulators of pathway specificity. Taken together, our findings provide the first regulatory link in yeast between components of the AMPK pathway and a MAPK pathway that controls cellular differentiation. PMID:22904036

  18. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    PubMed Central

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  19. WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.

    PubMed

    Yokotani, Naoki; Sato, Yuko; Tanabe, Shigeru; Chujo, Tetsuya; Shimizu, Takafumi; Okada, Kazunori; Yamane, Hisakazu; Shimono, Masaki; Sugano, Shoji; Takatsuji, Hiroshi; Kaku, Hisatoshi; Minami, Eiichi; Nishizawa, Yoko

    2013-11-01

    OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

  20. The filamentous growth MAPK Pathway Responds to Glucose Starvation Through the Mig1/2 transcriptional repressors in Saccharomyces cerevisiae.

    PubMed

    Karunanithi, Sheelarani; Cullen, Paul J

    2012-11-01

    In the budding yeast S. cerevisiae, nutrient limitation induces a MAPK pathway that regulates filamentous growth and biofilm/mat formation. How nutrient levels feed into the regulation of the filamentous growth pathway is not entirely clear. We characterized a newly identified MAPK regulatory protein of the filamentous growth pathway, Opy2. A two-hybrid screen with the cytosolic domain of Opy2 uncovered new interacting partners including a transcriptional repressor that functions in the AMPK pathway, Mig1, and its close functional homolog, Mig2. Mig1 and Mig2 coregulated the filamentous growth pathway in response to glucose limitation, as did the AMP kinase Snf1. In addition to associating with Opy2, Mig1 and Mig2 interacted with other regulators of the filamentous growth pathway including the cytosolic domain of the signaling mucin Msb2, the MAP kinase kinase Ste7, and the MAP kinase Kss1. As for Opy2, Mig1 overproduction dampened the pheromone response pathway, which implicates Mig1 and Opy2 as potential regulators of pathway specificity. Taken together, our findings provide the first regulatory link in yeast between components of the AMPK pathway and a MAPK pathway that controls cellular differentiation.

  1. Activator and repressor functions of the Mot3 transcription factor in the osmostress response of Saccharomyces cerevisiae.

    PubMed

    Martínez-Montañés, Fernando; Rienzo, Alessandro; Poveda-Huertes, Daniel; Pascual-Ahuir, Amparo; Proft, Markus

    2013-05-01

    Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Furthermore, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.

  2. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    PubMed

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  3. Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

    PubMed Central

    Buchberger, Elisabeth; El Harchi, Miriam; Payrhuber, Dietmar; Zommer, Anna; Schauer, Dominic; Simonitsch-Klupp, Ingrid; Bilban, Martin; Brostjan, Christine

    2013-01-01

    Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones. PMID:24146931

  4. Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

    PubMed Central

    Zhu, Li-Wen; Xia, Shi-Tao; Wei, Li-Na; Li, Hong-Mei; Yuan, Zhan-Peng; Tang, Ya-Jie

    2016-01-01

    This study was initiated to improve E. coli succinate production by engineering the E. coli global transcription factor, Cra (catabolite repressor/activator). Random mutagenesis libraries were generated through error-prone PCR of cra. After re-screening and mutation site integration, the best mutant strain was Tang1541, which provided a final succinate concentration of 79.8 ± 3.1 g/L: i.e., 22.8% greater than that obtained using an empty vector control. The genes and enzymes involved in phosphoenolpyruvate (PEP) carboxylation and the glyoxylate pathway were activated, either directly or indirectly, through the mutation of Cra. The parameters for interaction of Cra and DNA indicated that the Cra mutant was bound to aceBAK, thereby activating the genes involved in glyoxylate pathway and further improving succinate production even in the presence of its effector fructose-1,6-bisphosphate (FBP). It suggested that some of the negative effect of FBP on Cra might have been counteracted through the enhanced binding affinity of the Cra mutant for FBP or the change of Cra structure. This work provides useful information about understanding the transcriptional regulation of succinate biosynthesis. PMID:27811970

  5. Independent and interactive effects of DOF affecting germination 1 (DAG1) and the Della proteins GA insensitive (GAI) and Repressor of ga1-3 (RGA) in embryo development and seed germination.

    PubMed

    Boccaccini, Alessandra; Santopolo, Silvia; Capauto, Davide; Lorrai, Riccardo; Minutello, Emanuele; Belcram, Katia; Palauqui, Jean-Cristophe; Costantino, Paolo; Vittorioso, Paola

    2014-07-26

    The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.

  6. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

  7. CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

    PubMed Central

    Zhu, Quan; Gregg, Keqin; Lozano, Mary; Liu, Jinqi; Dudley, Jaquelin P.

    2000-01-01

    Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland. PMID:10864645

  8. Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors

    PubMed Central

    Ke, Jiyuan; Ma, Honglei; Gu, Xin; Thelen, Adam; Brunzelle, Joseph S.; Li, Jiayang; Xu, H. Eric; Melcher, Karsten

    2015-01-01

    TOPLESS (TPL) and TOPLESS-related (TPR) proteins comprise a conserved family of plant transcriptional corepressors that are related to Tup1, Groucho, and TLE (transducin-like enhancer of split) corepressors in yeast, insects, and mammals. In plants, TPL/TPR corepressors regulate development, stress responses, and hormone signaling through interaction with small ethylene response factor–associated amphiphilic repression (EAR) motifs found in diverse transcriptional repressors. How EAR motifs can interact with TPL/TPR proteins is unknown. We confirm the amino-terminal domain of the TPL family of corepressors, which we term TOPLESS domain (TPD), as the EAR motif–binding domain. To understand the structural basis of this interaction, we determined the crystal structures of the TPD of rice (Os) TPR2 in apo (apo protein) state and in complexes with the EAR motifs from Arabidopsis NINJA (novel interactor of JAZ), IAA1 (auxin-responsive protein 1), and IAA10, key transcriptional repressors involved in jasmonate and auxin signaling. The OsTPR2 TPD adopts a new fold of nine helices, followed by a zinc finger, which are arranged into a disc-like tetramer. The EAR motifs in the three different complexes adopt a similar extended conformation with the hydrophobic residues fitting into the same surface groove of each OsTPR2 monomer. Sequence alignments and structure-based mutagenesis indicate that this mode of corepressor binding is highly conserved in a large set of transcriptional repressors, thus providing a general mechanism for gene repression mediated by the TPL family of corepressors. PMID:26601214

  9. Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors.

    PubMed

    Ke, Jiyuan; Ma, Honglei; Gu, Xin; Thelen, Adam; Brunzelle, Joseph S; Li, Jiayang; Xu, H Eric; Melcher, Karsten

    2015-07-01

    TOPLESS (TPL) and TOPLESS-related (TPR) proteins comprise a conserved family of plant transcriptional corepressors that are related to Tup1, Groucho, and TLE (transducin-like enhancer of split) corepressors in yeast, insects, and mammals. In plants, TPL/TPR corepressors regulate development, stress responses, and hormone signaling through interaction with small ethylene response factor-associated amphiphilic repression (EAR) motifs found in diverse transcriptional repressors. How EAR motifs can interact with TPL/TPR proteins is unknown. We confirm the amino-terminal domain of the TPL family of corepressors, which we term TOPLESS domain (TPD), as the EAR motif-binding domain. To understand the structural basis of this interaction, we determined the crystal structures of the TPD of rice (Os) TPR2 in apo (apo protein) state and in complexes with the EAR motifs from Arabidopsis NINJA (novel interactor of JAZ), IAA1 (auxin-responsive protein 1), and IAA10, key transcriptional repressors involved in jasmonate and auxin signaling. The OsTPR2 TPD adopts a new fold of nine helices, followed by a zinc finger, which are arranged into a disc-like tetramer. The EAR motifs in the three different complexes adopt a similar extended conformation with the hydrophobic residues fitting into the same surface groove of each OsTPR2 monomer. Sequence alignments and structure-based mutagenesis indicate that this mode of corepressor binding is highly conserved in a large set of transcriptional repressors, thus providing a general mechanism for gene repression mediated by the TPL family of corepressors.

  10. Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

    PubMed

    Schiering, N; Tao, X; Murphy, J R; Petsko, G A; Ringe, D

    1994-12-16

    Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

  11. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses

    PubMed Central

    He, Jun-Xian; Gendron, Joshua M.; Sun, Yu; Gampala, Srinivas S. L.; Gendron, Nathan; Sun, Catherine Qing; Wang, Zhi-Yong

    2010-01-01

    Brassinosteroid (BR) homeostasis and signaling are crucial for normal growth and development of plants. BR signaling through cell-surface receptor kinases and intracellular components leads to dephosphorylation and accumulation of the nuclear protein BZR1. How BR signaling regulates gene expression, however, remains unknown. Here we show that BZR1 is a transcriptional repressor that has a previously unknown DNA binding domain and binds directly to the promoters of feedback-regulated BR biosynthetic genes. Microarray analyses identified additional potential targets of BZR1 and illustrated, together with physiological studies, that BZR1 coordinates BR homeostasis and signaling by playing dual roles in regulating BR biosynthesis and downstream growth responses. PMID:15681342

  12. SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

    PubMed Central

    Escudero, Jose Antonio; San Millan, Alvaro; Montero, Natalia; Gutierrez, Belen; Ovejero, Cristina Martinez; Carrilero, Laura

    2013-01-01

    Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones. PMID:23650171

  13. Cold denaturation of a repressor-operator complex: the role of entropy in protein-DNA recognition.

    PubMed

    Foguel, D; Silva, J L

    1994-08-16

    The mechanisms by which regulatory proteins recognize specific DNA sequences are not fully understood. Here we examine the basis for the stability of a protein-DNA complex, using hydrostatic pressure and low temperature. Pressure converts the DNA-binding Arc repressor protein from a native state to a denatured, molten-globule state. Our data show that the folding and dimerization of Arc repressor in the temperature range 0-20 degrees C are favored by a large positive entropy value, so that the reaction proceeds in spite of an unfavorable positive enthalpy. On binding operator DNA, Arc repressor becomes extremely stable against denaturation. However, the Arc repressor-operator DNA complex is cold-denatured at subzero temperatures under pressure, demonstrating that the favorable entropy increases greatly when Arc repressor binds tightly to its operator sequence but not a nonspecific sequence. We show how an increase in entropy may operate to provide the protein with a mechanism to distinguish between a specific and a nonspecific DNA sequence. It is postulated that the formation of the Arc-operator DNA complex is followed by an increase in apolar interactions and release of solvent which would explain its entropy-driven character, whereas this solvent would not be displaced in nonspecific complexes.

  14. Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

    SciTech Connect

    Safo, Martin K.; Ko, Tzu-Ping; Musayev, Faik N.; Zhao, Qixun; Archer, Gordon L.

    2006-04-01

    The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor. The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.

  15. A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

    PubMed Central

    León, Esther; Navarro-Avilés, Gloria; Santiveri, Clara M.; Flores-Flores, Cesar; Rico, Manuel; González, Carlos; Murillo, Francisco J.; Elías-Arnanz, Montserrat; Jiménez, María Angeles; Padmanabhan, S.

    2010-01-01

    Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription. PMID:20410074

  16. The Reduction of R1, a Novel Repressor Protein for Monoamine Oxidase A, in Major Depressive Disorder

    PubMed Central

    Johnson, Shakevia; Stockmeier, Craig A; Meyer, Jeffrey H; Austin, Mark C; Albert, Paul R; Wang, Junming; May, Warren L; Rajkowska, Grazyna; Overholser, James C; Jurjus, George; Dieter, Lesa; Johnson, Chandra; Sittman, Donald B; Ou, Xiao-Ming

    2011-01-01

    The novel transcriptional repressor protein, R1 (JPO2/CDCA7L/RAM2), inhibits monoamine oxidase A (MAO A) gene expression and influences cell proliferation and survival. MAO A is implicated in several neuropsychiatric illnesses and highly elevated in major depressive disorder (MDD); however, whether R1 is involved in these disorders is unknown. This study evaluates the role of R1 in depressed subjects either untreated or treated with antidepressant drugs. R1 protein levels were determined in the postmortem prefrontal cortex of 18 untreated MDD subjects and 12 medicated MDD subjects compared with 18 matched psychiatrically normal control subjects. Western blot analysis showed that R1 was significantly decreased by 37.5% (p<0.005) in untreated MDD subjects. The R1 level in medicated MDD subjects was also significantly lower (by 30% p<0.05) compared with control subjects, but was not significantly different compared with untreated MDD subjects. Interestingly, the reduction in R1 was significantly correlated with an increase (approximately 40% p<0.05) in MAO A protein levels within the MDD groups compared with controls. Consistent with the change in MAO A protein expression, the MAO A catalytic activity was significantly greater in both MDD groups compared with controls. These results suggest that reduced R1 may lead to elevated MAO A levels in untreated and treated MDD subjects; moreover, the reduction of R1 has been implicated in apoptotic cell death and apoptosis has also been observed in the brains of MDD subjects. Therefore, modulation of R1 levels may provide a new therapeutic target in the development of more effective strategies to treat MDD. PMID:21654740

  17. The Arabidopsis PR-1 Promoter Contains Multiple Integration Sites for the Coactivator NPR1 and the Repressor SNI1[W

    PubMed Central

    Pape, Sebastian; Thurow, Corinna; Gatz, Christiane

    2010-01-01

    Systemic acquired resistance is a broad-spectrum plant immune response involving massive transcriptional reprogramming. The Arabidopsis (Arabidopsis thaliana) PATHOGENESIS-RELATED-1 (PR-1) gene has been used in numerous studies to elucidate transcriptional control mechanisms regulating systemic acquired resistance. WRKY transcription factors and basic leucine zipper proteins of the TGA family regulate the PR-1 promoter by binding to specific cis-elements. In addition, the promoter is under the control of two proteins that do not directly contact the DNA: the positive regulator NONEXPRESSOR OF PR GENES1 (NPR1), which physically interacts with TGA factors, and the repressor SUPPRESSOR OF NPR1, INDUCIBLE1 (SNI1). In this study, we analyzed the importance of the TGA-binding sites LS5 and LS7 and the WKRY box LS4 for regulation by NPR1 and SNI1. In the absence of LS5 and LS7, NPR1 activates the PR-1 promoter through a mechanism that requires LS4. Since transcriptional activation of WRKY genes is under the control of NPR1 and since LS4 is not sufficient for the activation of a truncated PR-1 promoter by the effector protein NPR1-VP16 in transient assays, it is concluded that the LS4-dependent activation of the PR-1 promoter is indirect. In the case of NPR1 acting directly through TGA factors at its target promoters, two TGA-binding sites are necessary but not sufficient for NPR1 function in transgenic plants and in the NPR-VP16-based trans-activation assay in protoplasts. SNI1 exerts its negative effect in the noninduced state by targeting unknown proteins associated with sequences between bp −816 and −573. Under induced conditions, SNI1 negatively regulates the function of WRKY transcription factors binding to WKRY boxes between bp −550 and −510. PMID:20935179

  18. Overexpression of the chimeric gene of the floral regulator CONSTANS and the EAR motif repressor causes late flowering in Arabidopsis.

    PubMed

    Takase, Tomoyuki; Yasuhara, Masahiro; Geekiyanage, Sudarshanee; Ogura, Yasunobu; Kiyosue, Tomohiro

    2007-06-01

    The transcription factor CONSTANS (CO) plays a central role in the photoperiod pathway by integrating the circadian clock and light signals into a control for flowering time. CO induces flowering locus T (FT) and suppressor of overexpression of CO 1 (SOC1) expression, and thereby promotes flowering. The ethylene-responsive element-binding factor associated amphiphilic repression (EAR) motif was used to construct a CONSTANS-EAR motif repressor gene (CO-Rep), which was overexpressed in Arabidopsis under the control of the Cauliflower mosaic virus 35S promoter in order to test its potential for flowering time regulation under inductive long day conditions. Morphological abnormalities in the root and cotyledon formation, and dwarfness were frequently seen in the transgenic plants, suggesting that the proper timing, location, and/or level of CO-Rep expression are important for its application. In morphologically normal CO-Rep plants, both bolting and flowering times under inductive long day conditions were twofold greater than in controls. As a result of the delay in flowering, rosette leaf number at bolting, and rosette and cauline leaf number at flowering increased significantly in CO-Rep plants. RT-PCR analysis demonstrated that FT expression was greatly reduced in the CO-Rep plants, while endogenous CO and SOC1 expression levels were not markedly affected. Conservation of CO among a diverse range of plant species, and its involvement in a variety of photoperiodic responses including flowering, suggests a high potential for use of CO-Rep to manipulate such responses in an agronomically desirable manner.

  19. Cell cycle-related transformation of the E2F4-p130 repressor complex

    SciTech Connect

    Popov, Boris . E-mail: popov_478@hotmail.com; Chang, L.-S.; Serikov, Vladimir

    2005-10-28

    During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.

  20. ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

    PubMed

    Schuh, Anna H; Tipping, Alex J; Clark, Allison J; Hamlett, Isla; Guyot, Boris; Iborra, Francesco J; Rodriguez, Patrick; Strouboulis, John; Enver, Tariq; Vyas, Paresh; Porcher, Catherine

    2005-12-01

    Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

  1. Co-localization of a novel transcriptional repressor simiRP58 with RP58.

    PubMed

    Takahashi, Akiyo; Hirai, Shinobu; Ohtaka-Maruyama, Chiaki; Miwa, Akiko; Hata, Yutaka; Okabe, Shigeo; Okado, Haruo

    2008-04-11

    We have cloned a novel transcriptional repressor protein, termed simiRP58, which has high homology to RP58. Both simiRP58 and RP58 belong to the POZ domain and Kruppel Zn finger (POK) family of proteins. Using the luciferase assay system, we found that simiRP58 also has transcriptional repressor activity like RP58. Northern blotting and quantitative RT-PCR showed that simiRP58 was expressed in testes at the highest level. In situ hybridization of testes showed that simiRP58 is expressed by spermatocytes in only a portion of the seminiferous tubules. In contrast, expression of RP58 by spermatocytes was ubiquitous in all seminiferous tubules. Using COS-7 cells, we observed that simiRP58 was localized in the cytoplasm, which is in contrast to RP58 that was localized in the nucleus. Interestingly, co-transfection with simiRP58 and RP58 induced changes in the localization patterns of both proteins.

  2. Tetracycline-Tet Repressor Binding Specificity: Insights from Experiments and Simulations

    PubMed Central

    Aleksandrov, Alexey; Schuldt, Linda; Hinrichs, Winfried; Simonson, Thomas

    2009-01-01

    Abstract Tetracycline (Tc) antibiotics have been put to new uses in the construction of artificial gene regulation systems, where they bind to the Tet repressor protein (TetR) and modulate its affinity for DNA. Many Tc variants have been produced, both to overcome bacterial resistance and to achieve a broad range of binding strengths. To better understand TetR-Tc binding, we investigate a library of 16 tetracyclines, using fluorescence experiments and molecular dynamics free energy simulations (MDFE). The relative TetR binding free energies are computed by reversibly transforming one Tc variant into another during the simulation, with no adjustable parameters. The chemical variations involve polar and nonpolar substitutions along one entire edge of the elongated Tc structure, which provides many of the protein-ligand contacts. The binding constants span five orders of magnitude. The simulations reproduce the experimental binding free energies, when available, within the uncertainty of either method (±0.5 kcal/mol), and reveal many additional details. Contributions of individual Tc substituents are evaluated, along with their additivity and transferability among different positions on the Tc scaffold; differences between D- and B-class repressors are quantified. With increasing computer power, the MDFE approach provides an attractive complement to experiment and should play an increasing role in the understanding and engineering of protein-ligand recognition. PMID:19917238

  3. CO-REPRESSOR CBFA2T2 REGULATES PLURIPOTENCY AND GERMLINE DEVELOPMENT

    PubMed Central

    Tu, Shengjiang; Narendra, Varun; Yamaji, Masashi; Vidal, Simon E; Rojas, Luis Alejandro; Wang, Xiaoshi; Kim, Sang Yong; Garcia, Benjamin A; Tuschl, Thomas; Stadtfeld, Matthias; Reinberg, Danny

    2016-01-01

    SUMMARY Developmental specification of germ cells lies at the core of inheritance as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)1,2, precursors of sex specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own “latent” form of pluripotency. For example, PGCs express a number of transcription factors (TFs) in common with embryonic stem cells (ESCs), including OCT4, SOX2, NANOG and PRDM142–4. A biochemical mechanism by which these TFs converge on chromatin to produce the dramatic rearrangements underlying ESC- and PGC-specific transcriptional programs remains poorly understood. Here, we discover a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification. Cbfa2t2−/− mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional “passenger” role of a co-repressor, CBFA2T2 functions synergistically with TFs at the crossroads of the fundamental developmental plasticity between uni- and pluripotency PMID:27281218

  4. Inhibitory Role of Inducible cAMP Early Repressor (ICER) in Methamphetamine-Induced Locomotor Sensitization

    PubMed Central

    Han, Wenhua; Takamatsu, Yukio; Yamamoto, Hideko; Kasai, Shinya; Endo, Shogo; Shirao, Tomoaki; Kojima, Nobuhiko; Ikeda, Kazutaka

    2011-01-01

    Background The inducible cyclic adenosine monophosphate (cAMP) early repressor (ICER) is highly expressed in the central nervous system and functions as a repressor of cAMP response element-binding protein (CREB) transcription. The present study sought to clarify the role of ICER in the effects of methamphetamine (METH). Methods and Findings We tested METH-induced locomotor sensitization in wildtype mice, ICER knockout mice, and ICER I-overexpressing mice. Both ICER wildtype mice and knockout mice displayed increased locomotor activity after continuous injections of METH. However, ICER knockout mice displayed a tendency toward higher locomotor activity compared with wildtype mice, although no significant difference was observed between the two genotypes. Moreover, compared with wildtype mice, ICER I-overexpressing mice displayed a significant decrease in METH-induced locomotor sensitization. Furthermore, Western blot analysis and quantitative real-time reverse transcription polymerase chain reaction demonstrated that ICER overexpression abolished the METH-induced increase in CREB expression and repressed cocaine- and amphetamine-regulated transcript (CART) and prodynorphin (Pdyn) expression in mice. The decreased CART and Pdyn mRNA expression levels in vivo may underlie the inhibitory role of ICER in METH-induced locomotor sensitization. Conclusions Our data suggest that ICER plays an inhibitory role in METH-induced locomotor sensitization. PMID:21738744

  5. ADNP Is a Therapeutically Inducible Repressor of WNT Signaling in Colorectal Cancer.

    PubMed

    Blaj, Cristina; Bringmann, Agnes; Schmidt, Eva Marina; Urbischek, Manuela; Lamprecht, Sebastian; Fröhlich, Thomas; Arnold, Georg J; Krebs, Stefan; Blum, Helmut; Hermeking, Heiko; Jung, Andreas; Kirchner, Thomas; Horst, David

    2017-06-01

    Purpose: Constitutively active WNT signaling is a hallmark of colorectal cancers and a driver of malignant tumor progression. Therapeutic targeting of WNT signaling is difficult due to high pathway complexity and its role in tissue homeostasis. Here, we identify the transcription factor ADNP as a pharmacologically inducible repressor of WNT signaling in colon cancer.Experimental Design: We used transcriptomic, proteomic, and in situ analyses to identify ADNP expression in colorectal cancer and cell biology approaches to determine its function. We induced ADNP expression in colon cancer xenografts by low-dose ketamine in vivo Clinical associations were determined in a cohort of 221 human colorectal cancer cases.Results: ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Silencing ADNP expression increased migration, invasion, and proliferation of colon cancer cells and accelerated tumor growth in xenografts in vivo Treatment with subnarcotic doses of ketamine induced ADNP expression, significantly inhibited tumor growth, and prolonged survival of tumor-bearing animals. In human patients with colon cancer, high ADNP expression was linked to good prognosis.Conclusions: Our findings indicate that ADNP is a tumor suppressor and promising prognostic marker, and that ketamine treatment with ADNP induction is a potential therapeutic approach that may add benefit to current treatment protocols for patients with colorectal cancer. Clin Cancer Res; 23(11); 2769-80. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. DAX1 suppresses FXR transactivity as a novel co-repressor

    SciTech Connect

    Li, Jin; Lu, Yan; Liu, Ruya; Xiong, Xuelian; Zhang, Zhijian; Zhang, Xianfeng; Ning, Guang; Li, Xiaoying

    2011-09-09

    Highlights: {yields} DAX1 is co-localized with FXR and interacts with FXR. {yields} DAX1 acts as a negative regulator of FXR. {yields} Three LXXLL motifs in the N-terminus of DAX1 were required. {yields} DAX1 suppresses FXR transactivation by competing with co-activators. -- Abstract: Bile acid receptor FXR (farnesoid X receptor) is a key regulator of hepatic bile acid, glucose and lipid homeostasis through regulation of numerous genes involved in the process of bile acid, triglyceride and glucose metabolism. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains and acts primarily as a co-repressor of many nuclear receptors. Here, we demonstrated that DAX1 is co-localized with FXR in the nucleus and acted as a negative regulator of FXR through a physical interaction with FXR. Our study showed that over-expression of DAX1 down-regulated the expression of FXR target genes, whereas knockdown of DAX1 led to their up-regulation. Furthermore, three LXXLL motifs in the N-terminus of DAX1 were required for the full repression of FXR transactivation. In addition, our study characterized that DAX1 suppresses FXR transactivation via competing with co-activators such as SRC-1 and PGC-1{alpha}. In conclusion, DAX1 acts as a co-repressor to negatively modulate FXR transactivity.

  7. Crystal Structure of the CLOCK Transactivation Domain Exon19 in Complex with a Repressor.

    PubMed

    Hou, Zhiqiang; Su, Lijing; Pei, Jimin; Grishin, Nick V; Zhang, Hong

    2017-08-01

    In the canonical clock model, CLOCK:BMAL1-mediated transcriptional activation is feedback regulated by its repressors CRY and PER and, in association with other coregulators, ultimately generates oscillatory gene expression patterns. How CLOCK:BMAL1 interacts with coregulator(s) is not well understood. Here we report the crystal structures of the mouse CLOCK transactivating domain Exon19 in complex with CIPC, a potent circadian repressor that functions independently of CRY and PER. The Exon19:CIPC complex adopts a three-helical coiled-coil bundle conformation containing two Exon19 helices and one CIPC. Unique to Exon19:CIPC, three highly conserved polar residues, Asn341 of CIPC and Gln544 of the two Exon19 helices, are located at the mid-section of the coiled-coil bundle interior and form hydrogen bonds with each other. Combining results from protein database search, sequence analysis, and mutagenesis studies, we discovered for the first time that CLOCK Exon19:CIPC interaction is a conserved transcription regulatory mechanism among mammals, fish, flies, and other invertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Heat shock factor-4 (HSF-4a) is a repressor of HSF-1 mediated transcription.

    PubMed

    Zhang, Y; Frejtag, W; Dai, R; Mivechi, N F

    2001-01-01

    Heat shock transcription factors (HSFs) regulate the expression of heat shock proteins and other molecular chaperones that are involved in cellular processes from higher order assembly to protein degradation and apoptosis. Among the human HSFs, HSF-4 is expressed as at least two splice variants. One isoform (HSF-4b) possesses a transcriptional activation domain, but this region is absent in the other isoform (HSF-4a). We have recently shown that the HSF-4a isoform represses basal transcription from heterologous promoters both in vitro and in vivo. Here we show that HSF-4a and HSF-4b have dramatically different effects on HSF-1-containing nuclear bodies, which form after heat shock. While the expression of HSF-4b colocalizes with nuclear granules, the expression of HSF-4a prevents their formation. In addition, there is a concurrent reduction of HSF-1 in the nucleus, and there is reduction in its DNA binding activity and in HSE-dependent transcription of a reporter gene. To better understand the mechanism by which HSF-4a represses transcription, we inducibly expressed HSF-4a in cells and found that HSF-4a binds to the heat shock element (HSE) during attenuation of the heat shock response. Thus HSF-4a is an active repressor of HSF-1-mediated transcription. This repressor function makes the HSF-4a isoform unique within the HSF family.

  9. The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor.

    PubMed

    Tang, Tracy Tzu-Ling; Dowbenko, Donald; Jackson, Amanda; Toney, Lisa; Lewin, David A; Dent, Alexander L; Lasky, Laurence A

    2002-04-19

    The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated approximately 4-7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X(L), an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X(L) levels in AFX-expressing cells revealed enhanced down-regulation of the transcript ( approximately 1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X(L) promoter. Finally, macrophages isolated from BCL-6-/- mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.

  10. Transcriptional co-repressor function of the hippo pathway transducers YAP and TAZ.

    PubMed

    Kim, Minchul; Kim, Taekhoon; Johnson, Randy L; Lim, Dae-Sik

    2015-04-14

    YAP (yes-associated protein) and TAZ are oncogenic transcriptional co-activators downstream of the Hippo tumor-suppressor pathway. However, whether YAP and/or TAZ (YAP/TAZ) engage in transcriptional co-repression remains relatively unexplored. Here, we directly demonstrated that YAP/TAZ represses numerous target genes, including tumor-suppressor genes such as DDIT4 (DNA-damage-inducible transcript 4) and Trail (TNF-related apoptosis-inducing ligand). Mechanistically, the repressor function of YAP/TAZ requires TEAD (TEA domain) transcription factors. A YAP/TAZ-TEAD complex recruits the NuRD complex to deacetylate histones and alters nucleosome occupancy at target genes. Functionally, repression of DDIT4 and Trail by YAP/TAZ is required for mTORC1 (mechanistic target of rapamycin complex 1) activation and cell survival, respectively. Our demonstration of the transcriptional co-repressor activity of YAP/TAZ opens a new avenue for understanding the Hippo signaling pathway. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. A Transcriptional Repressor ZBTB1 Promotes Chromatin Remodeling and Translesion DNA Synthesis

    PubMed Central

    Kim, Hyungjin; Dejsuphong, Donniphat; Adelmant, Guillaume; Ceccaldi, Raphael; Yang, Kailin; Marto, Jarrod A.; D’Andrea, Alan D.

    2014-01-01

    SUMMARY Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for PCNA monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, we show that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for PCNA monoubiquitination, by localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a new ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair. PMID:24657165

  12. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation.

    PubMed Central

    Sheldon, C C; Burn, J E; Perez, P P; Metzger, J; Edwards, J A; Peacock, W J; Dennis, E S

    1999-01-01

    A MADS box gene, FLF (for FLOWERING LOCUS F ), isolated from a late-flowering, T-DNA-tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis. PMID:10072403

  13. Cif is negatively regulated by the TetR family repressor CifR.

    PubMed

    MacEachran, Daniel P; Stanton, Bruce A; O'Toole, George A

    2008-07-01

    We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

  14. Cif Is Negatively Regulated by the TetR Family Repressor CifR▿

    PubMed Central

    MacEachran, Daniel P.; Stanton, Bruce A.; O'Toole, George A.

    2008-01-01

    We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene. PMID:18458065

  15. cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

    PubMed Central

    Bodor, J; Spetz, A L; Strominger, J L; Habener, J F

    1996-01-01

    Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I. Images Fig. 3 Fig. 4 Fig. 5 PMID:8622971

  16. Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

    SciTech Connect

    Cherney, L.T.; Cherney, M.M.; Garen, C.R.; Lu, G.J.; James, M.N.G.

    2009-05-12

    The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

  17. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  18. Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor

    PubMed Central

    Brett, Paul J; Burtnick, Mary N; Fenno, J Christopher; Gherardini, Frank C

    2008-01-01

    Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a σ70-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroRTp) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroRTd) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroRTd is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroRTd possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroRTd. Based upon these findings, we propose that TroRTd represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete. PMID:18761626

  19. Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor.

    PubMed

    Brett, Paul J; Burtnick, Mary N; Fenno, J Christopher; Gherardini, Frank C

    2008-10-01

    Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a sigma(70)-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR(Tp)) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR(Td) is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td). Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.

  20. Decreased sucrase and lactase activity in iron deficiency is accompanied by reduced gene expression and upregulation of the transcriptional repressor PDX-1.

    PubMed

    West, Adrian R; Oates, Phillip S

    2005-12-01

    Disaccharidases are important digestive enzymes whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression, either by impairment to enterocyte differentiation or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron-deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested as well as the activity of intestinal alkaline phosphatase (IAP)-II because it is unrelated to carbohydrate digestion. mRNA levels of beta-actin, sucrase, lactase, and the associated transcription factors pancreatic duodenal homeobox (PDX)-1, caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridization, respectively. It was found that iron-deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower) but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9%, respectively, by iron deficiency, suggesting that enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5-fold higher in iron deficiency, whereas CDX-2, GATA-4, and HNF-1 levels were not significantly different. These data suggest that decreases in sucrase and lactase activities result from a reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.

  1. A general strategy for the production of difficult-to-express inducer-dependent bacterial repressor proteins in Escherichia coli.

    PubMed

    Christen, Erik H; Karlsson, Maria; Kämpf, Michael M; Weber, Cornelia C; Fussenegger, Martin; Weber, Wilfried

    2009-08-01

    Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As(3+), respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class.

  2. Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

    PubMed

    Spotts, R O; Chakerian, A E; Matthews, K S

    1991-12-05

    Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the

  3. Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

    PubMed Central

    Plumbridge, J

    1995-01-01

    The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS). Images PMID:7545108

  4. The activation domain of a basic helix-loop-helix protein is masked by repressor interaction with domains distinct from that required for transcription regulation.

    PubMed Central

    Jayaraman, P S; Hirst, K; Goding, C R

    1994-01-01

    While there are many examples of protein-protein interactions modulating the DNA-binding activity of transcription factors, little is known of the molecular mechanisms underlying the regulation of the transcription activation function. Using a two-hybrid system we show here that transcription repression of the basic domain/helix-loop-helix factor PHO4 is mediated by complex formation with the PHO80 repressor. In contrast to other systems, such as inhibition of GAL4 by GAL80 or of p53 by MDM2, where repression is mediated by direct interaction at regions overlapping the transcription activation domain, interaction with PHO80 involves two regions of PHO4 distinct from those involved in transcription activation or DNA-binding and dimerization. The possibility that repression of PHO4 by PHO80 may represent a general mechanism of transcription control, including regulation of the cell-type-specific transcription activation domain of c-Jun, is discussed. Images PMID:8187772

  5. Structure-based functional characterization of repressor of toxin (Rot), a central regulator of staphylococcus aureus virulence

    DOE PAGES

    Killikelly, April; Jakoncic, Jean; Benson, Meredith A.; ...

    2014-10-20

    Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure ofmore » Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R91, at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y66, predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. As a result, this work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.« less

  6. A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus

    PubMed Central

    Yu, Liansheng; Hisatsune, Junzo; Hayashi, Ikue; Tatsukawa, Nobuyuki; Sato’o, Yusuke; Mizumachi, Emiri; Kato, Fuminori; Hirakawa, Hideki; Pier, Gerald B.

    2017-01-01

    ABSTRACT Staphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148–159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus. PMID:28143981

  7. Water-mediated contacts in the trp-repressor operator complex recognition process.

    PubMed

    Wibowo, Fajar R; Rauch, Christine; Trieb, Michael; Wellenzohn, Bernd; Liedl, Klaus R

    2004-04-15

    Water-mediated contacts are known as an important recognition tool in trp-repressor operator systems. One of these contacts involves two conserved base pairs (G(6).C(-6) and A(5). T(-5)) and three amino acids (Lys 72, Ile 79, and Ala 80). To investigate the nature of these contacts, we analyzed the X-ray structure (PDB code: 1TRO) of the trp-repressor operator complex by means of molecular dynamics simulations. This X-ray structure contains two dimers that exhibit structural differences. From these two different starting structures, two 10 ns molecular dynamics simulations have been performed. Both of our simulations show an increase of water molecules in the major groove at one side of the dimer, while the other side remains unchanged compared to the X-ray structure. Though the maximum residence time of the concerned water molecules decreases with an increase of solvent at the interface, these water molecules continue to play an important role in mediating DNA-protein contacts. This is shown by new stable amino acids-DNA distances and a long water residence time compared to free DNA simulation. To maintain stability of the new contacts, the preferential water binding site on O6(G6) is extended. This extension agrees with mutation experiment data on A5 and G6, which shows different relative affinity due to mutation on these bases [A. Joachimiak, T. E. Haran, P. B. Sigler, EMBO Journal 1994, Vol. 13, No. (2) pp. 367-372]. Due to the rearrangements in the system, the phosphate of the base G6 is able to interconvert to the B(II) substate, which is not observed on the other half side of the complex. The decrease of the number of hydrogen bonds between protein and DNA backbone could be the initial step of the dissociation process of the complex, or in other words an intermediate complex conformation of the association process. Thus, we surmise that these features show the importance of water-mediated contacts in the trp-repressor operator recognition process.

  8. The histone-like nucleoid structuring protein (H-NS) is a repressor of Vibrio cholerae exopolysaccharide biosynthesis (vps) genes.

    PubMed

    Wang, Hongxia; Ayala, Julio C; Silva, Anisia J; Benitez, Jorge A

    2012-04-01

    The capacity of Vibrio cholerae to form biofilms has been shown to enhance its survival in the aquatic environment and play important roles in pathogenesis and disease transmission. In this study, we demonstrated that the histone-like nucleoid structuring protein is a repressor of exopolysaccharide (vps) biosynthesis genes and biofilm formation.

  9. Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

    SciTech Connect

    Safo,M.; Ko, T.; Musayev, F.; Zhao, Q.; Wang, A.; Archer, G.

    2006-01-01

    The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of {beta}-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Angstroms resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.

  10. The pathogenesis of ulnar polydactyly in humans.

    PubMed

    Al-Qattan, M M; Al-Motairi, M I

    2013-11-01

    The pathogenesis of ulnar polydactyly in humans is not known. There are numerous syndromes that are associated with ulnar polydactyly. We have noted that the genetic defects in these syndromes lead to a disturbance of the normal balance between the two forms of the Gli3 protein (the active and repressor forms of Gli3, which are known as Gli3-A and Gli3-R, respectively), leading to a relative increase in the Gli3-R protein. We offer the hypothesis of a unified pathogenesis of ulnar polydactyly through the relative predominance of Gli3-R.

  11. Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

    PubMed

    Kemmeren, Patrick; Sameith, Katrin; van de Pasch, Loes A L; Benschop, Joris J; Lenstra, Tineke L; Margaritis, Thanasis; O'Duibhir, Eoghan; Apweiler, Eva; van Wageningen, Sake; Ko, Cheuk W; van Heesch, Sebastiaan; Kashani, Mehdi M; Ampatziadis-Michailidis, Giannis; Brok, Mariel O; Brabers, Nathalie A C H; Miles, Anthony J; Bouwmeester, Diane; van Hooff, Sander R; van Bakel, Harm; Sluiters, Erik; Bakker, Linda V; Snel, Berend; Lijnzaad, Philip; van Leenen, Dik; Groot Koerkamp, Marian J A; Holstege, Frank C P

    2014-04-24

    To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.

  12. Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions.

    PubMed

    Chugha, Preeti; Sage, Harvey J; Oas, Terrence G

    2006-03-01

    Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

  13. Michaelis-Menten kinetics, the operator-repressor system, and least squares approaches.

    PubMed

    Hadeler, Karl Peter

    2013-01-01

    The Michaelis-Menten (MM) function is a fractional linear function depending on two positive parameters. These can be estimated by nonlinear or linear least squares methods. The non-linear methods, based directly on the defect of the MM function, can fail and not produce any minimizer. The linear methods always produce a unique minimizer which, however, may not be positive. Here we give sufficient conditions on the data such that the nonlinear problem has at least one positive minimizer and also conditions for the minimizer of the linear problem to be positive. We discuss in detail the models and equilibrium relations of a classical operator-repressor system, and we extend our approach to the MM problem with leakage and to reversible MM kinetics. The arrangement of the sufficient conditions exhibits the important role of data that have a concavity property (chemically feasible data).

  14. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    SciTech Connect

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-07-11

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.

  15. Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum?

    PubMed

    Thormann, K; Dürre, P

    2001-11-01

    The gene of Orf5 (SolR) of Clostridium acetobutylicum DSM 792 was subcloned and overexpressed in Escherichia coli. The protein was purified with Ni-NTA agarose and used for DNA binding assays. No DNA binding of Orf5 to regions upstream of the sol operon from C. acetobutylicum was observed. Overexpression of Orf5 in C. acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins. The Orf5 protein was localized in the cell membrane fraction and to a small extent in the supernatant medium. Based on these results Orf5 (SolR) appears not to act as a transcriptional repressor in C. acetobutylicum, but instead may be an enzyme involved in glycosylation or deglycosylation.

  16. Effect of helix length on the stability of the Lac repressor antiparallel coiled coil.

    PubMed

    Little, Wheaton; Robblee, James P; Dahlberg, Caroline L; Kokona, Bashkim; Fairman, Robert

    2015-07-01

    The helix length dependence of the stability of antiparallel four-chain coiled coils is investigated using eight synthetic peptides (Lac21-Lac28) whose sequences are derived from the tetramerization domain of the Lac repressor protein. Previous studies using analytical ultracentrifugation sedimentation equilibrium experiments to characterize Lac21 and Lac28 justifies the use of a two state model to describe the unfolding behavior of these two peptides. Using circular dichroism spectropolarimetry as a measure of tetramer assembly, both chemical and thermal denaturation experiments were carried out to determine thermodynamic parameters. We found that the hydrophobic core residues provide the greatest impact on stability and, as a consequence, must reorganize the register of the antiparallel helices to accommodate the burial of the nonpolar amino acids. Addition of noncore residues appears to have only a minor effect on stability, and in some cases, show a slight destabilization. © 2015 Wiley Periodicals, Inc.

  17. In vivo interactions of the Drosophila Hairy and Runt transcriptional repressors with target promoters.

    PubMed

    Jiménez, G; Pinchin, S M; Ish-Horowicz, D

    1996-12-16

    The Hairy and Runt pair-rule proteins regulate Drosophila segmentation by repressing transcription. To explore the ability of these proteins to function as promoter-bound regulators in vivo, we examined the effects of Hairy and Runt derivatives containing heterologous transcriptional activation domains (HairyAct and RunAct). Using this approach, we find that Hairy and Runt efficiently target such activation domains to specific segmentation gene promoters, leading to rapid induction of transcription. Our results strongly suggest that Hairy normally acts as a promoter-bound repressor of fushi tarazu, runt and odd-skipped, and that Runt directly represses even-skipped. We also show that expressing HairyAct in early blastoderm embryos causes ectopic Sex-lethal expression and male-specific lethality, implying that the Hairy-related denominator element Deadpan represses Sex-lethal during sex determination by directly recognizing the early Sex-lethal promoter.

  18. E. coli trp repressor forms a domain-swapped array in aqueous alcohol

    PubMed Central

    Lawson, Catherine L.; Benoff, Brian; Berger, Tatyana; Berman, Helen M.; Carey, Jannette

    2011-01-01

    The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA-binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 Å X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol. PMID:15274929

  19. Structure of the cro repressor from bacteriophage λ and its interaction with DNA

    NASA Astrophysics Data System (ADS)

    Anderson, W. F.; Ohlendorf, D. H.; Takeda, Y.; Matthews, B. W.

    1981-04-01

    The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage λ suggests how it binds to its operator DNA. We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA. A pair of 2-fold-related α-helices of the represser, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding. In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding.

  20. FBI-1 functions as a novel AR co-repressor in prostate cancer cells.

    PubMed

    Cui, Jiajun; Yang, Yutao; Zhang, Chuanfu; Hu, Pinliang; Kan, Wei; Bai, Xianhong; Liu, Xuelin; Song, Hongbin

    2011-03-01

    The pro-oncogene FBI-1, encoded by Zbtb7a, is a transcriptional repressor that belongs to the POK (POZ/BTB and Krüppel) protein family. In this study, we investigated a potential interaction between androgen receptor (AR) signaling and FBI-1 and demonstrated that overexpression of FBI-1 inhibited ligand-dependent AR activation. A protein-protein interaction was identified between FBI-1 and AR in a ligand-dependent manner. Furthermore, FBI-1, AR and SMRT formed a ternary complex and FBI-1 enhanced the recruitment of NCoR and SMRT to endogenous PSA upstream sequences. Our data also indicated that the FBI-1-mediated inhibition of AR transcriptional activity is partially dependent on HDAC. Interestingly, FBI-1 plays distinct roles in regulating LNCaP (androgen-dependent) and PC-3 cell (androgen-independent) proliferation.

  1. Overlapping repressor binding sites regulate expression of the Methanococcus maripaludis glnK1 operon

    PubMed Central

    Lie, Thomas J.; Hendrickson, Erik L.; Niess, Ulf M.; Moore, Brian C.; Haydock, Andrew K.; Leigh, John A.

    2011-01-01

    The euryarchaeal transcriptional repressor NrpR regulates a variety of nitrogen assimilation genes by 2-oxoglutarate-reversible binding to conserved palindromic operators. The number and positioning of these operators varies among promoter regions of regulated genes, suggesting NrpR can bind in different patterns. Particularly intriguing is the contrast between the nif and glnK1 promoter regions of Methanococcus maripaludis, where two operators are present but with different configurations. Here we study NrpR binding and regulation at the glnK1 promoter, where the two operator sequences overlap and occur on opposite faces of the double helix. We find that both operators function in binding, with a dimer of NrpR binding simultaneously to each overlapping operator. We show in vivo that the first operator plays a primary role in regulation and the second operator plays an enhancing role. This is the first demonstration of overlapping operators functioning in Archaea. PMID:20025661

  2. The transcriptional repressor Hes1 attenuates inflammation via regulating transcriptional elongation

    PubMed Central

    Shang, Yingli; Coppo, Maddalena; He, Teng; Ning, Fei; Yu, Li; Kang, Lan; Zhang, Bin; Ju, Chanyang; Qiao, Yu; Zhao, Baohong; Gessler, Manfred; Rogatsky, Inez; Hu, Xiaoyu

    2016-01-01

    Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription initiation steps and evidence for regulation of inflammatory gene expression post initiation remains scarce. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses production of CXCL1, a chemokine crucial for recruiting neutrophils. Hes1 negatively regulates neutrophil recruitment in vivo in a manner that is dependent on macrophage-produced CXCL1 and attenuates severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 does not involve modification of transcription initiation. Instead, Hes1 inhibits signal-induced recruitment of positive transcription elongation complex P-TEFb, thereby preventing phosphorylation of RNA polymerase II on serine-2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses which exerts its suppressive function by regulating transcription elongation. PMID:27322654

  3. Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex.

    PubMed

    Lewis, Peter W; Beall, Eileen L; Fleischer, Tracey C; Georlette, Daphne; Link, Andrew J; Botchan, Michael R

    2004-12-01

    The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription.

  4. Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex

    PubMed Central

    Lewis, Peter W.; Beall, Eileen L.; Fleischer, Tracey C.; Georlette, Daphne; Link, Andrew J.; Botchan, Michael R.

    2004-01-01

    The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription. PMID:15545624

  5. Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein

    SciTech Connect

    Lin, Jihjing; Thach, R.E. ); Patino, M.M.; Gaffield, L.; Walden. W.E. ); Smith, A. )

    1991-07-15

    Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. ({sup 14}C)Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H{sub 2}O{sub 2} is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.

  6. Waking up Streptomyces secondary metabolism by constitutive expression of activators or genetic disruption of repressors.

    PubMed

    Aigle, Bertrand; Corre, Christophe

    2012-01-01

    Streptomycete bacteria are renowned as a prolific source of natural products with diverse biological activities. Production of these metabolites is often subject to transcriptional regulation: the biosynthetic genes remain silent until the required environmental and/or physiological signals occur. Consequently, in the laboratory environment, many gene clusters that direct the biosynthesis of natural products with clinical potential are not expressed or at very low level preventing the production/detection of the associated metabolite. Genetic engineering of streptomycetes can unleash the production of many new natural products. This chapter describes the overexpression of pathway-specific activators, the genetic disruption of pathway-specific repressors, and the main strategy used to identify and characterize new natural products from these engineered Streptomyces strains.

  7. Regulation of nif expression in Methanococcus maripaludis: roles of the euryarchaeal repressor NrpR, 2-oxoglutarate, and two operators.

    PubMed

    Lie, Thomas J; Wood, Gwendolyn E; Leigh, John A

    2005-02-18

    The methanogenic archaean Methanococcus maripaludis can use ammonia, alanine, or dinitrogen as a nitrogen source for growth. The euryarchaeal nitrogen repressor NrpR controls the expression of the nif (nitrogen fixation) operon, resulting in full repression with ammonia, intermediate repression with alanine, and derepression with dinitrogen. NrpR binds to two tandem operators in the nif promoter region, nifOR(1) and nifOR(2). Here we have undertaken both in vivo and in vitro approaches to study the way in which NrpR, nifOR(1), nifOR(2), and the effector 2-oxoglutarate (2OG) combine to regulate nif expression, leading to a comprehensive understanding of this archaeal regulatory system. We show that NrpR binds as a dimer to nifOR(1) and cooperatively as two dimers to both operators. Cooperative binding occurs only with both operators present. nifOR(1) has stronger binding and by itself can mediate the repression of nif transcription during growth on ammonia, unlike the weakly binding nifOR(2). However, nifOR(2) in combination with nifOR(1) is critical for intermediate repression during growth on alanine. Accordingly, NrpR binds to both operators together with higher affinity than to nifOR(1) alone. NrpR responds directly to 2OG, which weakens its binding to the operators. Hence, 2OG is an intracellular indicator of nitrogen deficiency and acts as an inducer of nif transcription via NrpR. This model is upheld by the recent finding (J. A. Dodsworth and J. A. Leigh, submitted for publication) in our laboratory that 2OG levels in M. maripaludis vary with growth on different nitrogen sources.

  8. IRF2BP2 transcriptional repressor restrains naive CD4 T cell activation and clonal expansion induced by TCR triggering.

    PubMed

    Sécca, Cristiane; Faget, Douglas V; Hanschke, Steffi C; Carneiro, Mayra S; Bonamino, Martin H; de-Araujo-Souza, Patricia S; Viola, João P B

    2016-11-01

    CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iTregs but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli.

  9. Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum.

    PubMed

    Krug, Andreas; Wendisch, Volker F; Bott, Michael

    2005-01-07

    In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose. Here we show that this variation is caused by transcriptional regulation. In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C. glutamicum. Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression. DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C. glutamicum genome. Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from -11 to -28 relative to the transcription start. It thus presumably acts by interfering with the binding of RNA polymerase. The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions. Mutations within this motif inhibited AcnR binding. Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C. glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.

  10. The Coregulator, Repressor of Estrogen Receptor Activity (REA), Is a Crucial Regulator of the Timing and Magnitude of Uterine Decidualization

    PubMed Central

    Zhao, Yuechao; Park, Sunghee; Bagchi, Milan K.; Taylor, Robert N.

    2013-01-01

    Successful implantation and maintenance of pregnancy require the transformation of uterine endometrial stromal cells into distinct decidualized cells. Although estrogen and progesterone (P4) receptors are known to be essential for decidualization, the roles of steroid receptor coregulators in this process remain largely unknown. In this study, we have established a key role for the coregulator, repressor of estrogen receptor activity (REA), in the decidualization of human endometrial stromal cells (hESCs) in vitro and of the mouse uterus in vivo. Our studies revealed that the level of REA normally decreases to half as hESC decidualization proceeds and that uterine reduction of REA in transgenic heterozygous knockout mice or small interfering RNA knockdown of REA in hESC temporally accelerated and strongly enhanced the differentiation process, as indicated by changes in cell morphology and increased expression of biomarkers of decidualization, including P4 receptor. Findings in hESC cultured in vitro with estradiol, P4, and 8-bromo-cAMP over a 10-day period mirrored observations of enhanced decidualization response in transgenic mice with heterozygous deletion of REA. Importantly, gene expression and immunohistochemical analyses revealed changes in multiple components of the Janus kinase/signal transducer and activator of transcription pathway, including marked up-regulation of signal transducer and activator of transcription 3 and IL-11, master regulators of decidualization, and the down-regulation of several suppressor of cytokine signaling family members, upon reduction of REA. The findings highlight that REA physiologically restrains endometrial stromal cell decidualization, controlling the timing and magnitude of decidualization to enable proper coordination of uterine differentiation with concurrent embryo development that is essential for implantation and optimal fertility. PMID:23392257

  11. Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2.

    PubMed

    Clabby, Martha L; Robison, Trevor A; Quigley, Heather F; Wilson, David B; Kelly, Daniel P

    2003-02-21

    Dietary vitamin A and its derivatives, retinoids, regulate cardiac growth and development. To delineate mechanisms involved in retinoid-mediated control of cardiac gene expression, the regulatory effects of the retinoid X receptor alpha (RXR alpha) on atrial naturietic factor (ANF) gene transcription was investigated. The transcriptional activity of an ANF promoter-reporter in rat neonatal ventricular myocytes was repressed by RXR alpha in the presence of 9-cis-RA and by the constitutively active mutant RXR alpha F318A indicating that liganded RXR confers the regulatory effect. The RXR alpha-mediated repression mapped to the proximal 147 bp of the rat ANF promoter, a region lacking a consensus retinoid response element but containing several known cardiogenic cis elements including a well characterized GATA response element. Glutathione S-transferase "pull-down" assays revealed that RXR alpha interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR alpha and the second zinc finger of GATA-4. Liganded RXR alpha repressed the activity of a heterologous promoter-reporter construct containing GATA-response element recognition sites in cardiac myocytes but not in several other cell types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR alpha and the known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive repression of GATA-4 activity in ventricular myocytes. In addition, RXR alpha was found to bind FOG-2, in a 9-cis-RA-dependent manner. These data reveal a novel mechanism by which retinoids regulate cardiogenic gene expression through direct interaction with GATA-4 and its co-repressor, FOG-2.

  12. Aberrant spikelet and panicle1, encoding a TOPLESS-related transcriptional co-repressor, is involved in the regulation of meristem fate in rice.

    PubMed

    Yoshida, Akiko; Ohmori, Yoshihiro; Kitano, Hidemi; Taguchi-Shiobara, Fumio; Hirano, Hiro-Yuki

    2012-04-01

    Post-embryonic development depends on the activity of meristems in plants, and thus control of cell fate in the meristem is crucial to plant development and its architecture. In grasses such as rice and maize, the fate of reproductive meristems changes from indeterminate meristems, such as inflorescence and branch meristems, to determinate meristems, such as the spikelet meristem. Here we analyzed a recessive mutant of rice, aberrant spikelet and panicle1 (asp1), that showed pleiotropic phenotypes such as a disorganized branching pattern, aberrant spikelet morphology, and disarrangement of phyllotaxy. Close examination revealed that regulation of meristem fate was compromised in asp1: degeneration of the inflorescence meristem was delayed, transition from the branch meristem to the spikelet meristem was accelerated, and stem cell maintenance in both the branch meristem and the spikelet meristem was compromised. The genetic program was also disturbed in terms of spikelet development. Gene isolation revealed that ASP1 encodes a transcriptional co-repressor that is related to TOPLESS (TPL) in Arabidopsis and RAMOSA ENHANCER LOCUS2 (REL2) in maize. It is likely that the pleiotropic defects are associated with de-repression of multiple genes related to meristem function in the asp1 mutant. The asp1 mutant also showed de-repression of axillary bud growth and disturbed phyllotaxy in the vegetative phase, suggesting that the function of this gene is closely associated with auxin action. Consistent with these observations and the molecular function of Arabidopsis TPL, auxin signaling was also compromised in the rice asp1 mutant. Taken together, these results indicate that ASP1 regulates various aspects of developmental processes and physiological responses as a transcriptional co-repressor in rice. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  13. Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

    PubMed

    Bach, Anne-Sophie; Derocq, Danielle; Laurent-Matha, Valérie; Montcourrier, Philippe; Sebti, Salwa; Orsetti, Béatrice; Theillet, Charles; Gongora, Céline; Pattingre, Sophie; Ibing, Eva; Roger, Pascal; Linares, Laetitia K; Reinheckel, Thomas; Meurice, Guillaume; Kaiser, Frank J; Gespach, Christian; Liaudet-Coopman, Emmanuelle

    2015-09-29

    The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

  14. Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, Periplaneta americana.

    PubMed

    Blagburn, Jonathan M

    2007-01-01

    In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8-A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.

  15. XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta.

    PubMed

    Wang, Li; Zhang, Lili; Geng, Yunfeng; Xi, Wei; Fang, Rongxiang; Jia, Yantao

    2011-07-01

    We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.

  16. XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta

    PubMed Central

    Wang, Li; Zhang, Lili; Geng, Yunfeng; Xi, Wei; Fang, Rongxiang; Jia, Yantao

    2011-01-01

    We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc. PMID:21483448

  17. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation

    SciTech Connect

    Barbosa, Rosicler Lázaro; Rinaldi, Fábio Cupri; Guimarães, Beatriz Gomes Benedetti, Celso Eduardo

    2007-07-01

    In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.

  18. Brain REST/NRSF Is Not Only a Silent Repressor but Also an Active Protector.

    PubMed

    Zhao, Yangang; Zhu, Min; Yu, Yanlan; Qiu, Linli; Zhang, Yuanyuan; He, Li; Zhang, Jiqiang

    2017-01-01

    During neurogenesis, specific transcription factors are needed to repress neuronal genes in nonneuronal cells to ensure precise development. Repressor element-1 binding transcription factor (REST), or neuron-restrictive silencer factor (NRSF), has been shown to be an important regulator for the establishment of neuronal specificity. It restricts the expression of neuronal genes by binding to the neuron-restrictive silencer element (NRSE/RE1) domain in neuron-specific genes. REST/NRSF regulates many target genes in stem cells, nonneural cells, and neurons, which are involved in neuronal differentiation, axonal growth, vesicular transport, and release as well as ionic conductance. However, it is also regulated by some cytokines/regulators such as epigenetic factors (microRNAs) and even its truncated isoform. REST/NRSF is widely detected in brain regions and has been shown to be highly expressed in nonneuronal cells, but current findings also reveal that, at least in the human brain, it is also highly expressed in neurons and increases with ageing. However, its loss in expression and cytoplasmic translocation seems to play a pivotal role in several human dementias. Additionally, REST/NRSF knockdown leads to malformations in nerve and nonneural tissues and embryonic lethality. Altered REST/NRSF expression has been not only related to deficient brain functions such as neurodegenerative diseases, mental disorders, brain tumors, and neurobehavioral disorders but also highly correlated to brain injuries such as alcoholism and stroke. Encouragingly, several compounds such as valproic acid and X5050 that target REST/NRSF have been shown to be clinically effective at rescuing seizures or Niemann-Pick type C disease. Surprisingly, studies have also shown that REST/NRSF can function as an activator to induce neuronal differentiation. These findings strongly indicate that REST/NRSF is not only a classical repressor to maintain normal neurogenesis, but it is also a fine

  19. The Vibrio cholerae O139 Calcutta Bacteriophage CTXφ Is Infectious and Encodes a Novel Repressor

    PubMed Central

    Davis, Brigid M.; Kimsey, Harvey H.; Chang, William; Waldor, Matthew K.

    1999-01-01

    CTXφ is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXφ infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTXET, the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTXcalc). We found that the CTXcalc prophage gives rise to infectious virions; thus, CTXETφ is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTXcalcφ and CTXETφ are located within the phages’ repressor genes (rstRcalc and rstRET, respectively) and their RstR operators. RstRcalc is a novel, allele-specific repressor that regulates replication of CTXcalcφ by inhibiting the activity of the rstAcalc promoter. RstRcalc has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstRcalc renders a CTXcalc lysogen immune to superinfection by CTXcalcφ but susceptible (heteroimmune) to infection by CTXETφ. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTXETφ lysogen by CTXcalcφ. PMID:10542181

  20. Bacterial Effector Activates Jasmonate Signaling by Directly Targeting JAZ Transcriptional Repressors

    PubMed Central

    Jiang, Shushu; Yao, Jian; Ma, Ka-Wai; Zhou, Huanbin; Song, Jikui; He, Sheng Yang; Ma, Wenbo

    2013-01-01

    Gram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens. PMID:24204266

  1. Solution dynamics of the trp repressor: a study of amide proton exchange by T1 relaxation.

    PubMed

    Gryk, M R; Finucane, M D; Zheng, Z; Jardetzky, O

    1995-03-10

    The amide proton exchange rates of Escherichia coli trp repressor have been measured through their effects on the longitudinal relaxation rates of the amide protons. Three types of exchange regimes have been observed: (1) slow exchange (on a minute/hour time-scale), measurable by isotope exchange, but not by relaxation techniques in the core of the molecule; (2) relatively rapid exchange, with the rates on a T1 relaxation time-scale (seconds) in the DNA-binding region and (3) very fast exchange at the N and C termini. The results have been analyzed in terms of the two-site exchange model originally proposed by Linderstrøm-Lang, and of a three-site extension of the model. The values of the intrinsic exchange rates calculated using the two-state model agree with the values expected from the studies of Englander and co-workers for the very fast case of the chain terminals, but disagree with the literature values by two orders of magnitude in the intermediate case found in the DNA-binding region. The implication of these findings is that the "open" state of the two-state model in the DNA-binding region is not completely open and has an intrinsic exchange rate different from that of a random coil peptide. Alternatively, if the literature values of the intrinsic exchange rates are assumed to apply to the open states in all parts of the repressor molecule, two "closed" helical states have to be postulated, in slow exchange with each other, with only one of them in rapid exchange with the open state and hence with the solvent. Kinetically, the two models are indistinguishable.

  2. APETALA2 negatively regulates multiple floral organ identity genes in Arabidopsis by recruiting the co-repressor TOPLESS and the histone deacetylase HDA19.

    PubMed

    Krogan, Naden T; Hogan, Kendra; Long, Jeff A

    2012-11-01

    The development and coordination of complex tissues in eukaryotes requires precise spatial control of fate-specifying genes. Although investigations of such control have traditionally focused on mechanisms of transcriptional activation, transcriptional repression has emerged as being equally important in the establishment of gene expression territories. In the angiosperm flower, specification of lateral organ fate relies on the spatial regulation of the ABC floral organ identity genes. Our understanding of how the boundaries of these expression domains are controlled is not complete. Here, we report that the A-class organ identity gene APETALA2 (AP2), which is known to repress the C-class gene AGAMOUS, also regulates the expression borders of the B-class genes APETALA3 and PISTILLATA, and the E-class gene SEPALLATA3. We show that AP2 represses its target genes by physically recruiting the co-repressor TOPLESS and the histone deacetylase HDA19. These results demonstrate that AP2 plays a broad role in flower development by controlling the expression domains of numerous floral organ identity genes.

  3. Crystallization and preliminary X-ray crystallographic analysis of the CARD domain of apoptosis repressor with CARD (ARC).

    PubMed

    Kim, Seong Hyun; Park, Hyun Ho

    2015-01-01

    Apoptosis repressor with caspase-recruiting domain (ARC) is an apoptosis repressor that inhibits both intrinsic and extrinsic apoptosis signalling. Human ARC contains an N-terminal caspase-recruiting domain (CARD domain) and a C-terminal proline- and glutamic acid-rich (P/E-rich) domain. The CARD domain in ARC is the domain that is directly involved in inhibition of the extrinsic pathway. In this study, the N-terminal CARD domain of ARC was overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å and the crystals were found to belong to space group P6(1) or P65, with unit-cell parameters a=98.28, b=98.28, c=51.86 Å, α=90, β=90, γ=120°.

  4. An inhibitory RNA aptamer against the lambda cI repressor shows transcriptional activator activity in vivo.

    PubMed

    Ohuchi, Shoji; Suess, Beatrix

    2017-04-13

    An RNA aptamer is one of the promising components for constructing artificial genetic circuits. In this study, we developed a transcriptional activator based on an RNA aptamer against one of the most frequently applied repressor proteins, lambda phage cI. In vitro selection (SELEX), followed by in vivo screening identified an RNA aptamer with the intended transcriptional activator activity from an RNA pool containing a 40-nucleotide long random region. Quantitative analysis showed 35-fold elevation of reporter expression upon aptamer expression. These results suggest that the diversity of artificial transcriptional activators can be extended by employing RNA aptamers against repressor proteins to broaden the tools available for constructing genetic circuits. This article is protected by copyright. All rights reserved.

  5. The activities of the E3 ubiquitin ligase COP1/SPA, a key repressor in light signaling.

    PubMed

    Hoecker, Ute

    2017-06-01

    Light is a critical signal to integrate plant growth and development with the environment. Downstream of photoreceptors, the E3 ubiquitin ligase COP1/SPA is a key repressor of photomorphogenesis which targets many positive regulators of light signaling, mainly transcription factors, for degradation in darkness. In light-grown plants COP1/SPA activity is repressed, allowing light responses to occur. This review provides an overview on our current knowledge on COP1/SPA repressor function, focusing in particular on the roles of the respective protein domains and the mechanisms of light-induced inactivation of COP1/SPA. Moreover, we summarize how COP1 activity is regulated by other interacting proteins, such as a SUMO E3 ligase and Phytochrome-Interacting Factors (PIFs), as well as by hormones. At last, several novel functions of COP1 that were recently revealed are included. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping

    PubMed Central

    Normanno, Davide; Vanzi, Francesco; Pavone, Francesco Saverio

    2008-01-01

    Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between −0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI–DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes. PMID:18310101

  7. The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA

    PubMed Central

    Zentella, Rodolfo; Sui, Ning; Barnhill, Benjamin; Hsieh, Wen-Ping; Hu, Jianhong; Shabanowitz, Jeffrey; Boyce, Michael; Olszewski, Neil E.; Zhou, Pei; Hunt, Donald F.; Sun, Tai-ping

    2016-01-01

    Plant development requires coordination among complex signaling networks to enhance plant’s adaptation to changing environments. The transcription regulators DELLAs, originally identified as repressors of phytohormone gibberellin (GA) signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we showed that DELLA was mono-O-fucosylated by a novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid (BR)- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3), and PIF4. Consistently, spy mutants displayed elevated responses to GA and BR, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding has broader importance as SPY orthologs are conserved from prokaryotes to eukaryotes, suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms. PMID:28244988

  8. Sequence and expression analysis of the gene encoding inducible cAMP early repressor in tilapia.

    PubMed

    Chen, Ming; Wang, Rui; Gan, Xi; Lei, Aiying; Li, Chao; Yu, Xiaoli; Huang, Jun; Huang, Ting; Liang, Wanwen

    2010-06-01

    Suppression subtractive hybridization library was generated by comparison of cDNA populations isolated from peripheral leukocytes of pre- and post-immunized tilapia. One cDNA sequence encoding complete inducible cAMP early repressor was obtained from the library. The sequence was characterized by the presence of the basic structure of ICER IIgamma. Expression of ICER was in the tissues of four types of tilapia was decreased after infection with Streptococcus. After immunization, expression of ICER was initially decreased and then increased after 7 days. In addition, the order for the overall expression of ICER gene after infection and the increases of ICER expression later after immunization in these four types of tilapia was positively correlated to the disease resistance and productivity of these four species of tilapia. Our results provided molecular mechanisms for the different disease resistance capability in different species of tilapia. In addition, our results also provided reference molecular marker for breeding disease resistant tilapia, cAMP responsive element modulator.

  9. A cellular repressor regulates transcription initiation from the minute virus of mice P38 promoter.

    PubMed Central

    Krauskopf, A; Aloni, Y

    1994-01-01

    We previously reported that the P38 promoter of minute virus of mice (MVM) is trans activated by the viral nonstructural protein, NS1, through an interaction with a downstream promoter element designated DPE. In this communication we report the identification of a distinct downstream promoter element which inhibits transcription from the P38 promoter in vitro, in the absence of the DPE. Removal of 34 bp from the region between +95 and +129 downstream from the P38 initiation start site relieved inhibition of transcription in whole-cell extract. Inhibition was also relieved by the addition, to the transcription reaction, of excess DNA fragments which span the putative inhibiting element. This indicated the involvement of a trans-acting factor, in inhibition of transcription from the P38. Gel retardation experiments demonstrated the specific binding of a cellular protein to the inhibitory element. This P38 inhibitory element shows spacing and orientation dependence as well as promoter specificity. The regulation of viral transcription by a cellular repressor may play an important role in obtaining a fine temporal order of viral gene expression during the course of infection. Images PMID:8139925

  10. A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer.

    PubMed

    Davis, Brigid M; Kimsey, Harvey H; Kane, Anne V; Waldor, Matthew K

    2002-08-15

    CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.

  11. Backbone dynamics of the monomeric lambda repressor denatured state ensemble under nondenaturing conditions.

    PubMed

    Chugha, Preeti; Oas, Terrence G

    2007-02-06

    Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.

  12. KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions

    PubMed Central

    Lupo, Angelo; Cesaro, Elena; Montano, Giorgia; Zurlo, Diana; Izzo, Paola; Costanzo, Paola

    2013-01-01

    Zinc finger proteins containing the Kruppel associated box (KRAB-ZFPs) constitute the largest individual family of transcriptional repressors encoded by the genomes of higher organisms. KRAB domain, positioned at the NH2 terminus of the KRAB-ZFPs, interacts with a scaffold protein, KAP-1, which is able to recruit various transcriptional factors causing repression of genes to which KRAB ZFPs bind. The relevance of such repression is reflected in the large number of the KRAB zinc finger protein genes in the human genome. However, in spite of their numerical abundance little is currently known about the gene targets and the physiological functions of KRAB- ZFPs. However, emerging evidence links the transcriptional repression mediated by the KRAB-ZFPs to cell proliferation, differentiation, apoptosis and cancer. Moreover, the fact that KRAB containing proteins are vertebrate-specific suggests that they have evolved recently, and that their key roles lie in some aspects of vertebrate development. In this review, we will briefly discuss some regulatory functions of the KRAB-ZFPs in different physiological and pathological states, thus contributing to better understand their biological roles. PMID:24294107

  13. Moderately thermostable phage Phi11 Cro repressor has novel DNA-binding capacity and physicochemical properties.

    PubMed

    Das, Malabika; Ganguly, Tridib; Bandhu, Amitava; Mondal, Rajkrishna; Chanda, Palas K; Jana, Biswanath; Sau, Subrata

    2009-03-31

    The temperate Staphylococcus aureus phage Phi11 harbors cI and cro repressor genes similar to those of lambdoid phages. Using extremely pure Phi11 Cro (the product of the Phi11 cro gene) we demonstrated that this protein possesses a single domain structure, forms dimers in solution at micromolar concentrations and maintains a largely alpha-helical structure even at 45 degrees C. Phi11 Cro was sensitive to thermolysin at temperatures ranging from 55-75 degrees C and began to aggregate at ~63 degrees C, suggesting that the protein is moderately thermostable. Of the three homologous 15-bp operators (O1, O2, and O3) in the Phi11 cI-cro intergenic region, Phi11 Cro only binds efficiently to O3, which is located upstream of the cI gene. Our comparative analyses indicate that the DNA binding capacity, secondary structure and dimerization efficiency of thermostable Phi11 Cro are distinct from those of P22 Cro and lambda Cro, the best characterized representatives of the two structurally different Cro families.

  14. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models.

    PubMed

    Osman, Erkan Y; Miller, Madeline R; Robbins, Kate L; Lombardi, Abby M; Atkinson, Arleigh K; Brehm, Amanda J; Lorson, Christian L

    2014-09-15

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1(MO)-ASOs). A single intracerebroventricular injection in the relatively severe mouse model of SMA (SMNΔ7 mouse model) elicited a robust induction of SMN protein, and mean life span was extended from an average survival of 13 to 54 days following a single dose, consistent with large weight gains and a correction of the neuronal pathology. Additionally, E1(MO)-ASO treatment in an intermediate SMA mouse (SMN(RT) mouse model) significantly extended life span by ∼700% and weight gain was comparable with the unaffected animals. While a number of experimental therapeutics have targeted the ISS-N1 element of SMN2 pre-mRNA, the development of E1 ASOs provides a new molecular target for SMA therapeutics that dramatically extends survival in two important pre-clinical models of disease.

  15. Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in Clostridium acetobutylicum.

    PubMed

    Zhang, Lei; Nie, Xiaoqun; Ravcheev, Dmitry A; Rodionov, Dmitry A; Sheng, Jia; Gu, Yang; Yang, Sheng; Jiang, Weihong; Yang, Chen

    2014-11-01

    Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD(+) ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD(+) concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD(+) ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD(+) homeostasis in clostridia. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Redox-Responsive Repressor Rex Modulates Alcohol Production and Oxidative Stress Tolerance in Clostridium acetobutylicum

    PubMed Central

    Zhang, Lei; Nie, Xiaoqun; Ravcheev, Dmitry A.; Rodionov, Dmitry A.; Sheng, Jia; Gu, Yang; Yang, Sheng; Jiang, Weihong

    2014-01-01

    Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD+ ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD+ concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD+ ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD+ homeostasis in clostridia. PMID:25182496

  17. Transcription repressor Bach2 is required for pulmonary surfactant homeostasis and alveolar macrophage function

    PubMed Central

    Nakamura, Atsushi; Ebina-Shibuya, Risa; Itoh-Nakadai, Ari; Muto, Akihiko; Shima, Hiroki; Saigusa, Daisuke; Aoki, Junken; Ebina, Masahito; Nukiwa, Toshihiro

    2013-01-01

    Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), chiefly due to disruptions in the signaling of granulocyte macrophage colony–stimulating factor (GM-CSF). We found that mice deficient for the B lymphoid transcription repressor BTB and CNC homology 2 (Bach2) developed PAP-like accumulation of surfactant proteins in the lungs. Bach2 was expressed in AMs, and Bach2-deficient AMs showed alterations in lipid handling in comparison with wild-type (WT) cells. Although Bach2-deficient AMs showed a normal expression of the genes involved in the GM-CSF signaling, they showed an altered expression of the genes involved in chemotaxis, lipid metabolism, and alternative M2 macrophage activation with increased expression of Ym1 and arginase-1, and the M2 regulator Irf4. Peritoneal Bach2-deficient macrophages showed increased Ym1 expression when stimulated with interleukin-4. More eosinophils were present in the lung and peritoneal cavity of Bach2-deficient mice compared with WT mice. The PAP-like lesions in Bach2-deficient mice were relieved by WT bone marrow transplantation even after their development, confirming the hematopoietic origin of the lesions. These results indicate that Bach2 is required for the functional maturation of AMs and pulmonary homeostasis, independently of the GM-CSF signaling. PMID:24127487

  18. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    SciTech Connect

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  19. The transcription factor p53: Not a repressor, solely an activator

    PubMed Central

    Fischer, Martin; Steiner, Lydia; Engeland, Kurt

    2014-01-01

    The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway. PMID:25486564

  20. A Novel Function of δ Factor from Bacillus subtilis as a Transcriptional Repressor.

    PubMed

    Prajapati, Ranjit Kumar; Sur, Runa; Mukhopadhyay, Jayanta

    2016-11-11

    δ, a small protein found in most Gram-positive bacteria was, for a long time, thought to be a subunit of RNA polymerase (RNAP) and was shown to be involved in recycling of RNAP at the end of each round of transcription. However, how δ participates in both up-regulation and down-regulation of genes in vivo remains unclear. We have recently shown, in addition to the recycling of RNAP, δ functions as a transcriptional activator by binding to an A-rich sequence located immediately upstream of the -35 element, consequently facilitating the open complex formation. The result had explained the mechanism of up-regulation of the genes by δ. Here, we show that Bacillus subtilis δ could also function as a transcriptional repressor. Our results demonstrate that δ binds to an A-rich sequence located near the -35 element of the spo0B promoter, the gene involved in the regulatory cascade of bacterial sporulation and inhibits the open complex formation due to steric clash with σ region 4.2. We observed a significant increase in the mRNA level of the spo0B gene in a δ-knock-out strain of B. subtilis compared with the wild-type. Thus, the results report a novel function of δ, and suggest the mechanism of down-regulation of genes in vivo by the protein.

  1. A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1.

    PubMed

    Boylston, Jennifer A; Brenner, Charles

    2014-01-01

    Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

  2. Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) Enhances Multidrug Resistance of Hepatocellular Carcinoma Cells

    PubMed Central

    Fu, Rong-Jie; Lv, Ya-Ping; Jin, Wei; Meng, Chao; Chen, Guo-Qiang; Huang, Lei

    2016-01-01

    China accounts for almost half of the total number of liver cancer cases and deaths worldwide, and hepatocellular carcinoma (HCC) is the most primary liver cancer. Snail family transcriptional repressor 2 (SNAI2) is known as an epithelial to mesenchymal transition-inducing transcription factor that drives neoplastic epithelial cells into mesenchymal phenotype. However, the roles of endogenous SNAI2 remain controversial in different types of malignant tumors. Herein, we surprisingly identify that anchorage-independent growth, including the formation of tumor sphere and soft agar colony, is significantly increased when SNAI2 expression is inhibited by shRNAs in HCC cells. Suppression of SNAI2 suffices to up-regulate several cancer stem genes. Although unrelated to the metastatic ability, SNAI2 inhibition does increase the efflux of Hoechst 33342 and enhance multidrug resistance in vitro and in vivo. In agreement with this data, we demonstrate for the first time that decreasing SNAI2 level can transcriptionally upregulate several ATP binding cassette (ABC) transporter genes such as ABCB1. Moreover, ABC transporters’ inhibitor verapamil can rescue the multidrug resistance induced by SNAI2 inhibition. Our results implicate that SNAI2 behaves as a tumor suppressor by inhibiting multidrug resistance via suppressing ABC transporter genes in HCC cells. PMID:27760172

  3. DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation.

    PubMed

    Boedicker, James Q; Garcia, Hernan G; Johnson, Stephanie; Phillips, Rob

    2013-12-01

    As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

  4. DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

    NASA Astrophysics Data System (ADS)

    Boedicker, James Q.; Garcia, Hernan G.; Johnson, Stephanie; Phillips, Rob

    2013-12-01

    As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

  5. Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication.

    PubMed

    Molofsky, Ari B; Swanson, Michele S

    2003-10-01

    Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.

  6. Conversion of a gene-specific repressor to a regional silencer

    PubMed Central

    Rine, Laura N. Rusché and Jasper

    2001-01-01

    In Saccharomyces cerevisiae, gene silencing at the HMR and HML loci is normally dependent on Sir2p, Sir3p, and Sir4p, which are structural components of silenced chromatin. Sir2p is a NAD+-dependent histone deacetylase required for silencing. Silencing can be restored in cells lacking Sir proteins by a dominant mutation in SUM1, which normally acts as a mitotic repressor of meiotic genes. This study found that mutant Sum1-1p, but not wild-type Sum1p, associated directly with HM loci. The origin recognition complex (ORC) was required for Sum1-1p-mediated silencing, and mutations in ORC genes reduced association of Sum1-1p with the HM loci. Sum1-1p-mediated silencing also depended on HST1, a paralog of SIR2. Both Sum1-1p and wild-type Sum1p interacted with Hst1p in coimmunoprecipitation experiments. Therefore, the SUM1-1 mutation did not change the affinity of Sum1p for Hst1p, but rather relocalized Sum1p to the HM loci. Sum1-1–Hst1p action led to hypoacetylation of the nucleosomes at HM loci. Thus, Sum1-1p and Hst1p could substitute for Sir proteins to achieve silencing through formation of a compositionally distinct type of heterochromatin. PMID:11316790

  7. [PPARγ up-regulates TGFβ/smad signal pathway repressor c-Ski].

    PubMed

    Li, Gong-bo; Li, Jun; Zeng, Yi-jun; Zhong, Dan; Wu, Geng-ze; Fu, Xiao-hong; He, Feng-tian; Dai, Shuang-shuang

    2011-02-25

    TGFβ/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFβ/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.

  8. A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer

    PubMed Central

    Davis, Brigid M.; Kimsey, Harvey H.; Kane, Anne V.; Waldor, Matthew K.

    2002-01-01

    CTXφ is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXφ-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXφ. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders. PMID:12169626

  9. The transcriptional repressor Nab1 is a specific regulator of pathological cardiac hypertrophy.

    PubMed

    Buitrago, Monika; Lorenz, Kristina; Maass, Alexander H; Oberdorf-Maass, Silke; Keller, Ursula; Schmitteckert, Eva M; Ivashchenko, Yuri; Lohse, Martin J; Engelhardt, Stefan

    2005-08-01

    Hypertrophy represents the major physiological response of the heart to adapt to chronically enhanced workload, but is also crucial in the development of heart failure. Although we know of numerous inducers of cardiac hypertrophy, little is known about mechanisms that limit cardiac hypertrophy. Here, we describe the transcriptional repressor NAB1 as an endogenous regulator of cardiac growth. We identified NAB1 as being upregulated in both mouse and human heart failure. Nab1 is highly expressed in mammalian cardiac myocytes and it inhibited cardiomyocyte hypertrophy through repression of its targets, transcription factor Egr. Transgenic mice with cardiac-specific overexpression of Nab1 showed that Nab1 is a potent inhibitor of cardiac growth in response to pathological stimuli in vivo. Nab1 overexpression suppressed adrenergically induced and pressure overload-induced hypertrophy, whereas physiological growth during development and in response to exercise was not affected. These findings implicate the Nab1-Egr1 axis as a crucial regulator of pathological cardiac growth.

  10. NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

    PubMed Central

    Singer, Heike; Biswas, Arijit; Nuesgen, Nicole; Oldenburg, Johannes; El-Maarri, Osman

    2015-01-01

    Mutations in the maternal effect gene NLRP7 cause biparental hydatidiform mole (HYDM1). HYDM1 is characterized by abnormal growth of placenta and lack of proper embryonic development. The molar tissues are characterized by abnormal methylation patterns at differentially methylated regions (DMRs) of imprinted genes. It is not known whether this occurs before or after fertilization, but the high specificity of this defect to the maternal allele indicates a possible maternal germ line-specific effect. To better understand the unknown molecular mechanism leading to HYDM1, we performed a yeast two-hybrid screen against an ovarian library using NLRP7 as bait. We identified the transcriptional repressor ZBTB16 as an interacting protein of NLRP7 and verified this interaction in mammalian cells by immunoprecipitation and confocal microscopy. Native protein analysis detected NLRP7 and ZBTB16 in a 480kD protein complex and both proteins co-localize in the cytoplasm in juxtanuclear aggregates. HYDM1-causing mutations in NLRP7 did not show altered patterns of interaction with ZBTB16. Hence, the biological significance of the NLRP7-ZBTB16 interaction remains to be revealed. However, a clear effect of harvesting ZBTB16 to the cytoplasm when the NLRP7 protein is overexpressed may be linked to the pathology of the molar pregnancy disease. PMID:26121690

  11. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  12. Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.

    PubMed Central

    Ng, J; Li, R; Morgan, K; Simon, J

    1997-01-01

    The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins. PMID:9343430

  13. The BCG Moreau RD16 deletion inactivates a repressor reshaping transcription of an adjacent gene.

    PubMed

    Galvão, Teca Calcagno; Lima, Cristiane Rodrigues; Gomes, Leonardo Henrique Ferreira; Pagani, Talita Duarte; Ferreira, Marcelo Alves; Gonçalves, Antonio S; Correa, Paloma Rezende; Degrave, Wim Maurits; Mendonça-Lima, Leila

    2014-01-01

    The Brazilian anti-tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG) BCG Moreau is unique in having a deletion of 7608 bp (RD16) that results in the truncation of a putative TetR transcriptional regulator, the ortholog of Mycobacterium tuberculosis rv3405c, BCG_M3439c. We investigated the effect of this truncation on the expression of the rv3406 ortholog (BCG_M3440), lying 81 bp downstream in the opposite orientation. RT-PCR and western blot experiments show that rv3406 mRNA and Rv3406 accumulate in BCG Moreau but not in BCG Pasteur (strain that bears an intact rv3405c), suggesting this to be a result of rv3405c truncation. Recombinant Rv3405c forms a complex with the rv3405c-rv3406 intergenic region, which contains a characteristic transcription factor binding site, showing it to have DNA binding activity. Complementation of M. bovis BCG Moreau with an intact copy of rv3405c abolishes Rv3406 accumulation. These results show that Rv3405c is a DNA binding protein that acts as a transcriptional repressor of rv3406.

  14. Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

    NASA Astrophysics Data System (ADS)

    Lia, Giuseppe; Bensimon, David; Croquette, Vincent; Allemand, Jean-Francois; Dunlap, David; Lewis, Dale E. A.; Adhya, Sankar; Finzi, Laura

    2003-09-01

    The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of 40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

  15. Interactions between the leukaemia-associated ETO homologues of nuclear repressor proteins.

    PubMed

    Lindberg, Sofia Rondin; Olsson, André; Persson, Ann-Maj; Olsson, Inge

    2003-12-01

    The eight-twenty-one (ETO) homologues, represented by ETO, myeloid transforming gene-related protein 1 (MTGR1) and myeloid transforming gene chromosome 16 (MTG16), are nuclear repressor proteins. ETO is part of the fusion protein acute myeloid leukaemia (AML)1-ETO, resulting from the translocation (8;21). Similarly, MTG16 is disrupted to become part of AML1/MTG16 in t(16;21). The aberrant expression of these chimeras could affect interplay between ETO homologues and contribute to the leukaemogenic process. We investigated possible interactions between the ETO homologues. Ectopic co-expression in COS-cells resulted in heterodimerisation of the various ETO homologues suggesting that they may co-operate. Similarly, the chimeric oncoprotein AML1-ETO interacted with both MTGR1 and MTG16. However, results from cell lines endogenously expressing more than one ETO homologue did not demonstrate co-precipitation. Results from IP-Western and size determination by gel filtration of deletion mutants expressed in COS-cells, indicated an important role of the HHR domain for oligomerisation. A role was also suggested for the Nervy domain in the homologue interactions. Our results suggest that ETO homologues can interact with each other as well as with AML1-ETO, although it is unclear as to what extent these interactions occur in vivo.

  16. Direct imaging of LacI repressor protein sliding along DNA

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Mei

    2005-03-01

    LacI repressor protein was observed in 1970 to bind to its operator site 100 times faster than allowed by diffusion [1]. A facilitated diffusion model incorporating1-D sliding and 3-D diffusion of the nonspecifically bound protein has been suggested to explain this phenomenon [2]. We have imaged the nonspecific binding of GFP-LacI monomers to elongated DNA molecules using single molecule imaging techniques. Upon binding to DNA, LacI proteins were observed to either be stationary, or slide along DNA. The characteristics of the sliding motion fit that of 1-D Brownian motion (with and without drift). The 1-D diffusion constant of the sliding proteins is 104 nm2/s, and it is 104 times lower than a typical protein's 3- D diffusion constant, 108 nm2/s. The characteristic dissociation time for both the stationary and the sliding proteins is 6s, and it is 100 times longer than the known dissociation time of 0.08s. The sliding length (DNA length scanned by the protein, not counting repeatedly scanned bases) ranges from 300 bp to 3000 bp, and it is significantly higher than the calculated optimal sliding length of 100 bp. We will discuss how these abnormal parameters alter the LacI specific binding speed. [1] A. D Riggs, S. Bougeois and M Cohn, J. Mol. Biol. 53, 401- 417 (1970). [2] O. G. Berg and C. Blomberg, Biophys. Chem., 4, 367-381 (1976).

  17. ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression

    PubMed Central

    Aro, Nina; Ilmén, Marja; Saloheimo, Anu; Penttilä, Merja

    2003-01-01

    We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present. PMID:12513977

  18. The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

    SciTech Connect

    Tan, K.; Borovilos, M.; Zhou, M; Horer, S; Clancy, S; Moy, S; Volkart, LL; Sassoon, J; Baumann, U; Joachimiak, A

    2009-12-25

    Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.

  19. Repressor transcription factor 7-like 1 promotes adipogenic competency in precursor cells.

    PubMed

    Cristancho, Ana G; Schupp, Michael; Lefterova, Martina I; Cao, Shengya; Cohen, Daniel M; Chen, Christopher S; Steger, David J; Lazar, Mitchell A

    2011-09-27

    The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell-cell contact with the transcriptional repression required for adipogenic competency.

  20. Repressor transcription factor 7-like 1 promotes adipogenic competency in precursor cells

    PubMed Central

    Cristancho, Ana G.; Schupp, Michael; Lefterova, Martina I.; Cao, Shengya; Cohen, Daniel M.; Chen, Christopher S.; Steger, David J.; Lazar, Mitchell A.

    2011-01-01

    The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell–cell contact with the transcriptional repression required for adipogenic competency. PMID:21914845

  1. Transcriptional Repressor DAXX Promotes Prostate Cancer Tumorigenicity via Suppression of Autophagy*

    PubMed Central

    Puto, Lorena A.; Brognard, John; Hunter, Tony

    2015-01-01

    The DAXX transcriptional repressor was originally associated with apoptotic cell death. However, recent evidence that DAXX represses several tumor suppressor genes, including the DAPK1 and DAPK3 protein kinases, and is up-regulated in many cancers argues that a pro-survival role may predominate in a cancer context. Here, we report that DAXX has potent growth-enhancing effects on primary prostatic malignancy through inhibition of autophagy. Through stable gene knockdown and mouse subcutaneous xenograft studies, we demonstrate that DAXX promotes tumorigenicity of human ALVA-31 and PC3 prostate cancer (PCa) cells in vivo. Importantly, DAXX represses expression of essential autophagy modulators DAPK3 and ULK1 in vivo, revealing autophagy suppression as a mechanism through which DAXX promotes PCa tumorigenicity. Furthermore, DAXX knockdown increases autophagic flux in cultured PCa cells. Finally, interrogation of the OncomineTM database suggests that DAXX overexpression is associated with malignant transformation in several human cancers, including prostate and pancreatic cancers. Thus, DAXX may represent a new cancer biomarker for the detection of aggressive disease, whose tissue-specific down-regulation can serve as an improved therapeutic modality. Our results establish DAXX as a pro-survival protein in PCa and reveal that, in the early stages of tumorigenesis, autophagy suppresses prostate tumor formation. PMID:25903140

  2. The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA.

    PubMed

    Zentella, Rodolfo; Sui, Ning; Barnhill, Benjamin; Hsieh, Wen-Ping; Hu, Jianhong; Shabanowitz, Jeffrey; Boyce, Michael; Olszewski, Neil E; Zhou, Pei; Hunt, Donald F; Sun, Tai-Ping

    2017-05-01

    Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.

  3. Mutations in the TGF-β Repressor SKI Cause Shprintzen-Goldberg Syndrome with Aortic Aneurysm

    PubMed Central

    Doyle, Alexander J.; Doyle, Jefferson J.; Bessling, Seneca L.; Maragh, Samantha; Lindsay, Mark E.; Schepers, Dorien; Gillis, Elisabeth; Mortier, Geert; Homfray, Tessa; Sauls, Kimberly; Norris, Russell A.; Huso, Nicholas D.; Leahy, Dan; Mohr, David W.; Caulfield, Mark J.; Scott, Alan F.; Destrée, Anne; Hennekam, Raoul C.; Arn, Pamela H.; Curry, Cynthia J.; Van Laer, Lut; McCallion, Andrew S.; Loeys, Bart L.; Dietz, Harry C.

    2012-01-01

    Increased transforming growth factor beta (TGF-β) signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS)1-4. However, the location and character of many of the causal mutations in LDS would intuitively infer diminished TGF-β signaling5. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm6-8. We identified causative variation in 10 patients with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity9,10. Cultured patient dermal fibroblasts showed enhanced activation of TGF-β signaling cascades and increased expression of TGF-β responsive genes. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in SGS patients. These data support the conclusion that increased TGF-β signaling is the mechanism underlying SGS and contributes to multiple syndromic presentations of aortic aneurysm. PMID:23023332

  4. Optimization of Morpholino Antisense Oligonucleotides Targeting the Intronic Repressor Element1 in Spinal Muscular Atrophy.

    PubMed

    Osman, Erkan Y; Washington, Charles W; Kaifer, Kevin A; Mazzasette, Chiara; Patitucci, Teresa N; Florea, Kyra M; Simon, Madeline E; Ko, Chien-Ping; Ebert, Allison D; Lorson, Christian L

    2016-09-01

    Loss of Survival Motor Neuron-1 (SMN1) causes Spinal Muscular Atrophy, a devastating neurodegenerative disease. SMN2 is a nearly identical copy gene; however SMN2 cannot prevent disease development in the absence of SMN1 since the majority of SMN2-derived transcripts are alternatively spliced, encoding a truncated, unstable protein lacking exon 7. Nevertheless, SMN2 retains the ability to produce low levels of functional protein. Previously we have described a splice-switching Morpholino antisense oligonucleotide (ASO) sequence that targets a potent intronic repressor, Element1 (E1), located upstream of SMN2 exon 7. In this study, we have assessed a novel panel of Morpholino ASOs with the goal of optimizing E1 ASO activity. Screening for efficacy in the SMNΔ7 mouse model, a single ASO variant was more active in vivo compared with the original E1(MO)-ASO. Sequence variant eleven (E1(MOv11)) consistently showed greater efficacy by increasing the lifespan of severe Spinal Muscular Atrophy mice after a single intracerebroventricular injection in the central nervous system, exhibited a strong dose-response across an order of magnitude, and demonstrated excellent target engagement by partially reversing the pathogenic SMN2 splicing event. We conclude that Morpholino modified ASOs are effective in modifying SMN2 splicing and have the potential for future Spinal Muscular Atrophy clinical applications.

  5. The cytidine repressor participates in the regulatory pathway of indole in Pantoea agglomerans.

    PubMed

    Jia, Mengqi; Yu, Xuemei; Jiang, Jing; Li, Zihua; Feng, Yongjun

    2017-09-01

    Indole, an important signal molecule in both intraspecies and interspecies, regulates a variety of bacterial behaviors, but its regulatory mechanism is still unknown. Pantoea agglomerans YS19, a preponderant endophytic bacterium isolated from rice, does not produce indole, yet it senses exogenous indole. In this study, a mutant of YS19-Rp(r) whose target gene expression was downregulated by indole was selected through mTn5 transposon mutagenesis. Using the TAIL-PCR technique, the mutation gene was identified as a cytR homologue, which encodes a cytidine repressor (CytR) protein, a bacterial transcription factor involved in a complex regulation scheme. The negative regulation of indole in cytR, which is equivalent to the mutation in cytR, promotes the expression of a downstream gene deoC, which encodes the key enzyme deoxyribose-phosphate aldolase in participating in pentose metabolism. We found that DeoC is one of the regulatory proteins of P. agglomerans that is involved in counteracting starvation. Furthermore, the expression of deoC was induced by starvation conditions, accompanied by a decrease in cytR expression. This finding suggests that the indole signal and the mutation of cytR relieve inhibition of CytR in the transcription of deoC, facilitating better adaptation of the bacterium to the adverse conditions of the environment. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  6. The Transcriptional Repressor ZNF503/Zeppo2 Promotes Mammary Epithelial Cell Proliferation and Enhances Cell Invasion*

    PubMed Central

    Shahi, Payam; Slorach, Euan M.; Wang, Chih-Yang; Chou, Jonathan; Lu, Angela; Ruderisch, Aline; Werb, Zena

    2015-01-01

    The NET (nocA, Nlz, elB, TLP-1) subfamily of zinc finger proteins is an important mediator during developmental processes. The evolutionary conserved zinc finger protein ZNF503/Zeppo2 (zinc finger elbow-related proline domain protein 2, Zpo2) plays critical roles during embryogenesis. We found that Zpo2 is expressed in adult tissue and examined its function. We found that ZPO2 is a nuclearly targeted transcriptional repressor that is expressed in mammary epithelial cells. Elevated Zpo2 levels increase mammary epithelial cell proliferation. Zpo2 promotes cellular invasion through down-regulation of E-cadherin and regulates the invasive phenotype in a RAC1-dependent manner. We detect elevated Zpo2 expression during breast cancer progression in a MMTV-PyMT transgenic mouse model. Tumor transplant experiments indicated that overexpression of Zpo2 in MMTV-PyMT mammary tumor cell lines enhances lung metastasis. Our findings suggest that Zpo2 plays a significant role in mammary gland homeostasis and that deregulation of Zpo2 may promote breast cancer development. PMID:25538248

  7. Molecular mechanism of transcriptional repression of AhR repressor involving ANKRA2, HDAC4, and HDAC5

    SciTech Connect

    Oshima, Motohiko; Mimura, Junsei; Yamamoto, Masayuki; Fujii-Kuriyama, Yoshiaki

    2007-12-14

    The Aryl hydrocarbon receptor repressor (AhRR) has been proposed to inhibit Aryl hydrocarbon receptor (AhR) activity by competing with AhR for forming a heterodimer with AhR nuclear translocator (Arnt) and subsequently binding to the xenobiotic responsive elements (XRE). However, the precise mechanism of AhRR inhibitory activity remains unknown. Analysis of the inhibitory activity of AhRR on the expression of a TK promoter-driven reporter has localized a core repressor domain in the sequence of amino acid residue 555-701. The inhibitory activity of AhRR is sensitive to a histone deacetylase (HDAC) inhibitor, trichostatin A. By using the yeast two-hybrid screening method with the C-terminal sequence of AhRR as bait, we identified a binding partner, Ankyrin-repeat protein2 (ANKRA2), a protein known to interact with HDAC4 and HDAC5. RNA interference experiments using ANKRA2 and AhRR siRNAs indicate that ANKRA2 is important for transcriptional repression by AhRR. We have found that under normal conditions, CYP1A1 gene is kept silent in MEF cells by AhRR/Arnt heterodimer, which binds to the XRE sequence in its promoter and recruits ANKRA2, HDAC4, and HDAC5 as co-repressors.

  8. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

    PubMed

    Inui, Ken; Zhao, Zongpei; Yuan, Juan; Jayaprakash, Sakthidasan; Le, Le T M; Drakulic, Srdja; Sander, Bjoern; Golas, Monika M

    2017-02-20

    In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

  9. Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

    PubMed Central

    Rasmussen, Kim Krighaar; Frandsen, Kristian E. H.; Boeri Erba, Elisabetta; Pedersen, Margit; Varming, Anders K.; Hammer, Karin; Kilstrup, Mogens; Thulstrup, Peter W.; Blackledge, Martin; Jensen, Malene Ringkjøbing; Lo Leggio, Leila

    2016-01-01

    The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function. PMID:27403839

  10. Maternal Groucho and bHLH repressors amplify the dose-sensitive X chromosome signal in Drosophila sex determination.

    PubMed

    Lu, Hong; Kozhina, Elena; Mahadevaraju, Sharvani; Yang, Dun; Avila, Frank W; Erickson, James W

    2008-11-15

    In Drosophila, XX embryos are fated to develop as females, and XY embryos as males, because the diplo-X dose of four X-linked signal element genes, XSEs, activates the Sex-lethal establishment promoter, SxlPe, whereas the haplo-X XSE dose leaves SxlPe off. The threshold response of SxlPe to XSE concentrations depends in part on the bHLH repressor, Deadpan, present in equal amounts in XX and XY embryos. We identified canonical and non-canonical DNA-binding sites for Dpn at SxlPe and found that cis-acting mutations in the Dpn-binding sites caused stronger and earlier Sxl expression than did deletion of dpn implicating other bHLH repressors in Sxl regulation. Maternal Hey encodes one such bHLH regulator but the E(spl) locus does not. Elimination of the maternal corepressor Groucho also caused strong ectopic Sxl expression in XY, and premature Sxl activation in XX embryos, but Sxl was still expressed differently in the sexes. Our findings suggest that Groucho and associated maternal and zygotic bHLH repressors define the threshold XSE concentrations needed to activate SxlPe and that they participate directly in sex signal amplification. We present a model in which the XSE signal is amplified by a feedback mechanism that interferes with Gro-mediated repression in XX, but not XY embryos.

  11. Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages.

    PubMed

    Wang, Chenhui; Guo, Xingting; Dou, Kun; Chen, Hongyan; Xi, Rongwen

    2015-10-01

    In adult Drosophila midgut, intestinal stem cells (ISCs) periodically produce progenitor cells that undergo a binary fate choice determined primarily by the levels of Notch activity that they receive, before terminally differentiating into enterocytes (ECs) or enteroendocrine (EE) cells. Here we identified Ttk69, a BTB domain-containing transcriptional repressor, as a master repressor of EE cell specification in the ISC lineages. Depletion of ttk69 in progenitor cells induced ISC proliferation and caused all committed progenitor cells to adopt EE fate, leading to the production of supernumerary EE cells in the intestinal epithelium. Conversely, forced expression of Ttk69 in progenitor cells was sufficient to prevent EE cell specification. The expression of Ttk69 was not regulated by Notch signaling, and forced activation of Notch, which is sufficient to induce EC specification of normal progenitor cells, failed to prevent EE cell specification of Ttk69-depleted progenitors. Loss of Ttk69 led to derepression of the acheate-scute complex (AS-C) genes scute and asense, which then induced prospero expression to promote EE cell specification. These studies suggest that Ttk69 functions in parallel with Notch signaling and acts as a master repressor of EE cell specification in Drosophila ISC lineages primarily by suppressing AS-C genes. © 2015. Published by The Company of Biologists Ltd.

  12. The mammalian TRIM-NHL protein TRIM71/LIN-41 is a repressor of mRNA function.

    PubMed

    Loedige, Inga; Gaidatzis, Dimos; Sack, Ragna; Meister, Gunter; Filipowicz, Witold

    2013-01-07

    TRIM-NHL proteins are conserved regulators of development and differentiation but their molecular function has remained largely elusive. Here, we report an as yet unrecognized activity for the mammalian TRIM-NHL protein TRIM71 as a repressor of mRNAs. We show that TRIM71 is associated with mRNAs and that it promotes translational repression and mRNA decay. We have identified Rbl1 and Rbl2, two transcription factors whose down-regulation is important for stem cell function, as TRIM71 targets in mouse embryonic stem cells. Furthermore, one of the defining features of TRIM-NHL proteins, the NHL domain, is necessary and sufficient to target TRIM71 to RNA, while the RING domain that confers ubiquitin ligase activity is dispensable for repression. Our results reveal strong similarities between TRIM71 and Drosophila BRAT, the best-studied TRIM-NHL protein and a well-documented translational repressor, suggesting that BRAT and TRIM71 are part of a family of mRNA repressors regulating proliferation and differentiation.

  13. A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside.

    PubMed Central

    Baim, S B; Labow, M A; Levine, A J; Shenk, T

    1991-01-01

    LAP267 is a lacI activator protein (LAP) containing an insertion of the transcriptional activation domain of the herpes simplex virus virion protein 16 within the inducer-binding and dimerization domain of the lac repressor protein. LAP267 strongly induces expression in a conditional manner from a minimal simian virus 40 early promoter linked to lac operator sequences. LAP267 is temperature-sensitive, activating expression at 32 degrees C but not at 39.5 degrees C. It is allosterically regulated in a manner opposite that of wild-type lac repressor, in that LAP267 activity is rescued at the nonpermissive temperature by isopropyl beta-D-thiogalactopyranoside (IPTG). Stable mouse cell lines containing both the LAP267 gene and a LAP-inducible chloramphenicol acetyltransferase (CAT) reporter gene were readily established and exhibited up to a 1200-fold increase in CAT activity within 24 hr upon addition of IPTG. Thus, LAP267 is a powerful inducible switch in mammalian cells, imparting a regulatory stringency similar to that observed with lac repressor in Escherichia coli. Images PMID:2052587

  14. A Conserved Network of Transcriptional Activators and Repressors Regulates Anthocyanin Pigmentation in Eudicots[C][W][OPEN

    PubMed Central

    Albert, Nick W.; Davies, Kevin M.; Lewis, David H.; Zhang, Huaibi; Montefiori, Mirco; Brendolise, Cyril; Boase, Murray R.; Ngo, Hanh; Jameson, Paula E.; Schwinn, Kathy E.

    2014-01-01

    Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots. PMID:24642943

  15. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.

    PubMed

    Hiratsu, Keiichiro; Matsui, Kyoko; Koyama, Tomotsugu; Ohme-Takagi, Masaru

    2003-06-01

    The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. This chimeric repressor silencing technology (CRES-T), exploiting the EAR-motif repression domain, is simple and effective and can overcome genetic redundancy. Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors.

  16. The Cdk1 and Ime2 protein kinases trigger exit from meiotic prophase in Saccharomyces cerevisiae by inhibiting the Sum1 transcriptional repressor.

    PubMed

    Shin, Marcus E; Skokotas, Aikaterini; Winter, Edward

    2010-06-01

    The induction of middle meiotic promoters is a key regulatory event in the life cycle of Saccharomyces cerevisiae that controls exit from prophase, meiosis, and spore formation. The Sum1 repressor and Ndt80 activator proteins control middle promoters by binding to overlapping DNA elements. NDT80 is controlled by a tightly regulated middle meiotic promoter through a positive autoregulatory loop and is repressed in vegetative cells by Sum1. It has previously been shown that the meiosis-specific kinase Ime2 promotes the removal of Sum1 from DNA. Here, we show that Sum1 is also regulated by the cyclin-dependent kinase, Cdk1. While sum1 phosphosite mutants that are insensitive to Cdk1 or Ime2 complete meiosis and form spores, a mutant that is insensitive to both Ime2 and Cdk1 (sum1-ci) blocks meiotic development in prophase with an ndt80Delta-like phenotype. Ectopic expression of NDT80 or mutation of a Sum1-binding element in the NDT80 promoter bypasses the sum1-ci block. Hst1 is a NAD(+)-dependent histone deacetylase that is linked to Sum1 by the Rfm1 tethering factor. Deletion of HST1 or RFM1 also bypasses the sum1-ci block. These results demonstrate that Sum1 functions as a key meiotic brake through the NDT80 promoter and that Cdk1 and Ime2 trigger exit from meiotic prophase by inhibiting the Sum1 transcriptional repression complex.

  17. Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

    PubMed

    Pérez, Adam A; Gajewski, John P; Ferlez, Bryan H; Ludwig, Marcus; Baker, Carol S; Golbeck, John H; Bryant, Donald A

    2017-02-01

    Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn(2+)-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn(2+) uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster β-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002.

  18. The Cdk1 and Ime2 Protein Kinases Trigger Exit from Meiotic Prophase in Saccharomyces cerevisiae by Inhibiting the Sum1 Transcriptional Repressor

    PubMed Central

    Shin, Marcus E.; Skokotas, Aikaterini; Winter, Edward

    2010-01-01

    The induction of middle meiotic promoters is a key regulatory event in the life cycle of Saccharomyces cerevisiae that controls exit from prophase, meiosis, and spore formation. The Sum1 repressor and Ndt80 activator proteins control middle promoters by binding to overlapping DNA elements. NDT80 is controlled by a tightly regulated middle meiotic promoter through a positive autoregulatory loop and is repressed in vegetative cells by Sum1. It has previously been shown that the meiosis-specific kinase Ime2 promotes the removal of Sum1 from DNA. Here, we show that Sum1 is also regulated by the cyclin-dependent kinase, Cdk1. While sum1 phosphosite mutants that are insensitive to Cdk1 or Ime2 complete meiosis and form spores, a mutant that is insensitive to both Ime2 and Cdk1 (sum1-ci) blocks meiotic development in prophase with an ndt80Δ-like phenotype. Ectopic expression of NDT80 or mutation of a Sum1-binding element in the NDT80 promoter bypasses the sum1-ci block. Hst1 is a NAD+-dependent histone deacetylase that is linked to Sum1 by the Rfm1 tethering factor. Deletion of HST1 or RFM1 also bypasses the sum1-ci block. These results demonstrate that Sum1 functions as a key meiotic brake through the NDT80 promoter and that Cdk1 and Ime2 trigger exit from meiotic prophase by inhibiting the Sum1 transcriptional repression complex. PMID:20385771

  19. Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions

    PubMed Central

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Sun, Jing; Wang, Hang; Jiang, Li; Yang, Yongchun; Ma, Tiantian; Chen, Zhongwei; Yu, Jing; Fang, Weihuan; Song, Houhui

    2017-01-01

    Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti

  20. The role of GlpR repressor in Pseudomonas putida KT2440 growth and PHA production from glycerol.

    PubMed

    Escapa, I F; del Cerro, C; García, J L; Prieto, M A

    2013-01-01

    Pseudomonas putida KT2440 has evolved a tightly regulated system for metabolizing glycerol implying a prolonged growth lag-phase. We have learnt that this fact can be avoided by the addition of small amounts of some growth precursors. The addition of 1 mM octanoic acid as co-feeder completely eliminated the lag-phase, resulting in an improvement, in terms of invested time, of both growth and polyhydroxyalkanoates (PHA) accumulation. To investigate this phenomenon, we have followed co-metabolic approaches combined with mutations of the specific and global regulatory networks that connect glycerol catabolism and PHA synthesis. By using mutant strains in metabolic genes from the PHA and tricarboxylic acid (TCA) cycles, we have demonstrated that the co-feeding effect is independent of PHA accumulation, but driven on active glyoxylate shunt and Entner-Doudoroff (ED) routes. These findings suggested that the effect of octanoate on glycerol metabolism could rely, either on a global activation of the cell energy state, or on the generation of specific metabolites or cofactors needed for the activation of glycerol metabolism. Our results addressed GlpR as the key factor controlling the efficient utilization of glycerol as growth precursor in P. putida KT2440. Accordingly, a glpR knockout mutant of P. putida KT2440 showed an elimination of the lag-phase when cultured on glycerol in the absence of co-feeder. Besides, the production of PHA in this strain was increased near twofold, resulting in a higher final yield in terms of PHA accumulation. The repressor activity of the GlpR protein over the glp genes in the absence of glycerol was finally demonstrated by qRT-PCR. This work contributed to unravel the physiological causes of the long lag-phase produced by glycerol in the model strain P. putida KT2440 that hinders its use as carbon source in biotechnological applications for generating valuable products. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  1. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses.

    PubMed

    Gay, Gabrielle; Braun, Laurence; Brenier-Pinchart, Marie-Pierre; Vollaire, Julien; Josserand, Véronique; Bertini, Rose-Laurence; Varesano, Aurélie; Touquet, Bastien; De Bock, Pieter-Jan; Coute, Yohann; Tardieux, Isabelle; Bougdour, Alexandre; Hakimi, Mohamed-Ali

    2016-08-22

    An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.

  2. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

    PubMed

    Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

  3. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

    PubMed Central

    Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

  4. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses

    PubMed Central

    Brenier-Pinchart, Marie-Pierre; Bertini, Rose-Laurence; Varesano, Aurélie; De Bock, Pieter-Jan

    2016-01-01

    An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii–targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ–stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ–mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. PMID:27503074

  5. Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML

    PubMed Central

    Mak, P. Y.; Mak, D. H.; Mu, H.; Shi, Y.; Ruvolo, P.; Ruvolo, V.; Jacamo, R.; Burks, J. K.; Wei, W.; Huang, X.; Kornblau, S. M.; Andreeff, M.; Carter, B. Z.

    2014-01-01

    The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 versus 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2R null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy. PMID:24337870

  6. Ca2+-activated nucleotidase 1, a novel target gene for the transcriptional repressor DREAM (downstream regulatory element antagonist modulator), is involved in protein folding and degradation.

    PubMed

    Calì, Tito; Fedrizzi, Laura; Ottolini, Denis; Gomez-Villafuertes, Rosa; Mellström, Britt; Naranjo, Jose R; Carafoli, Ernesto; Brini, Marisa

    2012-05-25

    DREAM is a Ca(2+)-dependent transcriptional repressor highly expressed in neuronal cells. A number of genes have already been identified as the target of its regulation. Targeted analysis performed on cerebella from transgenic mice expressing a dominant active DREAM mutant (daDREAM) showed a drastic reduction of the amount of transcript of Ca(2+)-activated nucleotidase 1 (CANT1), an endoplasmic reticulum (ER)-Golgi resident Ca(2+)-dependent nucleoside diphosphatase that has been suggested to have a role in glucosylation reactions related to the quality control of proteins in the ER and the Golgi apparatus. CANT1 down-regulation was also found in neuroblastoma SH-SY5Y cells stably overexpressing wild type (wt) DREAM or daDREAM, thus providing a simple cell model to investigate the protein maturation pathway. Pulse-chase experiments demonstrated that the down-regulation of CANT1 is associated with reduced protein secretion and increased degradation rates. Importantly, overexpression of wtDREAM or daDREAM augmented the expression of the EDEM1 gene, which encodes a key component of the ER-associated degradation pathway, suggesting an alternative pathway to enhanced protein degradation. Restoring CANT1 levels in neuroblastoma clones recovered the phenotype, thus confirming a key role of CANT1, and of the regulation of its gene by DREAM, in the control of protein synthesis and degradation.

  7. Erwinia carotovora has two KdgR-like proteins belonging to the IciR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis.

    PubMed

    Thomson, N R; Nasser, W; McGowan, S; Sebaihia, M; Salmond, G P

    1999-07-01

    A novel Erwinia carotovora subsp. carotovora mutant designated RexZ, (regulator of exoenzymes) showed reduced production of the degradative exoenzymes. The rexZ gene product shows similarity of the KdgR regulatory protein from Erwinia chrysanthemi, described as the major repressor of the pectin catabolism pathway genes in the latter species. In vitro DNA-protein interaction experiments demonstrated that the synthesis of the RexZ protein is controlled by the cAMP-CRP (cAMP-receptor protein) complex. Western blot analysis also revealed the presence of a second KdgR homologue (distinct from RexZ) which, like RexZ, was present in all species of the genus Erwinia tested. The corresponding KdgR proteins from both E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica share a high level of sequence identity with the KdgR homologues from E. chrysanthemi and Escherichia coli. Although the E. carotovora subsp. carotovora rexZ regulatory region displayed specific interactions with both the purified E. chrysanthemi KdgR repressor and the partially purified E. carotovora subsp. carotovora KdgR, in vivo quantification revealed that the cellular level of RexZ protein was unaffected by the presence of pectic compounds. This study shows that the complex regulatory network governing virulence in the erwinias involves two totally distinct, but highly conserved, members of the IcIR class of DNA binding proteins: RexZ and KdgR.

  8. Functional Analysis of the Transcription Repressor PLU-1/JARID1B▿ †

    PubMed Central

    Scibetta, Angelo G.; Santangelo, Samantha; Coleman, Julia; Hall, Debbie; Chaplin, Tracy; Copier, John; Catchpole, Steve; Burchell, Joy; Taylor-Papadimitriou, Joyce

    2007-01-01

    The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G2/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B. PMID:17709396

  9. Investigation of Changes in Tetracycline Repressor Binding upon Mutations in the Tetracycline Operator

    PubMed Central

    2015-01-01

    The tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR–tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in Escherichia coli for a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild-type tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior. PMID:25308994

  10. Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia

    PubMed Central

    Nagel, Stefan; Pommerenke, Claudia; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G.; MacLeod, Roderick A.F.

    2016-01-01

    Recently, we identified deregulated expression of the B-cell specific transcription factor MEF2C in T-cell acute lymphoid leukemia (T-ALL). Here, we performed sequence analysis of a regulatory upstream section of MEF2C in T-ALL cell lines which, however, proved devoid of mutations. Unexpectedly, we found strong conservation between the regulatory upstream region of MEF2C (located at chromosomal band 5q14) and an intergenic stretch at 7q11 located between STAG3L4 and AUTS2, covering nearly 20 kb. While the non-coding gene STAG3L4 was inconspicuously expressed, AUTS2 was aberrantly upregulated in 6% of T-ALL patients (public dataset GSE42038) and in 3/24 T-ALL cell lines, two of which represented very immature differentiation stages. AUTS2 expression was higher in normal B-cells than in T-cells, indicating lineage-specific activity in lymphopoiesis. While excluding chromosomal aberrations, examinations of AUTS2 transcriptional regulation in T-ALL cells revealed activation by IL7-IL7R-STAT5-signalling and MEF2C. AUTS2 protein has been shown to interact with polycomb repressor complex 1 subtype 5 (PRC1.5), transforming this particular complex into an activator. Accordingly, expression profiling and functional analyses demonstrated that AUTS2 activated while PCGF5 repressed transcription of NKL homeobox gene MSX1 in T-ALL cells. Forced expression and pharmacological inhibition of EZH2 in addition to H3K27me3 analysis indicated that PRC2 repressed MSX1 as well. Taken together, we found that AUTS2 and MEF2C, despite lying on different chromosomes, share strikingly similar regulatory upstream regions and aberrant expression in T-ALL subsets. Our data implicate chromatin complexes PRC1/AUTS2 and PRC2 in a gene network in T-ALL regulating early lymphoid differentiation. PMID:27322685

  11. Plasticity in Repressor-DNA Interactions Neutralizes Loss of Symmetry in Bipartite Operators.

    PubMed

    Jain, Deepti; Narayanan, Naveen; Nair, Deepak T

    2016-01-15

    Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. To understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators, ORE1, ORX1, ORA1, and ORR3, the AraR-DNA binding domain (AraR-DBD) as well as full-length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07 and 1.97 Å resolution, respectively. These structures were compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with the symmetric operator, ORE1, are identical, but their interaction with the non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. ChIP-sequencing (ChIP-Seq) data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Tetracycline analogs affecting binding to Tn10-Encoded Tet repressor trigger the same mechanism of induction.

    PubMed

    Lederer, T; Kintrup, M; Takahashi, M; Sum, P E; Ellestad, G A; Hillen, W

    1996-06-11

    We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay. The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most tc analogs. While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold. A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively. Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction. Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction. The respective atc analog failed to induce as well, although binding was still observed. The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction. Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies. Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study. The equilibrium association constants of most TetR-[Mg-tc]+ and TetR-([Mg-tc]+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR. This suggests that these tc analogs share the same molecular mechanism of TetR induction.

  13. DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases

    PubMed Central

    Morales-Ruiz, Teresa; Ortega-Galisteo, Ana Pilar; Ponferrada-Marín, María Isabel; Martínez-Macías, María Isabel; Ariza, Rafael R.; Roldán-Arjona, Teresa

    2006-01-01

    Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-l-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants. PMID:16624880

  14. The RpiR-like repressor IolR regulates inositol catabolism in Sinorhizobium meliloti.

    PubMed

    Kohler, Petra R A; Choong, Ee-Leng; Rossbach, Silvia

    2011-10-01

    Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa, has the ability to catabolize myo-, scyllo-, and D-chiro-inositol. Functional inositol catabolism (iol) genes are required for growth on these inositol isomers, and they play a role during plant-bacterium interactions. The inositol catabolism genes comprise the chromosomally encoded iolA (mmsA) and the iolY(smc01163)RCDEB genes, as well as the idhA gene located on the pSymB plasmid. Reverse transcriptase assays showed that the iolYRCDEB genes are transcribed as one operon. The iol genes were weakly expressed without induction, but their expression was strongly induced by myo-inositol. The putative transcriptional regulator of the iol genes, IolR, belongs to the RpiR-like repressor family. Electrophoretic mobility shift assays demonstrated that IolR recognized a conserved palindromic sequence (5'-GGAA-N6-TTCC-3') in the upstream regions of the idhA, iolY, iolR, and iolC genes. Complementation assays found IolR to be required for the repression of its own gene and for the downregulation of the idhA-encoded myo-inositol dehydrogenase activity in the presence and absence of inositol. Further expression studies indicated that the late pathway intermediate 2-keto-5-deoxy-D-gluconic acid 6-phosphate (KDGP) functions as the true inducer of the iol genes. The iolA (mmsA) gene encoding methylmalonate semialdehyde dehydrogenase was not regulated by IolR. The S. meliloti iolA (mmsA) gene product seems to be involved in more than only the inositol catabolic pathway, since it was also found to be essential for valine catabolism, supporting its more recent annotation as mmsA.

  15. Plasticity in Repressor-DNA Interactions Neutralizes Loss of Symmetry in Bipartite Operators*

    PubMed Central

    Jain, Deepti; Narayanan, Naveen; Nair, Deepak T.

    2016-01-01

    Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. To understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators, ORE1, ORX1, ORA1, and ORR3, the AraR-DNA binding domain (AraR-DBD) as well as full-length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07 and 1.97 Å resolution, respectively. These structures were compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with the symmetric operator, ORE1, are identical, but their interaction with the non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. ChIP-sequencing (ChIP-Seq) data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits. PMID:26511320

  16. Biophysical Basis of the Promiscuous Binding of Bcl2 Apoptotic Repressor to BH3 Ligands

    PubMed Central

    Bhat, Vikas; Olenick, Max B.; Schuchardt, Brett J.; Mikles, David C.; McDonald, Caleb B.; Farooq, Amjad

    2013-01-01

    Bcl2 apoptotic repressor carries out its function by virtue of its ability to bind to BH3 domains of various pro-apoptotic regulators in a highly promiscuous manner. Herein, we investigate the biophysical basis of such promiscuity of Bcl2 toward its cognate BH3 ligands. Our data show that while the BH3 ligands harboring the LXXXAD motif bind to Bcl2 with submicromolar affinity, those with the LXXX[G/S]D motif afford weak interactions. This implies that the replacement of alanine at the fourth position (A+4)—relative to the N-terminal leucine (L0) within the LXXXAD motif—to glycine/serine results in the loss of free energy of binding. Consistent with this notion, the A+4 residue within the BH3 ligands harboring the LXXXAD motif engages in key intermolecular van der Waals contacts with A149 lining the ligand binding groove within Bcl2, while A+4G/S substitution results in the disruption of such favorable binding interactions. Of particular interest is the observation that while increasing ionic strength has little or negligible effect on the binding of high-affinity BH3 ligands harboring the LXXXAD motif, the binding of those with the LXXX[G/S]D motif in general experiences a varying degree of enhancement. This salient observation is indicative of the fact that hydrophobic forces not only play a dominant but also a universal role in driving the Bcl2-BH3 interactions. Taken together, our study sheds light on the molecular basis of the factors governing the promiscuous binding of Bcl2 to pro-apoptotic regulators and thus bears important consequences on the development of rational therapeutic approaches. PMID:23996493

  17. The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    PubMed Central

    Straub, Elke; Dreer, Marcel; Fertey, Jasmin; Iftner, Thomas

    2014-01-01

    Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8−), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8− and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in